Applicability of Multidimensional Fractionation to Affinity Purification Mass Spectrometry Samples and Protein Phosphatase 4 Substrate Identification

Wade Hampton Dunham

A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Molecular Genetics University of Toronto

Wade Dunham

Master of Science

Department of Molecular Genetics University of Toronto

2012 Abstract

Affinity-purification coupled to mass spectrometry (AP-MS) is gaining widespread

use for the identification of protein-protein interactions. It is unclear however,

whether typical AP sample complexity is limiting for the identification of all protein

components using standard one-dimensional LC-MS/MS. Multidimensional sample

separation is a useful for reducing sample complexity prior to MS analysis, and

increases peptide and protein coverage of complex samples, yet the applicability of

this approach to AP-MS samples remains unknown. Here I present work to show that

multidimensional separation of AP-MS samples is not a cost-effective method for

identifying increased peptide or protein coverage in these sample types. As such this

approach was not adapted for the identification of putative Phosphoprotein

Phosphatase 4 (PP4c) substrates. Instead, affinity purification coupled to one-

dimensional LC-MS/MS was used to identify putative PP4c substrates, and semi-

quantitative methods applied to identify possible PP4c targeted phosphosites in PP2A

subfamily phosphatase inhibited (okadaic acid treated) cells. iii

Acknowledgments

I would like to acknowledge and thank everyone in the Gingras lab, in particular my supervisor Anne-Claude, for allowing me to take on complex projects and to be an integral part in the pursuit of high impact publications. I would also like to thank our lab manager Marilyn, for patiently letting me know where I can find my much needed reagents, although initially, I likely asked her the same question at least once a month, and Brett for his initial training and work, which helped me to secure my first publication. I must also thank both of my committee members; Drs. Ben Blencowe and Thomas Kislinger for their helpful insight and guidance over the course of my projects. The past two and a half years have definitely been a great learning experience. Thanks to everybody! iv

Table of Contents

Chapter 1: Introduction…………………………………………………………..……..1 1.1 General Introduction and thesis overview……..………………………………….1 1.2 Identification of Proteins by Mass Spectrometry………………………………....2 1.3 Affinity-purification Coupled to Mass Spectrometry (AP-MS)………………...... 6 1.4 Affinity-purification using epitope tags…………………………………………...9 1.5 Background contaminants in AP-MS……………………………………………13 1.5.1 Strategies to remove the contaminants from the sample before mass spectrometry……………………………………………………………...14 1.5.2 Strategies to remove the contaminants during or after MS analysis: Label-free approaches……….…………………………………………..15 1.6 Fractionation of mass spectrometry samples………………………….…………17 1.7 Utilizing MS for identification of protein phosphorylation……………………...19 1.7.1 Phosphopeptide enrichment approaches and identification……..……….20 1.7.2 Label free phosphopeptide quantification and phosphosite localization…………………………………………………………..…...24 1.8 PP2A subfamily phosphatases…………………………………………..……….31 1.8.1 PP4c biology and regulation………...…………….…………………...... 31 1.8.2 PP4c interactions and substrate identification……..…………………….39 1.8.3 PP4c regulation of mRNA transcription and splicing……..……...……...49 1.9 Thesis objectives…………………………………………………………………52

Chapter 2: A cost-benefit analysis of multidimensional fractionation of affinity purification-mass spectrometry samples……………………………..….54 2.1 Methods………………………………………………………………………….55 2.1.1 Generation and culture of stably transfected Flp-In T-REx 293 cell lines……………………………………………………..55 2.1.2 Affinity purification……………………………………………………...56 2.1.3 One dimensional (1D) LC-MS/MS analysis……………………………..57 2.1.4 Multidimensional LC-MS/MS analysis……………………………….…57 v

2.1.4.1 MudPIT…………………………………………………………..58 2.1.4.2 RP/RP…………………………………………………………….58 2.1.4.3 GeLC……………………………………………………………..59 2.1.5 Data Analysis………………………………………………….……..…..59 2.2 Results……………………………………………………………………………60 2.2.1 Reproducibility of protein identifications made by AP-MS……………..64 2.2.2 Effect of fractionating affinity purified samples on spectral count, unique peptide, and protein identification…..…………………………………...69 2.2.3 Effect of fractionating affinity purified samples on protein complex component identification.………………………………………………..75 2.2.4 Applying Significance Analysis of INTeractome (SAINT) to fractionated affinity purified samples…………………………………………………82 2.3 Discussion………………………………………………………………………..84 2.3.1 Multidimensional fractionation of AP-MS samples appears to allow for a better depth of coverage of low level background proteins and not core protein complex components....……………………………………...…..84 2.3.2 Primary benefits and disadvantages of multidimensional fractionation of AP-MS samples…..……..……………………………………………….85 2.3.3 Applicability of multidimensional fractionation of AP-MS samples to the expansion of the PP4c network and substrate identification………….....86 2.3.4 Conclusions………………………………………………………………87

Chapter 3: PP4c interactor/subunit phosphosite identification…………………...... 88 3.1 Methods………………………………………………………………………….88 3.1.1 Generation and culture of stably transfected Flp-In T-REx 293 cell lines………………………………………………...... 88 3.1.2 Affinity purification……………………………………………………...91 3.1.3 Enrichment of phosphopeptides…………………………………….....…91 3.1.4 LC-MS/MS analysis…………………………………………………..…92 3.1.5 Data Analysis……………………………………………………….....…93 3.2 Results……………………………………………………………………………94 vi

3.2.1 PP4c interactor/subunit phosphosite identification………………...…….94 3.2.2 Phosphosite detection reproducibility……………….……………….....104 3.2.3 Changes in PP4c interactor phosphorylation upon PP2A subfamily phosphatase inhibition…………...………………….……………….…114 3.2.4 Reproducibility of phosphosite quantification across biological replicates…………….…………...………………….……………….…136 3.3 Discussion……………………………………………………………………....141 3.3.1 Discerning whether PP4c interactors are possible substrates for the enzyme……..…………………………………………………………...141

Chapter 4: Thesis Summary and Future Directions…………………………….….144 4.1 Thesis Summary……………………………………………………………...…144 4.2 Future Directions…………………………………………………………….....145 4.2.1 PP4c substrate identification……...……………...…………………...... 145 4.3 Conclusions………………………………………………………………….….150

References……………………………………………..…………………………….…152 vii

List of Tables

Table 1-1. Select peptide, protein, and dual affinity tags successfully used for purification of recombinant proteins in AP-MS studies………………………………………………10

Table 1-2. Classification of human protein phosphatases……………………………….32

Table 1-3. Gene and protein identifiers for PP4c, PP4c regulatory subunits, and interacting proteins investigated and discussed in detail in this thesis…………..………38

Table 2-1. Summary of the mass spectrometry data for this project.…....………..……..63

Table 2-2. Spectral counts, unique peptides, and non-redundant protein identification (A) for all proteins identified in COPS5 samples after background contaminant removal, (B) for the COPS5 interactors reported in BioGRID and detected in our samples, or (C) for all proteins prior to background contaminant removal..…………………………………65

Table 2-6. Proteins identified in "Core" interaction network of FLAG-COPS5 purifications, that are not annotated as COPS5 interactors in BioGRID…………...……76

viii

Table 2-7. Fold increase in spectral counts (A), or unique peptides (B) for BioGRID- annotated COPS5 interactors..……………………………………………………...……78

Table 2-8. A) Fold increase in spectral counts or unique peptides shown as the ratio of 2D/1D for BioGRID-annotated EIF4A2 interactors for each of the biological replicates analyzed (replicates annotated 1 and 2). B) Fold increase in spectral counts or unique peptides shown as the ratio of 2D/1D for BioGRID-annotated RAF1 interactors for each of the biological replicates analyzed (replicates annotated 1 and 2)...…….…………….80

Table 2-9. Fold increase in spectral counts or unique peptides shown as the ratio of 2D/1D for BioGRID-annotated MEPCE interactors………………………………..…...81

Table 3-1. FLAG tagged constructs and stable cell lines generated for identification of puataive PP4c substrates………………………………………………………………....90

Table 3-2. Summary table listing phosphopeptides identified for PP4c interacting proteins or regulatory subunits..…………………………………………….………………..…...98

Table 3-3. Quantification of phosphopeptides identified in biological replicate analysis of FLAG-DHX38…………………………………………………….……………………115

Table 3-4. Quantification of phosphopeptides identified in biological replicate analysis of FLAG-HTASF1..………………………………………………….……………………119

Table 3-5. Quantification of SUPT5H phosphopeptides identified in biological replicate analysis of FLAG-SUPT4H……………………………………….……………………124

Table 3-6. Fold change in DHX38 phosphosite abundance upon okadaic acid treatment………………………………………………….……….…………………....129

Table 3-7. Fold change in HTATSF1 phosphosite abundance upon okadaic acid treatment………………….……………………………………….……………………131

Table 3-8. Fold change in SUPT5H phosphosite abundance upon okadaic acid treatment………………….……………………………………….……………………134 ix

Table 3-9. Reproducibility of DHX38 phosphopeptide quantification across biological replicates………………….……………………………………….……………………137

Table 3-10. Reproducibility of HTATSF1 phosphopeptide quantification across biological replicates……....……………………………………….……………………138

Table 3-11. Reproducibility of SUPT5H phosphopeptide quantification across biological replicates………………….……………………………………….……………………139

List of Figures

Figure 1-1. Schematic overview of protein identification by mass spectrometry………...4

Figure 1-2. Affinity purification coupled to mass spectrometry……………………….…8

Figure 1-3. Peptide fragmentation induced by collision-induced dissociation (CID)...…22

Figure 1-4. Proteome Discoverer PhosphoRS algorithm phosphosite localization and scoring……………………………………..………..……………………………………29

Figure 1-5. Phylogenetic tree of PPP family of phosphatases…………………………...34

Figure 1-6. PP4c regulatory subunits………..…………………………...………………37

Figure 1-7. PP4c Network highlighting selective high-confidence interactions

(SAINT > 0.8)..…………………………………………………………………………..40

Figure 1-8. Network highlighting the interactions of SUPT5H-SUPT4H-RNGTT and PP4c-PP4R2-PP4R3A complex………………………………………………………….42

Figure 1-9. Network highlighting the interactions of DHX38-PRP19-U5 snRNP and PP4c-PP4R2-PP4R3A complex……………………………………………...…………..43

Figure 1-10. Network highlighting the interactions of SLC4A1AP-HTATSF1-U2 snRNP and PP4c-PP4R2-PP4R3A complex……………………………………………………..45

Figure 1-11. PP4R3A targeting of transcription and splicing factors occurs through its EVH1 domain……………………………………………………………………………47

Figure 1-12. Real-time PCR analysis of FOSB, JUNB mRNA expression.....…...……...51

Figure 2-1. Sample preparation…………………………………………………………..61

Figure 2-2. Venn diagrams showing protein identification overlap for COPS5…………68 xi

Figure 2-3. Venn diagram of SAINT result overlap in (A) EIF4A2 and (B) RAF1 sample analysis…………………………………………………………………………..83

Figure 3-1. Experimental workflow for PP4c interacting protein or regulatory subunit phosphopeptide identification…………………………………………………………...95

Figure 3-2. Venn diagrams showing phosphosite identification overlap for DHX38, HTATSF1, and SUPT5H………………………...……………………………………..105

Figure 3-3. Venn diagrams showing PhosphoSitePlus phosphosite identification overlap for DHX38, HTATSF1, and SUPT5H………………………………………………….107

Figure 3-4. DHX38 protein diagram illustrating phosphosites identified in biological replicate analysis of FLAG-DHX38………………………...………………………….108

Figure 3-5. HTATSF1 protein diagram illustrating phosphosites identified in biological replicate analysis of FLAG-HTATSF1…………...…………………………………….110

Figure 3-6. SUPT5H protein diagram illustrating phosphosites identified in biological replicate analysis of FLAG-SUPT4H………………...………………………………...112

Figure 3-7. Quantification of phosphopeptides reproducibly identified in biological replicate analysis of FLAG-DHX38. …………...……………………………………...117

Figure 3-8. Quantification of phosphopeptides reproducibly identified in biological replicate analysis of FLAG-HTATSF1……………..………………………………...... 122

Figure 3-9. Quantification of SUPT5H phosphopeptides reproducibly identified in biological replicate analysis of FLAG-SUPT4H...……………………...……………...126

Figure 3-10. Gel shift assay for identifying putative PP4c substrates by monitoring protein de-phosphorylation in the absence of PP4c…...………………………………..148 xii

List of Appendices

Appendix 1. Effect of siRNA directed depletion of PP4c on gene expression and RNA splicing………………………………………………………………………..….175 A1.1 Materials and Methods………………………………………………………….176 A1.1.1 Cell culture and siRNA directed PP4c depletion………….……………176 A1.1.2 Western blotting…………………………...……………………………177 A1.1.3 RNA isolation, reverse transcription, PCR, and qRT-PCR.……………177 A1.1.4 RNAseq analysis of alternate exon inclusion…………...………….…..180 A1.1.5 Splicing Assay….………………………………………………………180 A1.1.6 Data Analysis……..……………………………..…………………...…181 A1.2 Results……………………………………………………………………….…181 A1.2.1 Effect of PP4c depletion on gene expression……..……...……………..181 A1.2.2 Effect of PP4c depletion on RNA splicing……...…..…………………195 A1.3 Discussion…………………………………………………………………...….205 A1.3.1 siRNA off target effects and proper siRNA controls…..………………205 A1.4 Conclusions and Future Directions…………………………………………..…209 A1.4.1 PP4c regulation of Transcription and Splicing………..…..……………209

Appendix 1. List of Tables

Table A1-1. PCR primers used for gene expression analyses by qRT-PCR.....……..…179

Table A1-2. RNAseq genes demonstrated to be differentially expressed upon siPP4c ± EGF treatment selected for validation by qRT-PCR………..…..…………………...…190

Table A1-5. RNAseq genes demonstrated to be differentially spliced upon siPP4c ± EGF selected for validation by RT-PCR assay…………..…..………………………………197

Appendix 1. List of Figures

Figure A1-1. PP4c protein and mRNA levels after siRNA and EGF treatment..…..…..183

Figure A1-2. FOSB expression upon siRNA and epidermal growth factor (EGF) treatment..………..…..…………………………………………………………………186

Figure A1-3. JUNB expression upon siRNA and epidermal growth factor (EGF) treatment..………..………………………………………………………..……………187

Figure A1-4. NUMA1 and WBSCR1 alternate exon inclusion after siRNA and EGF treatment..……………………………………………………………..…..……………198

Figure A1-5. NUMA1 alternate exon inclusion after siRNA and EGF treatment..…….200

Figure A1-6. WBSCR1 alternate exon inclusion after siRNA and EGF treatment……..202

Appendix 2. A cost-benefit analysis of multidimensional fractionation of affinity purification-mass spectrometry samples……………………………………….……212 A2.1 Supplementary Tables Table S2-1. A) List of the proteins removed because they are listed as "frequent fliers" in our internal FLAG AP-MS HEK293 cell database (contains >1000 AP-MS analyses). B) Proteins removed from subsequent analysis because they were detected in FLAG alone negative Proteins identified in FLAG-COPS5 purifications after background removal……………...... …………………………212 Table S2-2. Proteins identified in FLAG-COPS5 purifications after background removal…………….....………………………………217 Table S2-3. Proteins identified in FLAG-EIF4A2 purifications after background removal….…...………………….……………………228 Table S2-4. Proteins identified in FLAG-RAF1 purifications after background removal……………………….………………………233 xiv

Table S2-5. Proteins identified in FLAG-MEPCE purifications after background removal…….…………………………………………238 Table S2-6. SAINT analysis of proteins identified in FLAG-EIF4A2 or FLAG-RAF1 purifications………………..……….……..……...... 240 1

Chapter 1 Introduction 1.1 General Introduction and Thesis Overview

In 2005, the Aebersold lab, where Dr. Gingras was a post doctoral fellow, discovered a novel mammalian trimeric complex containing protein phosphatase 4 (PP4c), a serine-threonine phosphatase conserved throughout eukaryotic evolution and involved in resistance to cisplatin, one of the oldest anticancer drugs [1]. This trimeric complex was demonstrated to consist of PP4c and the PP4c regulatory subunit PP4R2, in addition to one of two novel proteins PP4R3A or PP4R3B, now known to function in PP4c substrate targeting. Furthermore, Gingras et al. [1] went on to demonstrate that a similar complex is functional in yeast (comprised of the PP4c ortholog Pph3, the PP4R2 ortholog Ybl046w and the PP4R3A ortholog Psy2), deletion of which reduces cell viability following cisplatin induced DNA damage. In addition, they demonstrated that mammalian PP4R3A was able to revert the cisplatin hypersensitivity of a psy2∆ yeast strain indicating the human and yeast proteins are functionally equivalent and that a reduction in PP4R3A activity in Drosophila (flfl encodes the fly homolog of the PP4R3A and Psy2 proteins) renders them hypersensitive to cisplatin, indicating that this PP4c containing trimeric complex may function in a conserved role in DNA damage repair from yeast to higher eukaryotes, in addition to facilitating cisplatin resistance in mammalian cells. Hereafter this PP4c trimeric complex will be referred to as PP4c- PP4R2-PP4R3A.

To begin to understand how the PP4c-PP4R2-PP4R3A complex outlined above is linked to the cisplatin resistance phenotype and to uncover its physiological role in mammalian cells, a graduate student of the Gingras lab, Ginny Chen, extensively characterized the cellular context in which the components of this complex reside using affinity purification coupled to mass spectrometry (AP-MS). What Ginny discovered was that the components of the PP4c-PP4R2-PP4R3A trimeric complex associate with components of the splicing and transcription elongation machineries, especially PP4R3A, which was observed to localize to nuclear speckles (thought to be storage sites for the transcription and splicing machineries) in a transcription dependant manner. 2

Additionally, she demonstrated a positive role for PP4c in the regulation of mRNA transcription following epidermal growth factor (EGF) stimulation, in addition to a role for PP4c in the regulation of RNA splicing (both explained in more detail in section 1.8). Based on these discoveries, I postulated that in concert with its associated partners PP4R2 and PP4R3A, PP4c may serve as a master controller in the processes of splicing and transcription.

I begin this thesis with a general overview of protein identification by mass spectrometry and AP-MS (most of which is part of a review in Proteomics; in press), as these methods were used to generate the data presented in chapter 2 and 3, and by Ginny Chen to generate the PP4c interaction network presented in section 1.8. Next I move into the applicability of multidimensional fractionation methods (demonstrated to increase peptide and protein coverage of complex MS samples), to the identification of additional components of protein complexes in AP-MS samples (presented in chapter 2 and published in Proteomics [2]). At the onset of my studies, I was interested in testing whether fractionation of AP-MS samples could uncover new components of protein complexes. If this were the case, I would have re-interrogated the PP4c interactome in an attempt to expand the PP4c interaction network generated by Ginny Chen and further our understanding of PP4c regulation of transcription and splicing. Next I discuss methods for identifying protein phosphosites using mass spectrometry, and for label free quantification of changes in protein phosphorylation, techniques I used as an initial step towards determine the enzyme-substrate relationship between PP4c and its interaction partners (presented in chapter 3). Lastly, I end this introduction with background on PP4c and a summary of the work done by Ginny Chen, setting the stage for my thesis rational and a presentation of my thesis objectives. My thesis summary and future directions are presented in chapter 4. The role of PP4c in regulating mRNA transcription and splicing is further investigated using PCR-based assays and presented in appendix 1.

1.2 Identification of Proteins by Mass Spectrometry

Mass spectrometry (MS) has become the analytical technique of choice for the identification of proteins in biological samples. In general terms, mass spectrometers 3 measure the mass to charge (m/z) ratio of charged molecules in gas phase, allowing for the determination of their molecular masses after charge state resolution. Over the years, several improvements to mass spectrometry systems have made the analysis of large molecules, such as peptides or proteins, possible (e.g the advent of improved methods for large molecule ionization (e.g electrospray ionization), and separation and purification (e.g. high performance liquid chromatography), has allowed for increased gains in peptide/protein ionization and identification, allowing for sequencing of proteins/peptides in the femtomolar range; see [3-5] for a historical perspective). While mass spectrometric analysis of intact proteins is feasible [6-8], in most cases, identification of proteins by mass spectrometry first involves proteolysis with a site-specific enzyme to generate peptides. Most modern mass spectrometers measure m/z in two steps (tandem mass spectrometry): in a first step, also called survey scan or MS1 scan, the m/z of the intact ionized peptide (often referred to as the parent peptide) is monitored. Following isolation of peptide(s) of interest (most often based on their abundance in the survey scan), the peptides are fragmented and the m/z ratios of the resulting fragments are measured by the mass spectrometer. This second spectrum is referred to as MS/MS or MS2 spectrum. Fragmentation of the peptide ion along the peptide bond results in diagnostic fragments that can be used, with the assistance of software tools, to deduce the amino acid sequence of the parent peptide. These identified peptides are then employed, again with the help of specifically designed software, to reconstitute the protein composition of a sample (Figure 1-1).

Figure 1-1 Schematic overview of protein identification by mass spectromtery. (A) Protein mixtures are subjected to proteolytic digestion, usually by trypsin, resulting in a mixture of peptides. (B) MS analysis of the digested peptide mix produces a mass (m) to charge (z) ratio (m/z) for each peptide, and a measure of its relative abundance (approximately proportional to the signal intensity for the same peptide across samples). (C) Peptides selected on the basis of their abundance are fragmented by tandem mass spectrometry (MS/MS). (D) The identity of fragmented peptides is obtained in most cases by search engines which compare the pattern of fragment ions in the MS/MS spectrum to the theoretical patterns obtained following in silico fragmentation of all protein sequences in a reference database. Protein identification is obtained by mapping back identified peptides onto proteins.

Importantly, for interaction proteomics experiments, modern mass spectrometers are now very fast (with upwards of 20 fragmentation events per second), enabling the sequencing of multiple peptides as they enter the mass spectrometer. In most approaches, tandem mass spectrometry is directly coupled to high performance liquid chromatography, using nano-scale reversed-phase columns (50-100 microns in diameter). Peptide mixtures loaded onto these HPLC columns are eluted directly (as they are ionized) into the mass spectrometer. Together with the high sensitivity and fast duty cycle of mass spectrometers, liquid chromatography separation enables the identification of thousands of peptides and proteins in a single LC-MS analysis (see the excellent primer on proteomics by Steen and Mann for details [9]). When analysis of very complex samples is required, other fractionation approaches, either at the protein or peptide level, can be coupled to LC-MS/MS (discussed in more detail in section 1.6).

With modifications to purification protocols, mass spectrometry can be made fully compatible with affinity purification, enabling characterization of protein-protein interactions in a near-physiological context (see sections 1.3, 1.4). Combined with quantitative approaches, affinity purification coupled to mass spectrometry (AP-MS) can identify condition-specific interactions, allowing for a dynamic view of the interactome. Additionally, because AP-MS isolates and enriches proteins, it can allow for posttranslational modifications (PTMs) to be identified either with [10-15] or without [11, 12] further PTM enrichment, enabling in depth assessment of the role these modifications in regulating interactions (discussed in more detail, specifically in the context of protein phosphorylation in section 1.7).

1.3 Affinity-purification Coupled to Mass Spectrometry (AP-MS)

In general terms, “affinity purification” refers to the capture of biological material via specific enrichment with a ligand coupled to a solid support [15]. Many types of ligands can be used in affinity purification, including DNA and RNA molecules (most often oligonucleotides), chemicals [10, 13, 16], lipids, peptides or proteins [17-21], with some of the most widely used ligands for affinity purification being antibodies. For the purpose 7 of this thesis, I will focus on the detection of protein-protein interactions using antibodies targeting epitope-tagged proteins. However, in all cases, the general idea is to immobilize a ligand on a solid support (most often on agarose or magnetic beads), and to use this coupled ligand to capture target protein(s) from a soluble phase. Once purified, proteins can be processed for direct analysis by MS (Figure 1-2), or, fractionated to reduce sample complexity (discussed further in section 1.6).

Figure 1-2. Affinity purification coupled to mass spectrometry. In a generic AP-MS experiment: (A) cells expressing a protein of interest are cultured and harvested (alternatively, tissues are homogenized) allowing, (B) the purification of the protein by an affinity reagent targeting an epitope tag or the protein itself. (C) The targeted protein is then isolated from non-specific proteins through a series of washes before, (D) the protein is eluted and digested with a protease, or digested directly on the affinity reagent support matrix. (E) The resulting peptides are then separated by liquid chromatography and identified by mass spectrometry. Control experiments (e.g. cells expressing an epitope tag fused to an irrelevant protein) can be analyzed or processed in parallel to identify proteins that interact with the epitope tag or affinity matrix. For confidence in the interactions, the process is repeated to generate biological replicates of the dataset, including the negative controls.

1.4 Affinity-purification using epitope tags

A widespread technique that is particularly well-suited for high-throughput experiments, but also commonly used for projects of more limited scope, is the use of epitope-tagging. Epitope-tagging consists of fusing DNA sequences to an Open Reading Frame (ORF) of choice to express a peptide or protein tag that can be purified efficiently on a support material. The epitope tag may be fused to either the N or C-terminus of a protein (or even incorporated into the middle of the protein sequence [22]). The epitope tag can be translated directly into the affinity handle, or may be used for further modification, e.g. a biotinylatable sequence can be fused to a bait protein such that biotinylation can occur in vivo following co-expression of a biotin ligase such as the bacterial BirA protein [23]. In any case, the tagged protein can be purified using an affinity matrix that specifically recognizes the epitope [24, 25].

One main advantage of epitope tagging is that multiple proteins can be tagged with the same epitope and purified in an identical manner. Thus, background contaminants should be consistent across all purifications, enabling the use of streamlined control experiments. As shown in Table 1-1, many different types of tags (or tag combinations) have thus far been successfully applied to AP-MS, and it now seems that the main limitation is to fully characterize the affinity matrix / epitope tag interaction properties.

Table 1-1. Select peptide, protein, and dual affinity tags successfully used for purification of recombinant proteins in AP-MS studies.

PEPTIDE TAGS

Examples of tag Elution Original use in AP-MS Tag Sequence Affinity Resin Conditions* Reference Studies c-myc EQKLISEEDL Anti-c-myc Low pH [26] [27] (9E10)

FLAG DYKDDDDK Anti-FLAG FLAG [28] [2, 29, 30] (M1,M2,M5) peptide/pH/EDTA

HA YPYDVPDYA Anti-HA HA peptide [31] [32] (12CA5)

His-Tag HHHHHH Ni2+-NTA Imidazole/Low pH [33] [34] Co2+-CMA

Strep-tag II WSHPQFEK Streptavidin Desthiobiotin [35] [36, 37]

PROTEIN TAGS

Examples of tag Original use in AP-MS Tag Size Affinity Resin Elution Conditions Reference Studies

GST 26 kDA Glutathione Reduced [38] [39, 40] Glutathione

GFP 26.9 kDA Anti-GFP pH [41] [42-45]

Protein A 45 kDA** IgG Protein A/Low pH [46] [47, 48]

DUAL AFFINITY TAGS

Tag Examples of tag use in AP-MS Studies 11

FLAG- [49] STREPII- YFP

FLAG-CBP [50, 51]

FLAG-Biotin [52]

TAP [50, 53-57]

SF-TAP [58-60] (STREPII- FLAG)

GS-TAP (Prot [61, 62] G-STREPII)

SH [63, 64] (STREPII- HA)

His-GFP [65]

In an epitope-tagging experiment, important concerns relate to detrimental effects potentially mediated by the presence of a tag or the expression level of exogenously expressed recombinant proteins. Massively over-expressed or tagged proteins may exhibit protein misfolding, mislocalization or misregulation, leading to increases in both false positives and false-negatives [66-68].

To alleviate the issue of over-expression, a popular strategy in S. cerevisiae has consisted of inserting a protein tag at the endogenous locus using homologous recombination [69]. Homologous recombination provides the next best thing to an endogenous protein, as far as expression levels and regulation are concerned, since the tagged proteins remain under the regulation of the native promoter and many regulatory elements (note that in S. cerevisiae most tagging is at the C-terminus, leaving the promoter and 5’UTR intact, but destroying the 3’UTR). Another advantage of homologous recombination is that the entire population of the bait protein is now recombinant, and that the native protein cannot compete for association with interaction partners (this in theory would increase sensitivity of detection).

While it has gained prominence in yeast (Strain collections in which most yeast ORFs were epitope-tagged with a GFP tag or with a Tandem Affinity Purification tag by homologous recombination enabled comprehensive surveys of the yeast proteome to be done several years ago [54, 70, 71]), homologous recombination can also been applied to interaction studies in mammalian cells. For example, mouse knock-in technologies have been used to successfully identify interaction partners for the post-synaptic density protein PSD-95 [72], and for the tumor suppressor p53 [73]. However, these methods are quite complex and simpler alternative strategies have been developed. In the simplest implementation, a tagged protein can be expressed at near endogenous levels via the use of a weak promoter (often in the context of a viral infection) or following stable transfection [32, 74-76]. A variation on this theme consists of the use of inducible promoters to better control expression levels. While many different inducible systems have been used over the years for AP-MS, as in [17, 55, 63], a particularly robust inducible expression system is derived from the Flp-In T-REx system (Invitrogen), in which isogenic lines can be established and protein expression controlled via a 13 tetracycline-inducible promoter. This strategy, which results in a single integration of the tagged protein in a constant chromosomal location, permits establishment of relatively homogenous cell populations, and tunable expression. It has been used successfully by different groups, using different tagging systems, and was used to generate the cell lines described in this thesis [2, 63, 77].

It is important to keep in mind, however, that no matter how carefully controlled protein expression is, the absence of regulatory elements and the fact that the protein is affixed with a tag, will in some cases affect function and/or interactions. The fact that ~18% of yeast strains in which an essential gene is tagged at its C-terminus with the tandem-affinity purification (TAP) tag are non-viable point to potential problems associated with epitope tagging [54]. Whether this is due to the large size of the TAP tag or to its localization at the C-terminus of the proteins has not been systematically investigated. To control for the effects of protein tagging and over-expression, many of the interaction networks being generated at the moment include reciprocal purifications. In this context, a protein which was identified as an interaction partner for a given bait is in turn tagged and used as a bait. Thus, in one case (when used as bait) the protein is indeed tagged and expressed in the absence of physiological regulation; in the reciprocal purification, it is present at physiological levels and untagged. If, in both instances, an interaction can be detected between the proteins, it provides evidence that it is not the epitope tag or over-expression itself that is mediating the interaction. Of note, the high degree of complementarities detected between the interaction profiles of reciprocally tagged proteins strongly argues that whatever the effects of tagging and over-expression are, they are not such as to generate completely different networks [70, 74]. This approach was utilized by Ginny Chen to generate the Protein Phosphatase 4 interaction network (described in section 1.8).

1.5 Background contaminants in AP-MS

Although affinity purification exploits specific molecular recognition, background proteins are always also co-purified. For example, non-specific binding of proteins to the antibody or solid matrix can significantly contribute to the purification and identification 14 of proteins that do not specifically interact with a protein of interest. These “background contaminants” can make it difficult to distinguish true low-level interactors from non- specific interactors and, as such, effective methods for the identification of contaminants are constantly being developed. Contaminants in AP-MS experiments can be dealt with in several ways, broadly partitioned in two categories: up-front reduction of the contaminants and concomitant or post-analysis subtraction of contaminants.

1.5.1 Strategies to remove the contaminants from the sample before mass spectrometry

Up-front removal of contaminants includes all the biochemical approaches that are meant to prevent contaminant proteins from being identified in AP-MS. Classically, the inclusion of stringent washing steps as part of the purification procedures (e.g. salts and detergents) can help to minimize background. This is not always ideal however, as increasing the stringency of the purification procedures may lead to false negatives, whereby true weaker interactions are lost. Alternatively, approaches which should not directly impact true interactors can also be employed. For example, modification of the support matrix (e.g. sepharose versus magnetic beads, or the use of different antibodies) may also result in differences in contaminants, as support characteristics including size, hydrophobicity, porosity, and antibody density may all have an impact on purification specificity. Recent studies have also indicated that performing shorter purifications, and using less support matrix may also help enrich for specific interactions [42, 47, 78]. Another very useful strategy to decrease background contaminants is the incorporation of multiple enrichment steps in the purification procedure, such as in the Tandem Affinity Purification (TAP) protocol [57], which has notably been employed to characterize the S. cerevisiae interactome [70, 71]. However, it must be noted that performing multiple affinity purifications may be detrimental to the conservation of more transient and/or weaker interactions, as was demonstrated previously [79]. Importantly, it must also be noted that although the methods described above reduce background (sometimes drastically), it is virtually impossible to effectively eliminate all non-specific interactions that may occur during affinity purification; hence proper controls are still required. 15

1.5.2 Strategies to remove the contaminants during or after MS analysis - Label-free approaches

Even in the case of purifications in which a lot of background contaminants are detected, it may be feasible to use quantitative proteomics to assist in distinguishing true interactors from background contaminants. In recent years, multiple research groups have converged on the idea that simply removing proteins from an interaction list because they were detected in the control runs, or frequently seen across purifications, may have led to an over filtering of the data, and that a better way to proceed would be to monitor quantitative enrichment in the mass spectrometer (a true interactor should be enriched throughout purification).

Initial approaches for monitoring interactor enrichment relied on isotopic labeling. A popular approach to perform isotopic labeling and quantitative proteomics is referred to as stable Isotope Labeling with Amino acids in cell Culture (SILAC). SILAC is a type of metabolic labeling that has been successfully used for quantitative proteomics projects, including background subtraction in AP-MS experiments. In essence, cells are grown for several generations in the presence of isotopically-labeled amino acids (e.g. Lys or Arg containing 13C and/or 15N). These ‘heavy’ amino acids replace most of the naturally occurring corresponding light amino acids in the proteins. A major advantage of the SILAC approach is that it is generally compatible with most AP-MS approaches without modifications to the protocol. If cells expressing the bait proteins are labeled with the heavy isotope while the negative control cells are labeled with the light isotope, and AP- MS is performed on the combined sample, the difference in intensity between the light and heavy peaks should indicate whether a given protein is a contaminant or a specific interactor. In this scenario, non-specific binders will be present in equal amounts in light and heavy forms. Any specific interactions will have skewed intensity ratios for the heavy versus the light peptides [80]. Other strategies (both using metabolic and chemical labeling with isotopes) can also be employed; however as these methods were not employed here they will not be discussed in any further detail and the reader is referred to [81-85] for further review. 16

More recently, methods to identify background contaminants using label-free quantification have gained popularity and are being increasingly used in AP-MS analyses; these are reviewed briefly below [11, 76, 86-88]. In the simplest form, label free approaches use easily accessible information, such as spectral counts (which indicate how often peptides corresponding to the protein of interest have been sequenced in the mass spectrometer), or the scores from the search engine (for example, the Mascot protein score is representative of both the number of unique peptides and the confidence in their identification). Additional quantitative parameters can include intensity-based measurements (either the intensity of the parent ion in the MS1 scan, or the intensity of fragment ions, most often in the context of approaches known as selective reaction monitoring, or SRM). In all cases, the idea is to distinguish the “signal” corresponding to a given interactor (or prey) in the purification of the bait in relation to the “noise” corresponding to the same prey in the control analyses. Computational approaches to score these quantitative values can be very simple, e.g. by fixing a threshold for fold enrichment over background. Other approaches use statistical means to provide confidence that a protein is in the sample because it is a true interactor, as opposed to being a contaminant. While the diverse approaches were optimized for different types of datasets and vary somewhat in the proteins they classify as true interactors (or significant), they each are more effective than filtering that is not based on quantitative information. Established methods include a probabilistic method developed by Sardiu et al. [76] which was successfully applied to an analysis of the transcription machinery, the CompPASS approach [32, 89] successfully used to analyze the deubiquitinase proteome and the autophagy machinery proteome, a simple t-test in the QUBIC approach [44], and a method known as the Decontaminator which uses Mascot scores for discrimination [90]. Our lab also introduced, first in the context of a large interaction network of kinases and phosphatases, an approach based on spectral counting which we called SAINT (Significance Analysis of INTeractome). This method was further developed to accommodate dataset of different scales and topology [30, 91], and demonstrated to be able to correctly identify interactions even with frequent fliers in small datasets [30]. A recent implementation of SAINT [92] also enables the use of intensity data for scoring interactions. Of note, for the data presented in this thesis, SAINT (using spectral count 17 data) was utilized to assign a statistical confidence for the interactions detected and presented in the PP4c interaction network generated by Ginny Chen (section 1.8), and to determine whether more low level proteins can be identified as being enriched in AP-MS samples when multidimensional fractionation is applied, as compared to a sample analyzed by traditional 1D LC-MS/MS (chapter 2).

1.6 Fractionation of mass spectrometry samples

Fractionation of very complex samples (at the organelle, protein or peptide level) prior to mass spectrometric analysis has been shown to increase the depth of peptide and protein identifications made by MS analysis of complex samples [93-96]. In all methods, this is due to a reduction in sample complexity. When fractions of reduced complexity are analyzed independently of each other more peptides can be separated on a LC column using a specific gradient over a discrete time, than if the entire sample was to be analyzed together. Additionally, separation of peptides into less complex mixtures can also allow for detection of peptides whose levels fall within the lower detection limits of the MS and, theoretically, peptides which, when analyzed together, interfere with the ionization efficiency of each other [94, 97].

Historically, separation of proteins by SDS-PAGE, prior to in-gel digestion, has been the pre-eminent method for reducing sample complexity before MS analysis. In this case, separation of proteins by apparent molecular weight allows for proteins within a specific mass range to be isolated from the bulk of cellular proteins as a gel slice and to be analyzed independently by MS. In a variant of this method termed GeLC, which was used for work presented in chapter 2, proteins are separated by SDS-PAGE and the entire gel lane (containing all cellular proteins), rather than just a single gel slice, excised and divided into sections containing proteins of specific mass ranges. After partitioning of the gel slice into specific mass ranges, each range is analyzed independently of the other by MS allowing for a comprehensive view of all detectable proteins within the gel lane (not just the few that reside within a specific mass range as was originally the focus of SDS- PAGE fractionation). While other methods of protein fractionation do exist (e.g. 18 isoelectric focusing, size exclusion chromatography), they do not pertain to the work presented in the body of this thesis.

Separation of peptides into discrete fractions based on charge or hydrophobicity is also an effective way to reduce sample complexity prior to MS analysis. When separating peptides based on charge prior to reversed phase separation and MS analysis, termed MudPIT, peptides are first loaded onto a LC column containing strong cation exchange resin (SCX) where they can then be “bumped” onto the reversed phase column using discrete volumes of a salt solution of specific molarity (e.g. sodium acetate). After the first “bump” using low molarity salt solution, peptides exhibiting a weak or no positive charge are isolated from peptides bearing higher charge states and washed onto the reversed phase column as a discrete fraction of reduced complexity. These peptides are then separated based on hydrophobicity using an acetonitrile gradient, and analyzed by standard LC-MS/MS. Following this analysis the process is repeated, with each “bump” of salt solution having a higher molarity than the one preceding it, until all peptides have been analyzed. Importantly, because the two separation methods utilize different peptide properties for separation (i.e. are orthogonal to each other), peptides which co-elute from the SCX resin can be separated from each other based on their hydrophobicity during the reversed phase LC-MS/MS analysis.

The concept of separating peptides into discrete fractions based on hydrophobicity alone, before MS analysis, termed RP/RP, is also quite similar in principle to MudPIT. In this approach, peptides are first loaded onto a base stable reversed phase resin and separated into fractions based on hydrophobicity using “bumps” of increasing acetonitrile concentration at pH10. These fractions, once collected, are then analyzed independently of each other using standard LC-MS/MS at pH2. While each method separates peptides based on hydrophobicity, switching the pH of mobile phase in each of the two separation processes causes the charge state of acidic and basic residues to change which impacts peptide hydrophobicity and affinity for the reversed phase resin. In this case, peptides which share similar hydrophobic properties at pH10 and co-elute at a specific acetonitrile concentration, may have entirely different hydrophobic properties at pH2 and elute at 19 different points during the LC-MS/MS acetonitrile gradient allowing for effective separation and identification by MS.

1.7 Utilizing MS for identification of protein phosphorylation

Protein phosphorylation is a key posttranslational modification that regulates protein function and virtually all cellular processes, from signal transduction at the cell membrane, to gene expression within the nucleus, and everything in between [98-106]. The effect of phosphorylation on an individual protein can be wide ranged, and can impact such things as protein-protein interactions, protein activity, and protein localization, to name a few. These effects, then, in turn, dictate the proteins function at the cellular level and regulate the processes described above. Because protein phosphorylation plays such a vital role in regulating cellular functions, identification of protein phosphorylation sites (here referred to as phosphosites) and understanding their role in protein regulation has become an integral part of cellular biology.

In brief, in higher eukaryotes it has been demonstrated that most proteins are phosphorylated on serine, threonine, or tyrosine resides with greater than 98% of all cellular phosphorylation believed to occur on serine and threonine exclusively [102]. Although protein phosphorylation has been shown to regulate a wide variety of cellular processes, the actual stoichiometry of phosphorylation on each site at any one time is often very low and, as such, phosphoproteins and or peptides must often first be enriched in order for MS based approaches to be effective at site identification [107-109]. Phosphoprotein/peptide enrichment is useful in this context in that it reduces detection of non-phosphorylated peptides by the MS, which, because of their relatively high abundance, can result in suppression of less abundant phosphopeptide detection due to limitations in the dynamic range of the MS. Examples of enrichment approaches, alongside phosphopeptide identification, quantification, and site localization are discussed briefly in the sections that follow. 20

1.7.1 Phosphopeptide enrichment approaches and identification

A viable method for the identification of peptides containing phosphoserine and phosphothreonine resides, and one that was used in the experiments outlined in section 3, is the use of immobilized metal affinity chromatography (IMAC) [109-112]. In brief, IMAC resin consists of a stationary phase to which a metal cation (usually iron Fe3+, gallium Ga3+, aluminum Al3+, or zirconium Zr4+) has been coupled. Transfer of phosphopeptides through the stationary phase under acidic conditions allows for the capture of negatively charged phosphate groups by the metal cations by electrostatic interactions [109, 110]. Subsequent washing of the stationary phase allows for non- phosphorylated peptides to be removed and phosphopeptides isolated. In a variation of this technique, metal oxides such as titanium dioxide (TiO2) or zirconium dioxide (ZrO2) can be used [109, 112]. In comparison to IMAC, metal oxides have been shown to be more specific in the capture of phosphopeptides, reducing the carry over of non- phosphopeptides into subsequent analyses [110]. However, this does not mean they are better; in fact, different enrichment methods have been shown to differ in their specificity and the subset of phosphopeptides reproducibly identified [110, 113]. Indeed, the use of 3+ Ga IMAC and TiO2 for phosphopeptide enrichment resulted in different subpopulations of phosphopeptides being observed during the experiments described in chapter 3, and as such both methods were used in parallel and the results combined.

In addition to the IMAC and metal oxide enrichment approaches outlined above, alternate methods have also been developed and employed for enriching phosphopeptides. These include chemical trapping of the phosphorylated residues [107], and enrichment using antibodies directed against the modified site, the latter of which has been widely used for the capture of rarer phosphotyrosine containing peptides from biological samples [114, 115]. At this time however, generic anti-phosphoserine and/or phosphothreonine antibodies have not proven as adequate for this approach [109]. The use of antibodies directed against a consensus site for a specific kinase has been more successful; for example, employing a anti-phosphoSer/Gln or anti-phosphoThr/Gln 21 antibody has revealed numerous new substrates for the ATM/ATR and DNA-PK kinases involved in DNA damage signaling [116, 117].

Once isolated, phosphopeptides can be identified from their non-phosphorylated counterparts by MS through identification of peptides exhibiting a loss of phosphoric acid

(H3PO4) upon fragmentation, corresponding to a mass loss of 98 daltons (Da) from the parent ion (this occurs with both phosphoserine and phosphothreonine), or a loss of 2- hydrogen phosphate (HPO4 ), corresponding to a mass loss of 80 Da from the parent ion (which occurs with phosphothreonine) [108, 109, 118]. Termed “neutral loss”, the loss of a phosphate group during peptide fragmentation occurs because the linkage between the phosphate group and the phosphorylated amino acid residue is a relatively low energy bond compared to the chemical bonding which holds the peptide backbone together [118]. Upon peptide fragmentation (i.e. through collision induced dissociation CID), the phosphoester bond linking the phosphate group to the phosphorylated residue is fragmented disproportionately to the peptide backbone, resulting in an intense signal being detected in the MS (Figure 1-3).

Figure 1-3. Peptide fragmentation induced by collision-induced dissociation (CID). (A) Common CID peptide fragments are identified based on where the fragmentation occurs in the polypeptide backbone and localization of the charge. A, B, and C type ions result in cleavage of the polypeptide where indicated as do X, Y and Z type ions, however A,B, and C type ions retain the charge on the N terminus of the polypeptide while X,Y and Z type ions retain the charge on the C terminus. For clarity only four amino acids are shown here. CID results in prominent fragmentation at peptide bonds resulting in B and Y type ion detection. (B) The same polypeptide structure as shown in (A) however in this case R1 has been replaced with phosphoserine and R4 with phosphothreonine. Neutral loss is depicted by cleavage of the phosphoester bond linking the phosphate molecule to the amino acid residue (red line). The resulting intermediate is shown by dashed lines. Dissipation of kinetic energy through phosphoester bond fragmentation can result in poor fragmentation of the polypeptide backbone and B and Y ion detection (in addition to detection of the other ion types). 23

While useful for identifying phosphopeptides, the effect of neutral loss can be problematic when multiple phosphorylatable residues exist within a peptide sequence and site assignment is necessary. Because kinetic energy (from CID) is released predominantly though absolution of the phosphoester bond, less energy is transferred into peptide bonds and as a result, less ion fragments useful for peptide sequence assignment are generated. In fact, in some cases, the signal associated with neutral loss can be so predominant that peptide fragment ions become indistinguishable from background noise and no ions useful for assigning peptide sequence can be observed (due to limitations in the dynamic range of the MS) [118]. In this case a further round of fragmentation can be conducted on the ion exhibiting neutral loss (termed MS3), generating fragments necessary for peptide sequence identification, however the site of phosphorylation cannot be determined [119]. When sequence information is available, phosphosite assignment is highly dependent on the presence of site determining ions [120], and is facilitated through identification of fragment ions exhibiting a mass 80 Da greater (corresponding to mono- phosphorylation), than would be expected based on the amino acid sequence of the peptide alone [118]. Putative site assignment is usually done by the algorithm used to search the MS/MS data (e.g. SEQUEST, Mascot, X! Tandem), however further methods for statistical validation of correct site assignment have been developed (explained in section 1.7.2). However, if a phosphopeptide is detected that bears two phosphorylatable residues side by side and no observed fragmentation events are detected between them site assignment cannot be made [118].

To circumvent the issues associated with neutral loss from CID, and the problems associated with it for phosphosite identification as described above, other peptide fragmentation methods have been developed (e.g. electron transfer disassociation ETD, or electron capture dissociation ECD) [109, 110, 118]. In these methods, radical anions (ETD) or electrons (ECD) are introduced into the gas phase where they interact with charged peptides, causing a destabilization of the peptide and fragmentation without the predominant effect of neutral loss (i.e. the phosphate group stays attached to the peptide fragment allowing for better site assignment). While purported to be better than CID for phosphopeptide identification, these methods both also have their inherent pitfalls, such as the decreased efficiency of peptide fragmentation observed with ECD (resulting in the 24 need for a greater number of ions to be present for effective fragmentation which increases the overall duty cycle time and results in less MS/MS data acquisition); or the need for peptides that have a high charge density for effective ETD fragmentation (which may require modifications to the LC elution buffer to be made). Poor peptide fragmentation leads to sub-optimal database search results [118, 121]. In addition to ECD/ETD, another fragmentation method, termed higher energy collisional dissociation (HCD) exists, which allows for better peptide fragmentation than is observed with CID, in addition to allowing for the measurement of peptide fragment ion m/z (during MS/MS) to occur at high resolution. The increased peptide fragmentation and high resolution MS/MS scans obtained with HCD allow for better peptide fragment ion charge state resolution, MS/MS peak annotation, and peptide identification, in addition to phosphosite assignment [122]. Additionally, HCD was demonstrated to be an effective way of eliminating the gas-phase phosphate rearrangement reactions observed by CID, in which the phosphate shifts from a phosphorylated residue to a nearby non-phosphorylated S/T/Y in the gas phase, hindering correct phosphosite assignment [123, 124]. However, contrary to this Mischerikow et al. [125] observed that most ambiguous site assignment from CID was not due to gas phase transfer of phosphate group but rather due to poor fragmentation and spectrum quality. Similar to ECD, HCD suffers from lower sensitivity than CID because of longer duty cycle times and methods to address this have just begun to be developed [122]. While these methods were not used in the experiments outlined in this thesis, I would like to point out that in a comparison of the methods, ETD and ECD were found to be more effective at identifying phosphopeptides, yet CID (which was used here) was still observed to be able to identify phosphopeptides that were not observed using either ETD, ECD, or HCD [110, 122].

1.7.2 Label free phosphopeptide quantification and phosphosite localization

Phosphopeptide quantification without the use of isotopic labeling can be facilitated by using approaches such as spectral counting, or ion signal peak height/area analysis (referred to hereafter as an “area under the curve analysis”) [126, 127]. 25

In the spectral counting-based approach, peptide abundances are based solely on the number of peptide spectra acquired. While primarily used for measuring protein abundance (a strong linear correlation has been shown to exist between the number of peptide spectra acquired by MS, and protein abundance [86, 127]; i.e. as the molar amount of protein increases so too does the molar amount of tryptic peptides generated by enzymatic digest and available to be identified by MS), it makes intrinsic sense that the number of phosphopeptide spectra acquired would also correlate with the degree to which the protein is phosphorylated. As stated in the previous example, as the proportion of phosphoprotein increases it is expected that so too will number of phosphopeptides generated by enzymatic digest and available to be identified by MS. However, with this approach quantification of peptides which are observed only a few times is problematic, and as such protein quantification is usually done using all observed peptides for the protein.

For an area under the curve analysis, the concept is slightly different and more complex than that which was presented for the spectral counting method above. When monitoring peptide abundance using this approach, the ion signal for the peptide is recorded during MS1, while identifications are made during MS/MS, and the extracted ion current for the peptide used for quantification. In this scenario, when using electrospray ionization, the signal intensity for a specific ion at a specific time correlates to the abundance of the ion, and the peak areas of all peptides correlates to the protein concentration, even when analyzed in a complex mixture [126-128]. Additionally, and more specifically for phosphopeptide quantification, the peak area for a specific peptide has been shown to be proportional to its abundance even across multiple MS datasets [128]. Because of this, comparisons of phosphopeptide abundances across samples should be able to be made. To this effect, Choi et al., [129] showed that a comparison of phosphopeptide peak areas in four independent experiments resulted in less than 16.2% relative standard deviation, demonstrating a high degree of technical reproducibility for the quantification of a specific phosphopeptide. This approach has been shown to be more sensitive at peptide quantification than spectral counting, especially in the case when a peptide is only observed a few times by MS/MS, as the intensity of the parent ion can still be observed in MS1 and used for quantification, data that is lost when spectral 26 counting based approaches are utilized [92]. Aside from the need for a high mass accuracy instrument for the extraction of good MS1 information [92], a significant downfall of this method is, however, that because label free analyses require MS samples to be processed and analyzed independently of one another, the data must be “cleaned up” computationally to allow for accurate comparisons of peptide abundance to be made across multiple MS samples [126, 128, 130]. This data “clean up” involves such things as normalize for variance in sample preparation, injection, and chromatographic drift (e.g. due to column aging, variation in packing, or contamination) [131]. Normalization for sample preparation and injection can be performed through such means as normalizing to the sum, median, or average signal intensity for the entire MS run, while chromatographic drift can be controlled for by spiking in reporter ions of known m/z and retention time and comparing their intensities between runs [132]. While a detailed explanation of all these methods is outside the scope of this thesis, there are lots of commercial and open source software packages available for analyzing label free MS data which have the necessary data “clean up” algorithms built in [130-132]. While spectral counting based approaches also require MS samples to be processed and analyzed independently of one another, data normalization is generally simpler, and can be facilitated using things such as the total spectral counts for the sample (e.g. to correct for variation in starting protein concentration). Of note, the phosphopeptides listed in chapter 3 were quantified using an area under the curve analysis, using Proteome Discoverer software (V1.3.0.339; Thermo-Fisher Scientific) and phosphosite assignment was facilitated using the PhosphoRS algorithm [120].

In addition to the generic approaches outlined above, which can be used for the quantification of multiple peptides/proteins in a MS analysis, a more specific approach, Selected Reaction Monitoring (SRM) can be used for quantification of a select peptide/protein. In this approach a peptide of specific mass and a few of its fragment ions (selected manually) are monitored by the MS exclusively over the peptides retention time. The intensity of the peptide fragment ions are then used for quantification of peptide abundance (similar to area under the curve analysis). During SRM analysis both the parent ion and fragment ions must pass through mass filters increasing the signal to noise ratio and sensitivity of the analysis. This makes SRM useful for the quantification 27 of low level peptides which may be under sampled during data dependant acquisition. Additionally, because fragment ions are used for quantification rather than parent ions, this method can be used for quantification of isobaric peptides that co-elute, making it useful for the identification of co-eluting phosphopeptide positional isomers (peptides which exhibit phosphoryaltion at more that one site). While this type of quantification is more sensitive than spectral counting or label free area under the curve quantification, the experimental design is however much more complex, as suitable pairs of parent and fragment ions must first be identified for a protein of interest. For further information, the reader is directed to the following reviews [133, 134].

While phosphopeptide site assignment is facilitated by the search algorithm used (i.e. Mascot), the complex nature of matching MS/MS spectra to the theoretical spectra of a specific peptide sequence, especially when confounded by the additional mass shifts of post translation modifications (over 300 distinct types of which can occur) can lead to false positive or ambiguous site identification [135]. In fact most search engines are not optimized for PTM assignment and frequently fail to identify the correct site [120]. Because of this, spectra is often validated manually (i.e. by looking at each MS/MS spectra and ensuring that the site of phosphorylation can be pinpointed through the presence of specific peptide fragments) [136, 137], however, this can be quite time consuming for large scale analyses where thousands of MS/MS spectra may be observed, and is completely dependent on the expertise of whomever is doing the validation. To circumvent these issues several computer algorithms have been designed to statistically validate the assignment is correct (e.g. Ascore [138], Mascot Delta Score [139], PhosphoScore [140], PhoMSVal [141], PhosphoRS [120]). While each method works using different principles, the PhosphoRS algorithm (pRS), implemented in Proteome Discoverer (V1.3.0.339; Thermo-Fisher Scientific) was used for phosphosite assignment in chapter 3 and will be explained briefly here. In brief, the pRS algorithm works by first assuming that the peptide sequence generated by the search engine used (e.g. Mascot) is correct and that the MS/MS spectra contains no co-fragmenting isobaric peptides (demonstrating the need for a high mass accuracy/resolution instrument when conducting these analyses). The algorithm then filters the MS/MS spectra and a sequence probability is calculated for each possible peptide phospho-isoform based on the probability the 28 theoretical spectra for the phospho-isoform is a random match to the filtered MS/MS spectra, and the degree of difference between the best two matches. The probability a given phosphosite is phosphorylated is then calculated as the sum of the sequence probabilities for each possible phosphor-isoform in which the site is phosphorylated (Figure 1-4). This method was shown to be slightly better at phosphosite assignment than either Ascore [138] or the Mascot Delta Score [139], when CID spectra was used [120]. For a more in depth discussion of the statistical principles behind the algorithm the reader is referred to [120] and the Proteome Discoverer (V 1.3) user manual.

Figure 1-4. Proteome Discoverer PhosphoRS algorithm phosphosite localization and scoring. In brief, the PhosphoRS algorithm works by (A+B) retrieving a peptide sequence and associated spectra from the search engine used (e.g. Mascot). (C+D) The algorithm then filters the MS/MS spectra and a sequence probability is calculated for each possible peptide phospho-isoform based on the probability that the theoretical spectra for the phospho-isoform is a random match to the filtered MS/MS spectra, and the degree of difference between the best two matches. (E+F) The probability that a given phosphosite is phosphorylated is then calculated as the sum of the sequence probabilities for each possible phosphor-isoform in which the site is phosphorylated. Note in this case the search engine identified serines 6 and 13 as being phosphorylated, however the PhosphoRS algoritm identified serines 3 and 13 as being phosphorylated. Figure adapted from the Proteome Discoverer (V 1.3) user’s manual (Thermo-Fisher Scientific).

1.8 PP2A subfamily of phosphatases

Phosphatase regulation of cellular processes through the removal of phosphate molecules is one of the most key mechanisms by which cells have been shown to regulate their internal functions, such as the DNA damage response [142, 143], cell cycle [144, 145], and cell survival [146], to name a few. From cell signaling to protein activation, reversible protein phosphorylation has been demonstrated time and time again to be critical for proper cellular function, with many disease states such as cancer exhibiting misregulated phosphorylation events [146-150]. Indeed, kinases, which add phosphate molecules to proteins, and more recently phosphatases, which remove phosphate groups, have become viable therapeutic targets [148, 151-156]. In this section I will present a brief overview of the PP2A subfamily of serine and threonine phosphatases, and more specifically of PP4c, as it relates directly to the work presented in chapter 3 and appendix 1.

1.8.1 PP4c biology and regulation

Protein phosphatases are classified into two types based on their substrate preference, protein tyrosine phosphatases (PTP), and serine/threonine phosphatases. Serine/threonine phosphatases are further divided into 3 structurally distinct families, PPM (Protein Phosphatase Mg2+ or Mn2+ dependant), PPP (PhosphoProtein Phosphatase), or FCP/SCP (aspartate phosphatases; Table 1-2).

Table 1-2. Classification of human protein phosphatases. Phosphatase families, subfamilies and the number of genes encoding them are listed for Protein tyrosine phosphatases (PTP), and serine/threonine phosphatases. PPP (PhosphoProtein Phosphatase); PPM (Protein Phosphatase Mg2+ or Mn2+ dependant); Asp-based (aspartate phosphatases). Regulatory subunits, inhibitors and divalent metal ion cofactors are also listed. MC (microcystin); CA (calyculin A); OA (okadaic acid). Note: This table and legend is adapted from Ginny Chen’s Ph.D thesis [157].

, PP4R4

The PPP family includes PPP1, PPP2A, PPP2B (a.k.a. calcineurin), PPP4, PPP5, PPP6, and PPP7 [156]. Of these phosphatases PP2Ac, PP4c, and PP6c are classified as belonging to the PP2A subfamily of phosphatases due to their high degree of sequence identity at the amino acid level (PP4c and PP6c are 60-65% identical to PP2Ac at the amino acid level [158]), and sensitivity to inhibitors [159, 160], (Table 1-2; Figure 1-5).

Figure 1-5. Phylogenetic tree of PPP family of phosphatases. The tree shows the distribution of PPP phosphatase subfamilies across major phyla. Each of the subfamily is grouped and colour coded. Note: This figure and legend is adapted from Ginny Chen’s Ph.D thesis [157]. PP1, PP2A, PP4, and PP6 are referred to as PP1c, PP2Ac, PP4c, and PP6c throughout this thesis respectively. PP3 is referred to as PPP2B or calcineurin.

PP4c itself is an evolutionarily conserved [161], developmentally regulated, [162, 163] phosphatase in eukaryotes and has been shown to be involved in numerous cellular processes, most notably cell signaling [164-167] gene expression [168-170], DNA damage repair [143, 171-176], resistance to the DNA damaging agent and anticancer drug cisplatin [176, 177], proper progression of mitosis through regulation of microtubule growth and organization at centrosomes [178-181], and apoptosis [182, 183].

PP4c regulation of such temporal and spatially distinct processes is thought to be made possible at least in part by the presence of several mutually exclusive PP4c complexes, in which the catalytic (PP4c) subunit directly associates with a regulatory subunit, PP4R1 [177, 184], PP4R2 [172], or PP4R4 [185], to form a dimeric complex, or alternatively, associates with PP4R2 which then acts as a scaffolding subunit [163, 186] to form a trimeric complex with PP4R3 (two genes, PP4R3A and B exist in mammals) [173, 177]. It is the regulatory subunits of PP4c which are thought to confer cellular localization (PP4R1 and PP4R4 were observed to be localized to the cytoplasm, while PP4R2 and PP4R3A were localized in the nucleus. PP4c exhibited both nuclear and cytoplasmic staining but was more concentrated in the nucleus; G. Chen Ph.D thesis [157]), and substrate specificity to each of these complexes [187, 188]. For example, several studies have demonstrated different roles for each of the PP4c complexes, for instance the PP4c-PP4R1 complex was shown to have a role in regulating gene expression through HDAC3 activity [168], the PP4c-PP4R2 complex has been observed to have a role in DNA double strand break repair through targeting of replication protein A subunit RPA2 [172], the PP4c-PP4R2-PP4R3A complex was shown, when upregulated, to confer resistance to the anti cancer drug cisplatin in breast and lung tumors [177], and the PP4c-PP4R2-PP4R3B complex has been demonstrated to play an important role in DNA damage repair [143, 189]. To date no function has been attributed to PP4c-PP4R4, however Ginny was able to show that the PP4c-PP4R4 complex had reduced phosphatase activity towards a fluorogenic substrate (DiFMUP) and known substrate (γH2AX) as compared to the PP4c-PP4R2-PP4R3A complex (G. Chen Ph.D thesis [157]), indicating that the regulatory subunits of these complexs can regulate phosphatase activity, a view that is consistent with the effect of phosphatase regulatory subunits on phosphatase activity observed by others [190]. 36

In addition to the regulatory subunits outlined above, PP4c has also been shown to associate with the protein alpha 4 (α-4), as has PP2Ac and PP6c [158, 191], interactions with which, while not demonstrated to be necessary for substrate targeting, have been implicated in regulation of phosphatase activity [192, 193], stability [194-196], and assembly [196]. PP4c subunits and associated known functions are shown in Figure 1-6. HUGO gene names, protein names used throughout this thesis, Entrez Gene ID, and NCBI accession number for these regulatory subunits and other proteins discussed in detail in further sections are presented in Table 1-3.

Figure 1-6. PP4c regulatory subunits. PP4c regulatory subunits are displayed alongside cellular processes they have been implicated in regulating. Based on data generated in the Gingras lab it is believed PP4R3A is involved in targeting of substrates involved in transcription and splicing regulation (deletion of the first 115 amino acids of PP4R3A, a domain conserved from yeast to humans, resulted in a loss or decrease in PP4c association with most transcription and splicing factors detailed in section 1.8.2); Alpha 4 is involved in regulating phosphatase activity, complex assembly and stability.

Table 1-3. Gene and protein identifiers for PP4c, PP4c regulatory subunits, and interacting proteins investigated and discussed in detail in this thesis. Listed are the HUGO gene name “Gene Name”, protein name used throught the thesis “Protein Name”, Entrez Gene ID, and NCBI accession number “Accession Number” for proteins outlined in this thesis.

Gene Name Protein Name Entrez Gene ID Accession Number PPP4C PP4c 5531 NM_002720 PPP4R1 PP4R1 9989 BC060829 PPP4R2 PP4R2 151987 NP_777567 SMEK1 PP4R3A 55671 NM_032560 SMEK2 PP4R3B 57223 NM_020463 SUPT4H1 SUPT4H 6827 NM_003168.1 SUPT5H SUPT5H 6829 BC024203 DHX38 DHX38 9785 NM_014003.3 HTATSF1 HTATSF1 27336 NM_014500.3 RNGTT RNGTT 8732 NM_003800.3

1.8.2 PP4c interactions and substrate identification

While some information was known about PP4c regulation and its interacting proteins, no comprehensive assessment had been done to identify PP4c interacting proteins and PP4c regulatory subunit specific targeting of proteins until Ginny Chen begun her Ph.D work. As outlined in section 1.1, Ginny Chen extensively characterized the interactions occurring between PP4c and other cellular proteins using affinity purification coupled to mass spectrometry (AP-MS), to better understand how PP4c may be linked to the cisplatin resistance phenotype and to uncover the mechanism by which PP4c may regulate the various cellular processes outlined in section 1.8.1. Her discovery that PP4c associates with components of the transcription elongation and splicing machineries (Figure 1-7), and more specifically with what appear to be several mutually exclusive complexes (identified by hierarchical clustering; Figures 1-8 – 1-10), is presented below.

In particular, Ginny found that the transcription elongation components of the DRB Sensitivity Inducing Factor (DSIF) complex, SUPT4H and SUPT5H [197], together with the mRNA capping enzyme RNGTT (an interaction known to occur and to stimulate mRNA capping [198]), established an association with the PP4c-PP4R2-PP4R3A/B complex (Figure 1-8). These interactions were largely independent of another PP4c interacting complex, which contained the helicase DHX38 [199], as well as spliceosomal U5 (EFTUD2, DDX23, CD2BP2), and PRP19 (PRP19, BCAS2) components, which was in turn independent of another complex containing the human transcription elongation factor (HTATSF1), known to have a role in coupling the processes of transcription and splicing [200, 201], in association with spliceosomal U2 components and the uncharacterized protein SLC4A1AP (Figures 1-9 and 1-10 respectively). Notably, the majority of the interactions Ginny detected between the transcription and splicing machineries occurred with PP4c-PP4R2-PP4R3A, and not PP4c-PP4R1 or PP4c-PP4R4, consistent with the nuclear localization of PP4R2 and PP4R3A (PP4R2 and PP4R3A both contain nuclear localization signals while PP4c does not, indicating PP4c nuclear localization and these interactions may be facilitated by the regulatory subunits).

Figure 1-7. PP4c Network highlighting selective high-confidence interactions (SAINT > 0.8). Baits are displayed as large nodes and the nodes are functionally annotated and categorized by colour. Yellow= “signaling”; green = “splicing”; blue = “transcription or RNA capping”; pink = “DNA damage response, replication or chromatin”; purple = “protein degradation”; orange = “protein folding”; dark grey = “translation”; lighter grey = “unknown or other”. Arrow directionality indicates bait - prey relationship and the styles of the edges indicate the source of the reported interaction: dashed lines are from BioGRID [202], solid lines are interactions detected in this study. Edge thickness is mapped to the averaged spectral count per experiment. Note: This figure and legend is adapted from Ginny Chen’s Ph.D thesis [157].

Figure 1-8. Network highlighting the interactions of SUPT5H-SUPT4H-RNGTT and PP4c-PP4R2-PP4R3A complex. Baits are displayed as large nodes and the nodes are functionally annotated and categorized by colour. Yellow= “signaling”; green = “splicing”; blue = “transcription or RNA capping”; pink = “DNA damage response, replication or chromatin”; purple = “protein degradation”; orange = “protein folding”; dark grey = “translation”; lighter grey = “unknown or other”. Arrow directionality indicates bait - prey relationship and the styles of the edges indicate the source of the reported interaction: dashed lines are from BioGRID [202], solid lines are interactions detected in this study. Edge thickness is mapped to the averaged spectral count per experiment. Note: This figure and legend is adapted from Ginny Chen’s Ph.D thesis [157].

Figure 1-9. Network highlighting the interactions of DHX38-PRP19-U5 snRNP and PP4c-PP4R2-PP4R3A complex. Baits are displayed as large nodes and the nodes are functionally annotated and categorized by colour. Yellow= “signaling”; green = “splicing”; blue = “transcription or RNA capping”; pink = “DNA damage response, replication or chromatin”; purple = “protein degradation”; orange = “protein folding”; dark grey = “translation”; lighter grey = “unknown or other”. Arrow directionality indicates bait - prey relationship and the styles of the edges indicate the source of the reported interaction: dashed lines are from BioGRID [202], solid lines are interactions detected in this study. Edge thickness is mapped to the averaged spectral count per experiment. Note: This figure and legend is adapted from Ginny Chen’s Ph.D thesis [157].

Figure 1-10. Network highlighting the interactions of SLC4A1AP-HTATSF1-U2 snRNP and PP4c-PP4R2-PP4R3A complex. Baits are displayed as large nodes and the nodes are functionally annotated and categorized by colour. Yellow= “signaling”; green = “splicing”; blue = “transcription or RNA capping”; pink = “DNA damage response, replication or chromatin”; purple = “protein degradation”; orange = “protein folding”; dark grey = “translation”; lighter grey = “unknown or other”. Arrow directionality indicates bait - prey relationship and the styles of the edges indicate the source of the reported interaction: dashed lines are from BioGRID [202], solid lines are interactions detected in this study. Edge thickness is mapped to the averaged spectral count per experiment. Note: This figure and legend is adapted from Ginny Chen’s Ph.D thesis [157].

Ginny further went on to demonstrate that PP4R3A was needed for SUPT5H targeting by PP4c-PP4R2-PP4R3A and that the binding site of PP4R3A on SUPT5H overlapped with the binding site for RNA polymerase II binding, consistent with the lack of association between PP4c-PP4R2-PP4R3A and RNA polymerase II observed by AP- MS (Figure 1-8). In addition she found that PP4R3A co-localized with the nuclear speckle marker SC35 in a transcription dependent manner (nuclear speckles are thought to be storage sites for transcription and splicing components [203, 204] and SC35 is a component of the mRNA processing machinery), and that deletion of the first 115 amino acids of PP4R3A (the EVH1 domain conserved from yeast to human) eliminated or reduced most interactions between PP4c-PP4R2-PP4R3A and the transcription and splicing factors outlined in Figure 1-11. Interestingly, the EVH1 domain was not observed to be necessary for nuclear speckle localization, a fact attributed to PP4R3A interactions with PRP19 through its C-terminal domain (amino acids 653-820, G. Chen Ph. D thesis [157]) and PRP19 presence in nuclear speckles [205]. Based on the evidence presented here, Ginny hypothesized that PP4c-PP4R2-PP4R3A was the major PP4c containing complex involved in regulation of transcription and splicing, with PP4R3A possibly acting as a scaffold to bring different components of subcomplexes together (i.e. DHX38 through interactions with the EVH1 domain and PRP19 through interactions with the C-terminal domain). 47

Figure 1-11. PP4R3A targeting of transcription and splicing factors occurs through its EVH1 domain. (A) PP4R3A (SMEK1) interacting transcription and splicing factors (listed under gene name), were immunopurified using wild type (WT) FLAG-PP4R3A or FLAG-PP4R3A lacking the first 115 amino acids (the EVH1 domain). Deletion of the EVH1 domain of PP4R3A resulted in a decrease or loss of peptide spectra detection by MS for the majority of the transcription or splicing factors listed, indicating a loss of the interaction with PP4R3A. (B) Protein schematic of PP4R3A depicting the EVH1 domain and PP4c/PP4R2 binding domain. Transcription and splicing factors discussed in detail in this thesis are listed above the location binding is hypothesized to occur. This figure is adapted from Ginny Chen’s Ph.D thesis [157].

Of further note, while these proteins were observed to interact with PP4c-PP4R2- PP4R3A as distinct complexes, they are extensively interconnected in vivo for regulation of co-transcriptional splicing, indicating that PP4c could have a role in regulating the coupling of these processes as transcription and splicing have been demonstrated to be highly dependent on reversible protein phosphorylation [206-213]. SUPT4H, SUPT5H, and HTATSF1 have specifically been demonstrated to have roles both in the regulation of transcription elongation and splicing [200, 201, 214, 215], however, what role PP4c may play in this process has yet to be determined.

Interestingly, PPP phosphatase inhibiton has been shown to block both catalytic steps of splicing, with PP2Ac inhibition believed to be the major factor for inhibiton of the second catalytic step [216, 217]. However, the inhibitors used in these studies can also affect PP4c activity, and therefore cannot be used to rule out regulation by PP4c. DHX38 has been implicated in the regulation of the second catalytic step of splicing [218, 219] and the components of the DHX38-PRP19-U5 snRNP complex were only observed to purify with PP4c (they did not co-purify with either PP1c, PP2Ac, or PP6c), indicating that indeed PP4c may have a role in the regulation of splicing. However, Ginny was unable to demonstrate a role for PP4c-PP4R2-PP4R3A in the regulation of constitutive splicing at strong splice sites in vitro (G. Chen Ph. D Thesis [157]).

While the work reviewed above has greatly expanded the knowledge of PP4c protein-protein interactions in the cell, and presented a new role for PP4c-PP4R2- PP4R3A in the regulation of transcription and splicing, which if any of these PP4c interacting proteins is a substrate for PP4c is relatively unknown (Ginny did present some evidence that PP4R3A and CCDC6 are PP4c substrates). Aside from these, only a few other proteins have been identified as PP4c substrates, with the majority identified (aside from NDEL1, a microtubule/centrosome organizing protein [179, 220]), primarily involved in the DNA damage response (Rad53 [221], RPA [172], and H2AX [143, 175, 222]). Because of this, one of the goals of my project has been to try and identify which if any of the PP4c interacting proteins identified by Ginny could be possible PP4c substrates, and thereby directly involved in PP4c-PP4R2-PP4R3A regulation of transcription or splicing and not just acting as adaptor molecules to facilitate interactions 49 between PP4c and other proteins. It should be noted that Ginny validated all interactions by reciprocal AP-MS, and in doing so generated a number of constructs which I have been using for my studies.

1.8.3 PP4c regulation of mRNA transcription and splicing

Apart from her mass spectrometry work, Ginny further proposed a role for PP4c in regulation of transcription and splicing of mRNAs following epidermal growth factor (EGF) stimulation (using a combination of qRT-PCR analysis and RNA-seq). Initially, she performed siRNA-mediated depletion of PP4c and analyzed mRNA transcription of the AP-1 transcription factors JUNB and FOSB, known to be regulated at the level of transcription elongation by DSIF (comprised of SUPT4H and SUPT5H, and detected to interact with PP4c-PP4R2-PP4R3A) in a phosphodependant manner. In brief, DSIF in complex with negative elongation factor (NELF) can bind and hold RNA polymerase II (RNA Pol II) at the proximal region of the JUNB or FOSB promoters during transcription initiation [223-225], a phenomenon known as promoter proximal pausing and involved in the regulation of ~30% of human genes [226]. Upon cellular perturbation (i.e. EGF stimulation) transcription elongation progresses rapidly through phosphorylation of the poised RNA pol II C-terminal domain (CTD), on S2 of the heptapeptide repeat YSPTSPS). This phosphorylation event in addition to DSIF phosphorylation (both mediated through positive transcription elongation factor b, P-TEFb [227-229]), eliminates the repressive effect of DSIF on transcription elongation and turns it into an activator. Of note, HTATSF1, another protein found to complex with PP4c, has also been shown to have a stimulatory effect on P-TEFb mediated phosphorylation of RNA pol II and the DSIF complex [230], and needed for efficient transcription elongation of FOS [231]. Additionally, SUPT5H has been found to stimulate mRNA capping [198], a process needed to facilitate processive elongation [232], and stimulated by RNGTT binding of phosphorylated S5 of the RNA pol II CTD heptapeptide repeat [233]. Because these proteins were found to interact with PP4c and in part are dependent on phosphorylation for their activation, Ginny proposed PP4c could be involved in the regulation of JUNB and FOSB at the level of transcription elongation through its associations and possible phosphoregulation of these proteins. Initial RT-PCR assays by 50

Ginny demonstrated depletion of PP4c resulted in a decrease in JUNB and FOSB mRNA levels (Figure 1-12).

Figure 1-12. Real-time PCR analysis of FOSB, JUNB mRNA expression. HeLa cells were treated with RNAi targeting PP4c (N1: 5´-AAGACAAUCGACCGAAAGCAA-3´, N3: 5´-GACAAUCGACCGAAAGCAA-3´) and the cells stimulated with 0.1ug/ml EGF for 30 min. Total RNA was then prepared and analyzed by qRT-PCR to quantify the expression of (A) FOSB and (B) JUNB. (C) HPRT expression was used for normalization. PP4c depletion led to a decrease in both FOSB and JUNB expression compared to cells treated with a non-targeting siRNA (siScram). This figure and legend was adapted from Ginny Chen’s thesis [157]. Note the sequence of the N1 and N3 siRNA (which were thought by Ginny to be completely different) are in fact nearly identical; this is discussed in Appendix 1. 52

Knockdown of SUPT5H produced a similar decrease in both JUNB and FOSB expression, indicating that it functions in the same pathway as PP4c to regulate EGF induction of gene expression. Additionally, Ginny demonstrated that the EGF signaling cascade was not perturbed PP4c depletion (by looking at ERK1/2, and Elk-1 activation; Elk-1 activates FOS transcription downstream of ERK1/2), providing further evidence that PP4c mediation of JUNB and FOSB transcription may be through associations with DSIF. Prompted by these results, Ginny collaborated with the Blencowe lab to perform RNAseq analysis of four samples (siPP4c ± EGF and Control siRNA ± EGF). This approach was undertaken to generate unbiased lists of genes which demonstrate a change in either transcription or splicing in the absence of a functional PP4c holoenzyme (Presented in Appendix 1, Table A1-2). In summary, PP4c depletion was observed to significantly alter alternate exon inculusion (by ≥10%) in 95 genes, while only the expression of a handful of EGF inducible genes was altered. Interestingly, one of the genes identified to be upregulated in PP4c depleted cells (upon EGF stimulation) was 7SK RNA which in complex with MEPCE, LARP7 and HEXIM1/2 forms the 7SK snRNP which is a potent inhibitor of P-TEFb [234-237], and could be a means through which JUNB and FOSB expression are decreased upon PP4c depletion.

1.9 Thesis objectives

Based on Ginny’s discoveries, I postulated that PP4c may serve as a master controller in the processes of transcription and splicing, possibly regulating transcription elongation and coupled splicing through interactions with DSIF, HTATSF1 and DHX38 (or proteins that have yet to be identified). However, on which of these protein(s) it would act (or any of the others outlined above), and how it would mechanistically be able to control transcriptional elongation and splicing remains to be investigated. To this end, the goal of my project was to identify putative new substrates for PP4c (either SUPT5H, HTATSF1, DHX38, or others) by first identifying proteins that display an increased phosphorylation state upon cellular treatment with okadaic acid, a potent inhibitor of PP2A subfamily phosphatases [159, 238], and then quantifying differences in phosphopeptide abundance from each of these proteins by MS, with and without PP4c inhibition. Additionally, I sought to continue Ginny’s investigation into the functional consequences of siRNA 53 directed PP4c depletion on mRNA transcription and splicing, to determine whether 7SK RNA upregulation and inhibition of P-TEFb could be responsible for the decrease in JUNB and FOSB expression observed upon PP4c depletion (this work is presented in appendix 1).

My thesis hypothesis is that there may be as yet unidentified components of the transcription and splicing machineries that PP4c interacts with, and that the dephosphorylation of both these proteins, and the ones previously identified by Ginny Chen, by PP4c regulates mRNA transcription and splicing. I propose three aims to test my hypothesis:

Aims: 1) Investigate the applicability of multidimensional fractionation to AP-MS samples for identifying putative new PP4c substrates (or interactors).

2) Assess whether the phosphorylation of PP4c interaction partners involved in transcription and splicing could be modulated by PP4c in vivo.

3) Investigate the functional consequences of PP4c depletion on mRNA transcription and splicing upon EGF stimulation (outlined in Appendix 1).

Taken together, the approaches presented here will expand our understanding of PP4c mediated cellular regulation.

Chapter 2 A Cost-Benefit Analysis of Multidimensional Fractionation of Affinity Purification-Mass Spectrometry Samples

As a first step towards identifying putative new substrates for PP4c, I was interested in investigating whether the application of multidimensional fractionation (as described in section 1.6), a method known to increase the number of protein identifications made in complex samples [94, 239-241], would be beneficial when applied to affinity purified complexes (Aim 1). Currently, the Gingras lab utilizes single step FLAG affinity purification to identify interactors for proteins of interest by AP-MS in mammalian cells [74, 79, 242, 243], and it was unclear whether current one-dimensional LC-MS/MS approaches are able to effectively identify all of the components in this sample type. While many research groups have employed fractionation approaches for the analysis of mammalian AP-MS samples, including protein-level separation by SDS-PAGE [55, 244], or peptide-level separation via different types of multidimensional liquid chromatography [76, 245], a side-by-side comparison of the benefits of each type of fractionation approach for AP-MS protein identification has not been conducted.

To this end, I tested three commonly-used fractionation techniques: (a) protein- based separation by SDS-PAGE (GeLC) [96], (b) peptide-based separation by ion exchange chromatography (MudPIT) [245], or (c) an additional reversed-phase separation step (RP-RP). I also assessed the performance of a new generation fast- scanning instrument, the AB SCIEX TripleTOF 5600 in relation to a linear ion trap. These methods were benchmarked for their effectiveness at identifying interaction partners in AP-MS samples of known complexity. To this end, I selected bait proteins (COPS5, eIF4A2, RAF1, MEPCE) for which multiple binding partners are annotated in the protein interaction database BioGRID [32, 202]. Aside from having a list of known interaction partners for these proteins by which I could assess the effectiveness multidimensional separation approaches, these baits were selected because they have different biological roles and/or subcellular localization, allowing for these techniques to be assessed with different types of cellular proteins. COPS5 is a core component of the COP9 signalosome, a well-defined multifunctional 8-subunit complex involved in the 55 ubiquitin-proteasome system, in part via deneddylation of the cullins with which it physically interacts [32, 246]. RAF1 is a serine-threonine kinase which binds to and is activated by GTP-loaded Ras, leading to the activation of the MAP kinase ERK pathway. Importantly, several chaperones (Hsp90, Cdc37 and immunophilins) and 14-3-3 proteins have been identified as physically interacting with and critical for proper activation of RAF1 [247, 248], eIF4A2 is a translation initiation factor which is part, together with eIF4E and eIF4G, of the biochemically-stable eIF4F complex; through eIF4G, eIF4F associates with all members of the eIF3 multisubunit complex, which in turn binds to the 40S ribosomal subunit [249]. Association with eIF4E and eIF4G also enables association with capped mRNAs, and coincident recruitment of other mRNA binding proteins. Lastly, MEPCE, the 7SK snRNA methylphosphate capping enzyme also known as BCDIN3, was recently identified by interaction proteomics screens as a component of an snRNP complex containing both RNA processing and transcription factors [55]. My studies on the usefulness of multidimensional separation on AP-MS analysis were published in Proteomics [2].

2.1 Materials and Methods 2.1.1 Generation and culture of stably transfected Flp-In T- REx 293 cell lines

The vector pcDNA5-FRT-FLAG, engineered to inducibly express fusion proteins with a single N-terminal FLAG epitope, was constructed from the parent vector pcDNA5-FRT- TO (Invitrogen) and the vector pcDNA3-FLAG [1] as follows: A HindIII/XhoI cassette from pcDNA3-FLAG (containing the FLAG and the multiple cloning site) was subcloned into the pcDNA5-FRT-TO vector also digested with HindIII/XhoI. An internal EcoRI site was subsequently destroyed by mutagenesis, and the modified versions of the resulting vector were sequenced. The coding sequences of COPS5, eIF4A2, RAF1, and MEPCE were amplified by PCR from Mammalian Gene Collection constructs BC001187, BC015842, BC018119 and BC018935 respectively, and cloned into pcDNA5-FRT-FLAG (using EcoRI/NotI for COPS5 and RAF1, and AscI/NotI for eIF4A2 and MEPCE), and the junctions sequenced. The resulting vectors were stably co- transfected with the Flp-recombinase expressing vector pOG44 into Flp-In T-REx 293 56 cells (Invitrogen). Selection was performed by plating transfected cells at low density in Dulbecco’s Modified Eagles Medium (high glucose) supplemented with 5% FBS, 5% calf serum, 100U/mL penicillin/streptomycin, and 200 g/mL hygromycin. Individual hygromycin-resistant clones were picked (after ~ 2 weeks in culture), amplified in selection medium, and recombinant protein expression induced by the addition of tetracycline (200 ng/mL, HyClone) to cell media for 24 hrs. This work, with the exception of cell culture, protein induction and cell harvesting, was not performed by me, but rather Marilyn Goudreault and Beatriz Gonzalez, technicians in the Gingras lab.

2.1.2 Affinity Purification

Cells expressing FLAG tagged proteins were harvested at a confluence of 90% by scraping with a rubber spatula. Cells were pelleted by centrifugation, washed once with PBS, and frozen at -80°C. Cell lysis and FLAG immunoprecipitation (IP) on M2- sepharose was performed essentially as previously described [250], with the following modifications. Briefly, to the frozen cell pellet (from six 150 mm plates), a 1:4 (pellet weight:volume) ratio of lysis buffer was added. Lysis buffer was 50 mM Hepes-KOH pH 8.0, 100 mM KCl, 2 mM EDTA, 0.1% NP40, 10% glycerol, 1 mM PMSF, 1 mM DTT and protease inhibitor cocktail (Sigma; P8340; 1:500). Cells were lysed on ice, subjected to one freeze-thaw cycle, and lysate cleared by centrifugation (20,800 x g, 20 minutes, 4°C). Cleared lysate was incubated with 30 l bed volume of pre-washed FLAG M2 agarose beads (Sigma; A2220), for 2 hours at 4°C. FLAG M2-sepharose was washed four times with lysis buffer (minus 1mM PMSF, 1mM DTT and protease inhibitor cocktail), followed by two washes with ammonium bicarbonate rinsing buffer (50 mM NH4HCO3 pH 8.0, 75 mM KCl), before bound proteins were eluted (3x) by incubating the resin for 15 minutes at 4°C with 150 l of freshly-prepared ammonium hydroxide solution (pH 11-

12, prepared by diluting 500 l NH4OH in 5 ml HPLC-grade H2O), per elution. Pooled elutions were evaporated in a speed vac until dryness, after which HPLC-grade water (100 l) was added to each tube, and lyophilization repeated. Except for GeLC, samples were digested overnight with Sigma Trypsin Singles (Sigma; T7575; 1 g per sample) in

50 mM NH4HCO3, pH 8.0 at 37 ºC. After overnight digestion, samples were spiked with an additional 0.25 µg trypsin (in 50 mM NH4HCO3, pH 8.0) and incubated for 3 hours at 57

37 ºC before acidification to 1% formic acid and lyophilization. Digested samples were stored at -40°C. COPS5 samples coming from the same batch purification are indicated by superscript numbering in Table 1. All eIF4A2, RAF1, and MEPCE RP and RP-RP analyses were done using samples from the same batch purification.

2.1.3 One dimensional (1D) LC-MS/MS analysis

For 1D LC-MS/MS analysis, affinity purified, digested, and lyophilized FLAG alone or FLAG-COPS5, eIF4A2, RAF1, or MEPCE samples were re-suspended in 5% formic acid before direct loading onto fused silica capillary columns (0.75 m id) packed in-house with 10 cm Zorbax C18 (ZorbaxSB, 3.5 m) and pre-equilibrated with HPLC buffer A. The amount of affinity purified material loaded on column was equivalent to two 90% confluent 150 mm plates. Loaded columns were placed in-line with a LTQ mass spectrometer equipped with an Agilent 1100 capillary HPLC, an LTQ-Orbitrap mass spectrometer equipped with an Eksigent Ultra HPLC or a TripleTOF 5600 equipped with an Eksigent nanoLC. On all platforms, HPLC gradients were delivered at 200 nl/min using a split flow arrangement or nanoflow, respectively. Buffer A was 3% acetonitrile, 0.1% formic acid; buffer B was 80% acetonitrile, 0.1% formic acid. The HPLC gradient program delivered an acetonitrile gradient over 120 min (1-5% buffer B over 4 min, 5- 40% buffer B over 100 min, 40-60% buffer B over 5 min, 60-100% buffer B over 5 min, hold buffer B at 100% 3 min, and 100-0% B in 2 min). The parameters for data dependent acquisition on the LTQ mass spectrometer were: 1 centroid MS (mass range 400-2000) followed by MS/MS on the 5 most abundant ions. On the Orbitrap, 1 high resolution MS scan followed by 3 low resolution MS/MS scans of the most abundant ions. On the TripleTOF 5600, data dependent acquisition was done with 1 high resolution MS scan followed by 20 high resolution MS/MS scans over a 1.3 second cycle time. Data acquisition on the TripleTOF 5600 was not performed by me, but rather Stephen Tate, an employee at AB SCIEX.

2.1.4 Multidimensional LC-MS/MS analysis

Because sample fractionation allows for the analysis of more digested material, peptide level fractionation (RP-RP or MudPIT) of FLAG-COPS5 samples was done with the 58 equivalent of four 90% confluent 150 mm plates, and at the protein level (GeLC) with the equivalent of six 90% confluent plates. However, to directly assess whether changes in the number of spectra, peptides and proteins are truly due to the multidimensional separation and not the amount of starting material used, a true parallel comparison for FLAG-eIF4A2, RAF1, and MEPCE was also performed in by using the same amount of material for 1D LC-MS and RP-RP, namely two 90% confluent 150 mm plates.

2.1.4.1 MudPIT

MudPIT was performed essentially as described [251], pre-columns (150µm id) and analytical columns (75 µm id) were fused silica packed in-house with Phenomenex SCX resin and Magic C18, respectively. MudPIT samples were desalted by loading onto a capillary column (200 m id), packed in house with 5 cm Zorbax C18 (ZorbaxSB, 3.5 m), and washing with buffer A. Desalted samples were eluted using 50% acetonitrile, 50% buffer A and lyophilized before loading onto SCX resin in buffer A. Loaded samples were bumped sequentially onto the RP resin using 8 L salt bumps of 100 mM, 200 mM, 300 mM, and 500 mM sodium acetate. The loaded column was placed in-line with an LTQ-Orbitrap XL mass spectrometer equipped with a Proxeon Easy-LC. Buffer A is 0.1% formic acid; buffer B is 99.9% acetonitrile, 0.1% formic acid. The HPLC gradient program delivered an acetonitrile gradient over 120 min at 400nL/min, (0-7% buffer B over 7 min, 7-40% buffer B over 98 min, 40-80% buffer B over 2 min, hold buffer B at 80% 13 min). Data dependent acquisition on the mass spectrometer was: 1 MS scan followed by 5 data dependent MS/MS scans. Data acquisition for MudPIT analysis was not performed by me, but rather Thomas Kislinger, a faculty member in the Department of Medical Biophysics, University of Toronto.

2.1.4.2 RP/RP

For RP-RP fractionation, samples were re-suspended in 20 mM ammonium formate

(NH4HCO2, pH10) and bomb loaded onto a capillary column (200 m id), packed in house with 6 cm X-Bridge base stable C-18 resin (3 µm, Waters), pre-equilibrated with

NH4HCO2, (pH10). Sample flow-through was collected and the column washed using 20

L of 20 mM NH4HCO2 (pH10). Sample fractions were eluted using serial 20 L 59

injections of 5, 10, 15, 20, or 50% acetonitrile in 20 mM NH4HCO2 (pH10) and lyophilized. Lyophilized flow-through and sample fractions were reconstituted in 5% formic acid, and analyzed using LC-MS/MS as described above.

2.1.4.3 GeLC

For GeLC, undigested affinity purified and lyophilized FLAG-COPS5 or FLAG alone samples were resuspended in Laemmli sample buffer, subjected to SDS-PAGE, colloidal blue staining (Thermo-Fisher Scientific; 24592), and in-gel digestion essentially as described [96, 252]. Sample lanes were divided into 20 proportionally sized fractions and sequential fractions pooled before elution solvent evaporation in a speed vac. Fraction eluates were reconstituted in 10 L of 5% formic acid, centrifuged at 16,000 rcf to sediment any gel debris and 6 L analyzed using LC-MS/MS as described above.

2.1.5 Data Analysis

All ThermoFinnigan RAW files were saved in our local interaction proteomics LIMS, ProHits [253]. mzXML files were generated from ThermoFinnigan RAW files using the ProteoWizard converter [254], implemented within ProHits (--filter “peakPicking true2” - -filter “msLevel2”). TripleTOF 5600 .wiff files were converted to .mgf format using ProteinPilot software before being saved into ProHits for analysis. The searched database contained the human complement of the RefSeq protein database (version 37; 38097 entries searched). mzXML files were searched with Mascot version 2.2 using the following parameters: one missed cleavage site, methionine oxidation and asparagine/glutamine deamidation as variable modifications. The fragment mass tolerance was 0.6 Da (monoisotopic mass) for LTQ and LTQ-Orbitrap data, and the mass window for the precursor was 3 Da average mass in the case of LTQ , and 12 ppm (monoisotopic) in the case of LTQ-Orbitrap. TripleTOF 5600 fragment mass tolerance was 0.2 Da (monoisotopic) and precursor 40ppm. Search results were further analyzed using the PeptideProphet and ProteinProphet TransProteomics Pipeline tools [255, 256]. Peptides which did not meet the minimum PeptideProphet significance p value of 0.05 were filtered out. To combine multiple analyses from the fractionation experiments, pepXML files were combined into a single mzXML using TPP. The increase in the 60 number of spectra, unique peptide, and protein identifications made after RP-RP analysis of FLAG-eIF4A2, RAF1, and MEPCE samples was assessed for statistical significance using a paired t-test implemented by GraphPad Software (http://www.graphpad.com/quickcalcs/ttest1.cfm). All raw files have been deposited in Tranche, hash: OSV877I4YeXnaQQnBk2BjOxRFOIuyT6qh1Nl8K1mnf413ufsMk2m5kTH30VgJzEut w KXLITEEY3vU2fvbuBgWmyzlagAAAAAAABT4a55..

SAINT analysis

SAINT calculates, for each prey protein identified in a purification, the probability of true interaction by using spectral counting (semi-supervised clustering, using a number of negative control runs) [30, 91, 257]. SAINT analysis of eIF4A2 and RAF1 was done using two biological replicates per bait and condition (1D versus 2D). BAIT protein samples were analyzed alongside 7 negative control runs, consisting of purifications from cells expressing the FLAG tag alone that had been analyzed using either one or two dimensional separation. Probability scores, indicating the likelihood for a true protein interaction to exist, were first computed for each prey protein in independent biological replicates before a final probability score for said prey protein was calculated as the average of its probabilities in the two individual replicates (AvgP); final results with AvgP≥0.9 are reported.

2.2 Results

To determine whether applying multidimensional separation methods to AP-MS samples, increases coverage for this type of sample (both in terms of total number of proteins identified and number of unique peptides or spectral counts observed), FLAG-tagged COPS5, eIF4A2, RAF1, MEPCE, and a negative control consisting of the FLAG tag alone, were purified by immunoprecipitation from inducible cell lines, as in [250] (Figure 2-1A). Samples were analysed by one-dimensional LC-MS/MS on an LTQ, an LTQ- Orbitrap (Thermo-Fisher Scientific), or a TripleTOF 5600 instrument (AB SCIEX), and/or subjected to an additional fractionation step prior to mass spectrometric identification (see Methods for details; Figure 2-1B). 61

Figure 2-1. Sample preparation. A) Western blot showing expression of FLAG-COPS5 (lanes 1,2), RAF1 (lanes 3,4), eIF4A2 (lanes 5,6), and MEPCE (lanes 7,8), using an inducible system in 293 Flp-In T-REX cells. Lanes 1,3,5,7, show un-induced cells, and lanes 2,4,6,8, induced. B) Immunopurified FLAG-COPS5 was subjected to LC-MS/MS on either an LTQ, LTQ-Orbitrap, or TripleTOF 5600 mass spectrometer. FLAG-eIF4A2, RAF1, and MEPCE were subjected to LC-MS/MS on an LTQ mass spectrometer (B steps 1-7). Samples were fractionated for multidimensional analysis using, reverse phase liquid chromatography (RP) at pH10 (B, step 8), strong cation exchange (SCX) liquid chromatography (B, step 9), or SDS-PAGE (B, step 10), prior to RP separation at pH2 (B, step 6).

After data acquisition as described above, proteins present on a list of common AP-MS contaminants (Supplementary Table S2-1A; n = 232) or proteins detected in any of the FLAG alone control runs (Supplementary Table S2-1B, n=126), were removed from subsequent analysis as these are suspected contaminant proteins (as outlined in section 1.5). Additionally, of the proteins remaining after background filtering, only those proteins identified with at least 2 unique peptides, and a ProteinProphet [256] probability of 0.95 were used for comparison. This was done to increase confidence that the protein identifications being made by Mascot, which are based on the spectra acquired, are correct, reducing the rate of false positive identifications. Details on the number of identifications made pre- and post-filtering are presented in Table 2-1.

Table 2-1. Summary of the mass spectrometry data for this project. Total MS/MS acquired, spectra with PeptideProphet ≥ 0.05, and high quality spectra with PeptideProphet ≥ 0.9 are listed for (A) FLAG-tagged COPS5 in the different replicates of 1D RP LTQ, (B) FLAG-tagged COPS5 in other fractionation methods, and (C) FLAG- tagged EIF4A2, RAF1, MEPCE bait proteins. "ORB" refers to analysis on the LTQ- Orbitrap, "3TOF" is the analysis on the AB-SCIEX 5600 TripleTOF, number in parentheses refer to different biological replicates. All other abbreviations are defined in the text. For the remainder of the analysis only those proteins detected with ProteinProphet ≥ 0.95 and 2 unique peptides are displayed. A. 1D RP LTQ 1D RP LTQ 1D RP LTQ 1D RP LTQ BAIT, Parameter (A) (B) (C) (D)

COPS5, MS/MS acquired 30262 37098 40949 32609 COPS5, PepP≥ 0.05 5047 4586 7988 3491 COPS5, PepP≥ 0.9 1554 1723 1844 612

B. BAIT, Parameter 1D RP ORB 1D RP 3TOF 2D RP/RP MudPIT GeLC

COPS5, MS/MS acquired 8357 39554 125922 40100 195668 COPS5, PepP≥ 0.05 3281 7586 11933 5957 11669 COPS5, PepP≥ 0.9 1186 3793 2657 1351 1053

C. 1D RP LTQ 1D RP LTQ 2D RP/RP 2D RP/RP BAIT, Parameter (1) (2) LTQ (1) LTQ (2)

eIF4A2, MS/MS acquired 12571 12878 66653 70447 eIF4A2, PepP≥ 0.05 1957 1781 3177 4841 eIF4A2, PepP≥ 0.9 391 341 661 805

RAF1, MS/MS acquired 12902 13379 70696 69715 RAF1, PepP≥ 0.05 1447 2269 3692 4066 RAF1, PepP≥ 0.9 228 437 659 860

MEPCE, MS/MS acquired 12768 N/A 70197 N/A MEPCE, PepP≥ 0.05 1904 N/A 3239 N/A MEPCE, PepP≥ 0.9 349 N/A 611 N/A 64

2.2.1 Reproducibility of Protein Identifications made by AP- MS

To begin my investigation into whether multidimensional fractionation of AP-MS samples could be useful for the identification of new PP4c interaction partners and expansion of the PP4c interaction network outlined in section 1.8, I first wanted to assess the reproducibility of our standard AP-MS approach through analysis of four independent biological replicates of FLAG-COPS5 (i.e. APs from four different cell pellets performed at different times) on an LTQ. After subtraction of likely contaminants, an average of 2045 spectra, 576 unique peptides, and 45 proteins were identified per biological replicate (Table 2-2A, first row; unfiltered data is in Table 2-2C, first row).

Table 2-2. Spectral counts, unique peptides, and non-redundant protein identification, A) for all proteins identified in COPS5 samples after background contaminant removal (COPS4 and DDB1 spectra were detected in FLAG alone controls, but these proteins were not filtered out due to substantial increase in spectra in FLAG-COPS5 purifications), B) for the COPS5 interactors reported in BioGRID and detected in our samples, or, C) for all proteins prior to background contaminant removal. Immunopurified FLAG-COPS5 was subjected to LC-MS/MS either on an LTQ, an LTQ-Orbitrap (ORB), or a TripleTOF 5600. Samples were fractionated for multidimensional analysis using RP-RP (pH10/pH2), GeLC, or MudPIT. Only proteins identified with 2 or more unique peptides and a ProteinProphet probability of at least 0.95 are considered. Sample batch purifications are indicated in superscript.

Separation Method Spectral Fold Unique Fold Fold (Instrument) Counts Change pep Change Proteins Change A) All after background removal 1D RP Merged (LTQ)1,2,3,4 2045±691 576±189 45±19

1D RP (LTQ)1 1944 626 43 RP/RP (LTQ)5 3234 1.66 723 1.15 51 1.19 GeLC (LTQ)6 2712 1.40 675 1.08 47 1.09 1D RP (Triple TOF 5600)5 2892 1.49 657 1.05 49 1.14

1D RP (ORB)7 1365 736 40 MudPIT (ORB)7 2013 1.47 515 0.70 83 2.08

B) BioGRID Annotated Interactors 1D RP Merged (LTQ)1,2,3,4 1511±435 433±126 20±2

1D RP (LTQ)1 1501 483 22 RP/RP (LTQ)5 2649 1.76 541 1.12 22 1.00 GeLC (LTQ)6 2233 1.49 540 1.12 24 1.09 1D RP (Triple TOF 5600)5 2327 1.55 514 1.06 23 1.05

1D RP (ORB)7 1066 581 24 MudPIT (ORB)7 1675 1.57 313 0.54 25 1.04

C) All before background removal 1D RP Merged (LTQ)1,2,3,4 2718±1102 892±381 111±53

1D RP (LTQ)1 2500 923 111 RP/RP (LTQ)5 4502 1.80 1219 1.32 148 1.33 GeLC (LTQ)6 4247 1.70 1223 1.33 123 1.11 1D RP (Triple TOF 5600)5 4687 1.87 1370 1.48 160 1.44

1D RP (ORB)7 2080 1340 143 MudPIT (ORB)7 3390 1.63 1273 0.95 238 1.66

Examining the reproducibility of protein identifications in the four different COPS5 biological replicates revealed that 23 proteins were detected in all four samples, 5 in three samples, 11 in two samples, and 54 proteins were observed in a single sample (36 of which were associated with COPS5 sample C; Figure 2-2A).

Figure 2-2. Venn diagrams showing protein identification overlap for COPS5. A) All proteins identified across four 1D LC-MS/MS analyses on an LTQ. B) Protein identification overlap for the samples outlined in (A) when only BioGRID-annotated COPS5 interactors are considered. C) Protein identification overlap when all 4 one dimensional runs are merged together and compared to samples subjected to multidimensional separation and analysis on an LTQ, or analysis on a TripleTOF 5600. D) Protein identification overlap for the samples outlined in (C) when only BioGRID- annotated COPS5 interactors were considered. E) Average spectral count per protein identified in either 4, 3, 2, or 1 samples. Venn diagrams were created using the web application Venny [258]. 69

The database BioGRID [202] (v3.0.67) reports 52 COPS5 interacting partners identified by a variety of different approaches, including 35 which were previously identified by AP-MS [32]. A total of 24 of these proteins were detected in at least one of our four 1D LC-MS/MS analyses. Importantly, 18 of these previously reported COPS5 interactors were detected across all four samples (Figure 2-2B). The proteins observed in only 1 or 2 runs yielded low spectral counts (Figure 2-2E). These data indicate that our standard AP-MS method is robust for the identification of known interactors, but that there is some degree of variation in the recovery of a given interacting partner between biological replicates, especially for proteins present in lower abundance in the sample.

2.2.2 Effect of fractionating affinity purified samples on spectral count, unique peptide, and protein identification

Next I analyzed the effect of applying multidimensional separation to the analysis of AP samples. As expected, additional sample fractionation resulted in an increase in spectral counts for almost all COPS5 interacting proteins detected by MS analysis after background removal; RP-RP, GeLC, and MudPIT increased average spectral counts by 1.66, 1.4 and 1.47-fold, respectively (Table 2-2A).

A similar increase (1.49-fold) was observed when the samples were analyzed on a new generation instrument with a faster scan rate, the AB SCIEX TripleTOF 5600 (of note, this result was obtained with only 50% of the sample volume loaded on the 1D LTQ, and 25% of the sample volume analyzed by multidimensional separation).

To analyze whether these increases in spectral counts could be reproduced with different AP samples, I performed RP-RP analysis on eIF4A2, RAF1 and MEPCE samples. RP-RP analysis of biological replicate eIF4A2 and RAF1 samples increased the average spectral counts observed after background removal by 2.34 and 2.48 fold respectively (Table 2-3A).

Table 2-3. Spectral counts, unique peptides, and non-redundant protein identification for two biological replicate analyses of EIF4A2 and RAF1 (A) for all proteins after background contaminant removal, (B) for the interaction partners reported in BioGRID, or (C) for all protein hits prior to background contaminant removal. Immunopurified FLAG-EIF4A2 or RAF1 were subjected to LC-MS/MS on an LTQ mass spectrometer. Samples were fractionated for multidimensional analysis using RP-RP (pH10/pH2). Only proteins identified with 2 or more unique peptides and a ProteinProphet probability of at least 0.95 are considered. 1D RP(1,2) is the 1D sample, RP/RP(1,2) is the 2D sample and Ratio (1,2) is the enrichment ratio in the 2D sample over the 1D sample. 1D RP 1D RP RP/RP RP/RP BAIT/Instrument/Parameter (1) (2) (1) (2) Ratio (1) Ratio (2) A) All after background removal EIF4A2/Spectral counts 523 626 1491 1147 2.85 1.83 EIF4A2/Unique peptides 363 381 513 460 1.41 1.21 EIF4A2/Proteins Identified 42 40 62 60 1.48 1.50

RAF1/Spectral counts 248 488 747 949 3.01 1.94 RAF1/Unique peptides 139 319 272 444 1.96 1.39 RAF1/Proteins Identified 23 51 63 104 2.74 2.04

B) BioGRID Annotated Interactors EIF4A2/Spectral counts 233 318 689 543 2.96 1.71 EIF4A2/Unique peptides 158 189 187 180 1.18 0.95 EIF4A2/Proteins Identified 10 12 10 11 1.00 0.92

RAF1/Spectral counts 80 136 278 301 3.48 2.21 RAF1/Unique peptides 45 78 74 76 1.64 0.97 RAF1/Proteins Identified 7 8 8 9 1.14 1.13

C) All before background Removal EIF4A2/Spectral counts 932 1070 2612 1855 2.80 1.73 EIF4A2/Unique peptides 725 715 1077 892 1.49 1.25 EIF4A2/Proteins Identified 202 177 278 266 1.38 1.50

RAF1/Spectral counts 701 1114 1937 2370 2.76 2.13 RAF1/Unique peptides 509 791 862 1331 1.69 1.68 RAF1/Proteins Identified 180 244 348 582 1.93 2.39 71

Analysis of one MEPCE sample resulted in an increase in average spectral count of 1.71 fold (Table 2-4A).

Table 2-4. Spectral counts, unique peptides, and non-redundant protein identifications for MEPCE samples (A) for all proteins identified after background contaminant removal, (B) for the interactors reported in BioGRID, and (C) for all protein hits prior to background contaminant removal. Immunopurified FLAG- MEPCE was subjected to LC-MS/MS on an LTQ mass spectrometer. Samples were fractionated for multidimensional analysis using RP/RP (pH10/pH2) or analyzed by 1D LC-MS/MS. Only proteins identified with 2 or more unique peptides and a ProteinProphet probability of at least 0.95 are considered. 1D RP(1) is the 1D sample, RP/RP(1) is the 2D sample and Ratio (1) is the enrichment ratio in the 2D sample over the 1D sample.

BAIT/Parameter 1D RP (1) RP/RP (1) Ratio (1) A) All Interactors after background removal MEPCE/Spectral counts 379 647 1.71 MEPCE//Unique peptides 199 315 1.58 MEPCE/Proteins Identified 29 54 1.86

B) BioGRID Annotated Interactors MEPCE/Spectral counts 200 310 1.55 MEPCE/Unique peptides 145 187 1.29 MEPCE/Proteins Identified 18 20 1.11

C) All Hits before background removal MEPCE/Spectral counts 960 1716 1.79 MEPCE/Unique peptides 617 976 1.58 MEPCE/Proteins Identified 187 308 1.65

More importantly however, the increase in spectral counts observed for the baits analyzed was only accompanied, in all cases, by a more modest increase in the number of unique peptides detected, with an average of 68% of the gain detected for spectral counts observed for unique peptides across all baits, methods, and instruments analyzed, respectively (Table 2-2A, 2-3A, 2-4A).

When the same parameters were analyzed for only the previously annotated COPS5, eIF4A2, RAF1, and MEPCE interactors, the same trend was observed: i.e. an overall > 2-fold increase in spectral counts in response to an additional degree of separation, and a more modest change in the recovery of known interactors, and in the number of unique peptides assigned to each of these interactors (Table 2-2B, 2-3B, 2- 4B). Notably, the increase in unique peptides and proteins observed in the RAF1 RP-RP, MEPCE RP-RP, and COPS5 MudPIT analyses after background removal (Table 2-2A, 2- 3A, 2-4A), were reduced to levels comparable to those observed across our other samples when only these previously annotated interacting proteins were considered. These observations indicate that conventional 1D-LC MS/MS may be sufficient for the detection of most interactions. Additionally, a paired T-test using RP-RP LTQ data for bait proteins which had paired samples separated in 1D (eIF4A2, RAF1, MEPCE), revealed that interactors annotated in BioGRID had a statistically significant ~2.2-fold spectral enrichment (P = 0.0178) while unique peptides and number of unique proteins were only mildly enriched (1.15 and 1.05 fold respectively; not statistically significant; Table 2-5).

Table 2-5. Paired t-test analysis comparing enrichment of spectra, unique peptides and protein identification by RP-RP analysis of FLAG-eIF4A2, RAF1, and MEPCE samples, after background removal (A), or for BioGRID annotated interactors only (B). Immunopurified FLAG-eIF4A2, RAF1, or MEPCE were subjected to LC-MS/MS on an LTQ mass spectrometer. Samples were fractionated for multidimensional analysis using RP-RP (pH10/pH2). Only proteins identified with 2 or more unique peptides and a ProteinProphet probability of at least 0.95 are considered. STDEV: standard deviation, SEM: standard error of the mean, N: number samples analyzed. Statistically significant*, Very Statistically Significant**, Extremely Statistically Significant***

P value Mean Mean STDEV STDEV SEM SEM N RP-RP RP RP-RP RP RP-RP RP A) Proteins after background removal Spectral counts 0.0092** 996 453 337 145 151 65 5 Unique peptides 0.0005*** 401 280 102 106 46 47 5 Proteins Identified 0.0082** 69 37 20 11 9 5 5

B) BioGRID Annotated Interactors Spectral counts 0.0178* 424 193 183 91 82 41 5 Unique peptides 0.146 141 123 60 60 27 27 5 Proteins Identified 0.305 12 11 5 4 2 2 5

2.2.3 Effect of fractionating affinity purified samples on protein complex component identification

To investigate whether different fractionation approaches led to the recovery of specific subsets of COPS5 binding partners, I compared the protein identifications made in 1D- LTQ (by merging non-redundant protein identifications in the 4 samples) to those made in 2D RP-RP, GeLC, and by the TripleTOF 5600. Similar to what I observed in the four independent 1D LTQ samples, I saw 31 proteins identified in all methods; 5 additional proteins observed across three methods, 16 proteins across two methods, and 69 by a single method (Figure 2-1C). Of the 26 known COPS5 interactors that I detected in our studies, the majority (20) were detected by all approaches, 2 proteins by three approaches, 2 by two approaches and only one by a single technique (Figure 2-1D). Notably, 8 of the 10 proteins detected in our analyses across all methods, but not listed as COPS5 interactors in BioGRID, are annotated interactors of other COP9 signalosome components, indicating that the majority of these proteins are indeed bona fide COPS5 binding partners (Table 2-6).

Table 2-6. Proteins identified in "Core" interaction network of FLAG-COPS5 purifications, that are not annotated as COPS5 interactors in BioGRID. Most of them have evidence for COP9 signalosome interaction in BioGRID [202] (v3.0.67).

Hit Gene Name Hit Entrez Gene ID Evidence for COP9 signalosome interaction MYEOV2 150678 Interacts with COPS6 KLHDC5 57542 Interacts with COPS6 SKP1 6500 Interacts with CUL1 TCEB1 6921 Interacts with COPS6, CUL2 CRBN 51185 Interacts with COPS6, CUL4A DTL 51514 Interacts with COPS6, CUL4A, CUL4B CUL5 8065 Interacts with COPS6, NEDD8, RBX1 MEPCE 56257 None LARP7 51574 None KLHL8 57563 Interacts with COPS6

Not surprisingly, the trends described above for the entire population of proteins remained in place for individual known COPS5, eIF4A2, RAF1, and MEPCE interactors, with the main benefit of multidimensional fractionation appearing to be an increase in spectral counts, followed by a modest increase in unique peptide identification (Tables 2- 7A, B, 2-8A, B, 2-9, S2-2 – S2-5).

Table 2-7. Fold increase in spectral counts (A), or unique peptides (B) for BioGRID- annotated COPS5 interactors. RP-RP, GeLC, 1D RP (Triple TOF) fold increase calculated relative to 1D LTQ sample A. MudPIT fold increase calculated relative to 1D ORB. **Number of peptides detected in RP/RP sample, none detected in 1D RP sample; ND (#) - Not detected in RP/RP sample (Number peptides detected in 1D RP sample). ***COPS4 and DDB1 spectra were detected in FLAG alone controls, but these proteins were not filtered out due to substantial increase in spectra in FLAG-COPS5purifications.

A. 1D RP Fold Interactor RP/RP (LTQ) GeLC (LTQ) (TripleTOF) MudPIT (ORB) Difference COPS1 1.57 2.61 0.87 0.83 >5 COPS2 1.67 1.37 1.16 1.95 4.01-5.00 COPS3 1.90 1.66 3.67 1.71 3.01-4.00 COPS4*** 1.60 1.64 2.40 1.80 2.01-3.00 COPS5-bait 1.04 0.91 1.39 0.31 1.01-2.00 COPS6 2.55 3.42 2.68 2.33 1 COPS7A 1.79 1.10 1.17 3.59 0.51-0.99 COPS7B 10.00 4.39 3.17 3.52 0.01-0.50 COPS8 1.28 0.45 0.88 0.26 ND (#) CUL1 1.78 1.32 2.11 1.08 CUL2 1.20 1.18 0.61 0.97 CUL3 2.34 1.13 1.62 1.40 CUL4A 1.67 1.47 1.09 1.27 CUL4B 1.27 1.16 0.93 2.38 DDB1*** 2.14 1.72 1.34 1.10 DDB2 1.77 1.45 1.23 1.35 VPRBP 3.00 5.00 2.25 1.67 LRRC14 0.77 3.23 1.31 1.00 DCAF11 1.27 0.58 1.38 0.83 BTBD2 3.00 5.67 2.67 4.00 PPIL5 ND (9) 1.11 0.77 ND (4) ERCC8 0.4 ND (5) ND (5) 4** FBX017 1.67 0.67 0.78 0.44

B. 1D RP Fold Interactor RP/RP (LTQ) GeLC (LTQ) (TripleTOF) MudPIT (ORB) Difference COPS1 1.14 1.59 0.90 0.31 >5 COPS2 0.98 0.72 1.02 0.26 4.01-5.00 COPS3 1.16 0.94 1.10 0.43 3.01-4.00 COPS4*** 1.13 0.74 0.94 0.73 2.01-3.00 COPS5-bait 0.87 0.55 0.80 0.24 1.01-2.00 COPS6 0.76 1.18 1.35 0.15 1 COPS7A 1.24 0.56 0.88 0.68 0.51-0.99 COPS7B 2 1.75 1.08 1.50 0.01-0.50 COPS8 0.84 0.39 0.74 0.15 ND (#) CUL1 1.44 1.75 1.56 0.53 CUL2 1.00 1.33 0.92 0.45 CUL3 1.12 1.15 1.39 0.59 CUL4A 1.06 1.39 1.33 0.44 CUL4B 1.14 1.27 1.04 0.86 DDB1*** 1.18 1.28 0.96 0.45 DDB2 1.44 1.44 1.33 0.50 VPRBP 2.67 4.33 2.00 0.93 LRRC14 0.75 3.25 1.38 0.53 DCAF11 1.18 0.82 1.27 0.47 BTBD2 1.33 1.67 1.67 1.50 PPIL5 ND (4) 2 1.00 ND (4) ERCC8 0.50 ND (4) ND (4) 2** FBX017 1.00 0.50 0.67 0.29 80

Table 2-8. A) Fold increase in spectral counts or unique peptides shown as the ratio of 2D/1D for BioGRID-annotated eIF4A2 interactors for each of the biological replicates analyzed (replicates annotated 1 and 2). **Number of peptides detected in RP/RP sample, none detected in 1D RP sample; ND (#) - Not detected in RP/RP sample (Number peptides detected in 1D RP sample). B) Fold increase in spectral counts or unique peptides shown as the ratio of 2D/1D for BioGRID-annotated RAF1 interactors for each of the biological replicates analyzed (replicates annotated 1 and 2). **Number of peptides detected in RP/RP sample, none detected in 1D RP sample; ND (#) - Not detected in RP/RP sample (Number peptides detected in 1D RP sample). Spectral Spectral Unique Unique A. Count Count Peptides Peptides Fold Interactor RP/RP (1) RP/RP (2) RP/RP (1) RP/RP (2) Increase EIF4A2-bait 1.60 1.76 0.92 1.26 >5 EIF4G1 2.90 2.03 1.18 0.89 4.01-5.00 EIF3A 2.60 1.38 1.00 0.95 3.01-4.00 EIF3L 3.33 2.43 1.25 1.11 2.01-3.00 EIF4G3 4.30 1.97 1.50 1.33 1.01-2.00 PDCD4 3.84 2.13 1.25 0.69 1 EIF4G2 2.25 0.89 1.25 0.75 0.51-0.99 EIF3H 3.56 2.45 1.43 1.00 0.01-0.50 EIF3F 1.38 0.57 1.17 0.57 ND (#) EIF3D 2.14 2.00 1.17 1.13 EIF3M ND (5) ND (8) ND (4) ND (4) DDX3X 4** 1.75 2** 1.33

Spectral Spectral Unique Unique B. Count Count Peptides Peptides Interactor RP/RP (1) RP/RP (2) RP/RP (1) RP/RP (2) RAF1-bait 1.42 1.50 0.63 0.66 YWHAE 2.85 2.90 1.43 1.08 YWHAG 3.71 1.60 1.55 0.90 YWHAZ 4.22 1.81 2.00 0.82 YWHAB 4.25 1.93 2.00 0.80 KRAS 1.14 0.80 0.80 0.75 YWHAH 4.28 1.62 1.33 0.89 YWHAQ 6.50 1.83 2.50 1.00 NRAS 9** 7** 5** 5** HRAS ND (0) ND (6) ND (0) ND (2) RAP1A ND (0) 2** ND (0) 2**

Table 2-9. Fold increase in spectral counts or unique peptides shown as the ratio of 2D/1D for BioGRID-annotated MEPCE interactors. **Number of peptides detected in RP/RP sample, none detected in 1D RP sample; ND (#) - Not detected in RP/RP sample (Number peptides detected in 1D RP sample). Spectral Unique Fold Interactor Counts Peptides Difference MEPCE-bait 1.41 1.00 >5 LARP7 1.70 1.20 4.01-5.00 SART3 1.64 1.33 3.01-4.00 PRPF4 0.89 0.71 2.01-3.00 CDK9 1.00 0.86 1.01-2.00 PRPF3 2.08 1.18 1 SNRNP200 2.58 1.90 0.51-0.99 SART1 1.09 1.00 0.01-0.50 CCNT1 1.00 0.89 ND (#) HEXIM1 1.25 0.83 EFTUD2 2.33 2.00 HNRNPUL1 0.40 0.67 METT10D 1.20 1.00 LSM4 ND (5) ND (2) KPNA2 1.20 1.25 PPIH ND (4) ND (3) DDX23 4.50 3.50 USP39 ND (3) ND (2) CSDA 3.00 2.50 CCNT2 3** 3** SNRPD2 3** 3** KPNB1 3** 3** LUC7L3 2** 2** SNRNP27 2** 2**

To further analyze what are the new proteins indentified after 2D separation, I analyzed the spectral count distribution of the proteins detected in each of the COPS5 samples. Analysis of the average spectral count per protein detected across all COPS5 1D LTQ runs or across all methods revealed an average of 83 spectral counts per protein (Figure 2-1E). This number dropped to an average of 12 spectral counts or less per protein once proteins were restricted to those detected in 3 or fewer samples (and were further reduced for proteins detected in only one or two samples), suggesting that these proteins are low stoichiometry interactors or low level contaminants that have escaped detection in the FLAG control experiments.

2.2.4 Applying Significance Analysis of INTeractome (SAINT) to fractionated affinity purified samples

Lastly, I was intrigued by the possibility that the increase in spectral counts brought about by applying multidimensional fractionation techniques to AP-MS samples may be beneficial in conjunction with methods that use spectral counts for scoring whether an observed protein is an interactor or contaminant. To assess this possibility I utilized SAINT (Significance Analysis of INTeractome – described in section 1.5), which uses spectral count information for a given prey protein across a dataset containing negative controls to calculate the probability that a protein is detected in a purification because it is an interactor and not a contaminant, [11, 91] to investigate whether more proteins would be identified as eIF4A2 or RAF1 interactors when RP-RP fractionation was used. In brief, I used 7 negative control runs consisting of purifications from cells expressing the FLAG tag alone which had been analyzed using either one or two dimensional separation for the SAINT semi-supervised modelling, and analysed the effects of performing RP-RP fractionation for identifying interactors for eIF4A2 and RAF1 (I used the AvgP, averaged probability, across both biological replicates). In both cases, SAINT identified a higher number of putative interactors with AvgP ≥0.9, an average of 65 vs. 48 and 118 vs. 62 when RP-RP fractionation was employed for either eIF4A2 or RAF1 respectively (Figure 2-3; Table S2-6), indicating that the increase in spectral count can be useful, depending on the downstream analysis.

Figure 2-3. Venn diagram of SAINT result overlap in (A) eIF4A2 and (B) RAF1 sample analysis. Protein identified with AvgP ≥0.9 are shown. BioGRID data is from interactions identified in our dataset prior to SAINT filtering. The presence of 2 RAF1 BioGRID annotated interactors not being identifying in either the 1D or 2D analysis although they were present in the unfiltered data indicates the data may have been overfiltered. Venn diagrams were created using the web application Venny [258].

2.3 Discussion 2.3.1 Multidimensional fractionation of AP-MS samples appears to allow for a better depth of coverage of low level background proteins and not core protein complex components

Multidimensional separation is typically applied to high complexity samples, and results in the identification of both more peptides and proteins than standard 1D LC-MS/MS in these sample types [93, 94, 97]. Importantly, for the work presented in this thesis, both MudPIT and RP/RP as described above, have been demonstrated to increase the number of peptides and proteins identified in a complex biological mixture when compared to one dimensional (1D) LC-MS/MS using reversed phase resin. Additionally, aside from identifying additional proteins, both these methods identified 95-100% of the proteins present in the 1D data set with the added benefit of having better sequence coverage [94]. However, here I found that when applied to lower complexity AP samples, the benefit of multidimensional separation was not as evident.

I observed that multidimensional separation robustly increased the number of spectra observed by MS; however, the effect on the number of unique peptides or proteins observed was more modest. While this trend was present, in most cases, when all proteins identified were analyzed together before background removal (Table 2-2C, 2-3C, 2-4C), or after background removal (Table 2-2A, 2-3A, 2-4A), it was most evident when only the interaction partners annotated BioGRID database, proteins known to be key components of complexes formed by the bait protein used, were considered (Table 2-2B, 2-3B, 2-4B). To this effect, the number of spectra, unique peptides, and proteins identified after RP-RP fractionation were all found to be significantly more than were observed by standard 1D LC-MS/MS after background removal alone (P = 0.0092, 0.0005, 0.0082 respectively), however, when only BioGRID annotated interactors were considered, only the number of spectra acquired was found to be significant (P = 0.0178), (Table 2-5). Additionally, when looking at the effect of multidimensional fractionation on the number of spectra and unique peptides acquired for individual known interactors (Tables 2-7 – 2-9), the same trend was evident, with the main benefit of sample 85 fractionation appearing to be an increase in the number of spectra acquired. This data leads me to believe that, although multidimensional fractionation of these sample types does lead to modest increases in unique peptide and protein identifications being made, what is being observed is not an increased depth of coverage of the core protein complex of interest but rather of the low level background proteins. In fact most “core” COPS5 proteins (Figure 2-2C), eIF4A2 and RAF1 interacting proteins (Figure 2-3) were able to be identified using 1D LC-MS/MS, indicating sample complexity for these sample types may not be limiting and they can be adequately resolved using 1D LC-MS/MS alone.

2.3.2 Primary benefits and disadvantages of multidimensional fractionation of AP-MS samples

The main benefit for fractionation of AP-MS samples observed during these experiments was an increase in the number of peptide spectra acquired. Notably, the increase in the number of spectra detected by multidimensional separation can be useful if spectral counts are used either for quantification (described briefly in section 1.7) [257] or statistical noise filtering (described in section 1.6, Figure 2-3) [76]. In these cases, additional spectral observations may increase the confidence that an identified protein is a biologically relevant interactor, and/or provide a better estimate of its relative abundance in a given sample. Note, however, that there is a significant level of variation in the spectral counts (before any filtering is applied) across our biological replicates, even in 1D LC-MS/MS (Table 2-2C first row, up to ~40% variation; the standard deviation between technical replicates is lower, at ~16%, Table S2-2). This variability will need to be considered with approaches based on spectral counting. In the SAINT example shown above, I have solely focused, for example, on those proteins that were statistically enriched in both of the eIF4A2 or RAF1 biological replicates.

Unfortunately, the increased sensitivity brought by multidimensional fractionation comes at the cost of increased handling and instrument time. For example, to obtain the average 2.17-fold increase in spectral counts yielded by RP-RP across all replicates of all 4 baits analyzed, mass spectrometer analysis time was increased from 2 to 12 hr per sample (6 fractions), and the handling time was increased from 4 to 7 hr. For GeLC the 1.40-fold increase in sensitivity for COPS5 was attained with an increase in instrument 86 time from 2 to 20 hr (10 gel fractions analyzed), and an increase in sample preparation time from 4 to 8 hr. Additionally, the loading of multiple sample fractions required for some multidimensional separation techniques (i.e. RP-RP, GeLC) increases the probability of sample loss due to the increased complexity of the platform which can lead to column clogging, column leakage, or loss of spray. In fact, these effects resulted in some of the samples needing to be repeated.

Lastly, I note that in some cases, fractionation can induce selective loss of some of the proteins / peptides, if, during the first separation, they bind to the stationary phase and are unable to be recovered by the elution conditions used. Additionally, splitting peptides into too many fractions can result in the reduction of lower abundance peptides such that they may drop below the MS detect limit [97].

2.3.3 Applicability of multidimensional fractionation of AP-MS samples to the expansion of the PP4c network and substrate identification

While the increase in the number of spectra detected by multidimensional separation allowed more proteins to pass background filtering using SAINT (Figure 2-3, Supplemental Table S2-6), and thus would indicate multidimensional fractionation is viable method for expanding the PP4c network when background filtering based on spectral counts is employed (Figure 1-7), the evidence presented above did not lead me to believe that it would be particularly useful for the identification of additional core components of, or achieving a better depth of coverage for the components already identified in, the PP4c complexes outlined in section 1.8. Based on this finding, the fact that I was able to identify the majority of the BioGRID annotated interactors for the baits examined without sample fractionation (Figure 2-2D, 2-3), and the additional amount of work/instrument time sample fractionation entails (four 1D LC-MS/MS analyses of FLAG-COPS5 took 8h of instrument time vs. 12h for RP-RP and 20h for GeLC, and identified 77% of all proteins after background removal Figure 2-2C and 96% of all BioGRID annotated interactors Figure 2-2D), I did not think fractionation of affinity purified PP4c-PP4R2-PP4R3A/B and PP4c interactor samples would provide enough useful data to justify its use for expanding this network. Based on my results, it appears 87 likely that the interaction network generated by Ginny using traditional 1D LC-MS/MS already encompasses the majority of the PP4c interactors that can be isolated and detected using MS based approaches.

2.3.4 Conclusions

In summary, in this study I have employed four baits with different cellular functions and/or intracellular localization, and for which several interaction partners were already annotated in the BioGRID interaction database (to enable benchmarking). These also tend to lean toward the highest level of complexity for AP samples, with fairly large protein complexes being co-precipitated [32, 55, 202]. It must be noted however, that, while the robust increase in spectral counts, and more modest increase in unique peptide and protein identifications made upon sample fractionation was consistent across these samples, it is still possible that some baits bringing down an even more complex set of associating proteins may benefit from the multidimensional fractionation approaches outlined herein to a larger degree. However, at this point I did not believe the application of these methods to PP4c or its interactors would justify its use.

Additionally, employing an instrument with a faster scan rate (TripleTOF 5600) enabled me to achieved results similar to those obtained by multidimensional separation on older mass spectrometers (Table 2-2, 2-7, Figure 2-2). These analyses were conducted using a reduced sample volume, which allows for smaller scale experiments to be performed at reduced cost, or additional technical replicates to be conducted in the same amount of time as a single multidimensional analysis. With the increased availability of these fast-scanning mass spectrometers, I conclude it is likely that the advantages of multidimensional fractionation for AP-MS samples will be further decreased.

Chapter 3 PP4c interactor phosphosite identification

To address my second aim, which was to assess whether the proteins involved in transcription and splicing, shown to interact with the PP4c-PP4R2-PP4R3A trimeric complex (Figure 1-7) could have their phsophorylation modulated by PP4c in vivo, FLAG tagged versions of these proteins were affinity purified from cells treated with okadaic acid or vehicle (H2O) alone, digested, and phosphopeptides isolated using IMAC or TiO2 and identified by MS/MS. Okadaic acid was selected as an initial treatment as no specific PP4c inhibitor is currently known, and okadaic acid has been shown to be a potent inhibitor of PP2A subfamily phosphatases (PP4c included) [159, 238], allowing for a maximal differential in the level of phosphorylation between treated and untreated samples to be observed. Isolated phosphopeptides were identified using a high resolution mass spectrometer, and their relative abundance in both okadaic acid and vehicle treated samples quantified using parent ion signal intensity during the MS1 scan with Proteome Discoverer software (V1.3.0.339; Thermo-Fisher Scientific). Phosphosite localization was determined using the PhosphoRS algorithm incorporated into the Proteome Discoverer software, as described previously. As an initial approach, I focused primarily on the proteins DHX38, SUPT5H, and HTATSF1, as these proteins were identified to be core components of PP4c interacting complexes outlined in section 1.8 (Figures 1-8 – 1.9), and implicated in the coupling of transcription and splicing (SUPT5H, HTATSF1). This initial discovery phase was used to identify phosphopeptides that demonstrate an increase in abundance upon okadaic acid treatment, indicating they could be regulated by PP4c, and can be investigated further in follow up experiments.

3.1 Materials and Methods 3.1.1 Generation and culture of stably transfected Flp-In T- REx 293 cell lines

To allow for a continuing investigation into which PP4c interaction partners (outlined in section 1.8 and Figure 1-7) are substrates for the enzyme, stable, inducible cell lines expressing PP4c, PP4c regulatory subunits, or interacting proteins were generated (Table 89

3-1). Selection, cell culture, and recombinant protein expression was performed as outlined in section 2.1.1; in this instance however, protein expression was induced by the addition of 1 µg/mL tetracycline (HyClone) to cell media for 24 hrs. Cloning of the DSIF component SUPT5H was unsuccessful.

Table 3-1. FLAG tagged constructs and stable cell lines generated for identification of puataive PP4c substrates. Listed are the HUGO gene name “Gene Name”, protein name used throught the thesis “Protein Name”, amino acid substitutions, or deletions constructs “Mutation” (∆SPG is lacking a series of SPG repeats in the C-terminal tail of PP4R3A), Entrez Gene ID, NCBI accession number “Accession Number”, the construct and cell line identifiers in open freezer [259], as well as the 5´ and 3´ cloning sites used in vector generation and whether a given bait was analyzed by AP-MS and if it purified well.

Gene Protein Entrez Accession Construct Cloning Site Cell Line AP- Purifies Name Name Mutation Gene ID Number Identifier 5´/3´ Identifier MS Well PPP4C PP4c 5531 NM_002720 V6686 EcoRI/XhoI C481 YES YES PPP4C PP4c R86A 5531 NM_002720 V6824 EcoRI/XhoI C492 YES NO PPP4R1 PP4R1 9989 BC060829 V6831 EcoRI/NotI C490 YES YES PPP4R2 PP4R2 151987 NP_777567 V6819 EcoRI/XhoI C487 YES YES PPP4R2 PP4R2 R103A 151987 NP_777567 V6820 BgIII/XhoI C486 YES YES SMEK1 PP4R3A 55671 NM_032560 V4845 EcoRI/NotI C483 YES YES SMEK1 PP4R3A 1-653 55671 NP_115949 V6834 EcoRI/NotI C494 YES YES SMEK1 PP4R3A 115-653 55671 NP_115949 V6833 EcoRI/NotI C493 NO N/A SMEK1 PP4R3A 115-820 55671 NM_032560 V4846 EcoRI/NotI C480 NO N/A SMEK1 PP4R3A ∆SPG 55671 NM_032560 V6832 EcoRI/NotI C495 YES YES SMEK2 PP4R3B 57223 NM_020463 V6822 AscI/NotI C491 YES YES SUPT4H1 SUPT4H 6827 NM_003168.1 V4843 EcoRI/NotI C476 YES NO SUPT5H SUPT5H 6829 BC024203 V5421 EcoRI/NotI N/A N/A N/A DHX38 DHX38 9785 NM_014003.3 V4847 EcoRI/NotI C473 YES YES HTATSF1 HTATSF1 27336 NM_014500.3 V4842 EcoRI/NotI C474 YES YES CCDC6 CCDC6 8030 NM_005436.2 V4849 BamHI/NotI C475 YES YES CDC5L CDC5L 988 NM_001253.2 V4848 AscI/NotI C477 YES NO

3.1.2 Affinity Purification

Cells expressing FLAG tagged proteins were harvested and proteins affinity purified essentially as described in section 2.1.2; however, after protein induction cells were treated with 100ng/mL okadaic acid (to induce protein phosphorylation), or vehicle

(H2O), for 3 hours before harvesting. Lysis buffer is as described in section 2.1.2, with the addition of 15nM okadaic acid, 15nM calyculin A, phosphatase inhibitor cocktail set II 1:100 (Calbiochem; 524625), and 50mM β-glycerol phosphate. Cleared lysate corresponding to 670-980mg of pellet was incubated with 25 l bed volume of pre- washed anti-FLAG M2 magnetic beads (Sigma; M8823). Affinity purifications from treated and untreated samples, for a specific bait, were conducted from an equivalent volume of cell lysate to ensure that direct comparisons of phosphopeptide abundance across samples could be made. After affinity purification, cell lysates were aspirated and beads washed once with lysis buffer (without 1mM DTT, 1mM PMSF, protease inhibitor cocktail 1:500, 15nM okadaic acid, 15nM calyculin A, phosphatase inhibitor cocktail set II 1:100, and 50mM β-glycerol phosphate), followed by one wash with 20mM Tris-HCl

(pH 8.0) + 2mM CaCl2, before bound proteins were subject to on bead digestion overnight with 1µg trypsin (Sigma; T6567) in 20mM Tris-HCl (pH 8.0) at 37 ºC. After overnight digestion, samples were removed from beads and incubated with an additional 0.25µg trypsin in 20mM Tris-HCl (pH 8.0) for 4 hours at 37 ºC. After protein digest samples were acidified to 2% formic acid (FA). Affinity purification was done using material from 6 x 150mm plates; this material, after digest and acidification, was pooled to 12 x 150mm plates for subsequent phosphopeptide enrichment. Samples were stored at -40°C if necessary, pending enrichment.

3.1.3 Enrichment of Phosphopeptides

Phosphopeptides were enriched from affinity purified digested material using IMAC

(Pierce Phosphopeptide isolation kit Cat#89853) and TiO2 (GL Sciences MonoTip spin columns cat# 5010-21311). Briefly, affinity purified digested material from 12 x 150mm plates (either treated with okadaic acid or not) was split in half and 6 x 150mm plates worth enriched using IMAC and 6 x 150mm plates worth enriched by TiO2. IMAC enrichment was performed as per the manufacturers protocol, with peptides incubated 92 with the IMAC resin for the maximum time indicated for each step of the enrichment procedure. IMAC elution was done using 100mM ammonium bicarbonate (pH9) and the elutions pooled. TiO2 enrichment was done using our in-house protocol as follows: TiO2 spin tips were first equilibrated by adding 100µL H2O and centrifuging at 1000 x g for 5 minutes followed by 3000 x g for 1 minute. Next 100µL MeOH was added and the process repeated, then 100µL of lactic acid solution (25% lactic acid, 60% acetonitrile (ACN), 2.5% trifluoroacetic acid (TFA)) was added, and the process repeated. After equilibration, 6 x 150mm plates worth of affinity purified digested material, in 2% FA (see section 3.1.2) was mixed with 100µL of lactic acid solution and loaded onto the spin tip. Samples were centrifuged at 1000 x g for 10 minutes, after which the sample flowthrough was collected and reloaded onto the spin tip. This process was repeated 3 times. Samples were then spun at 3000 x g for 1 minute, to clear out loading, and washed once with 50µL lactic acid solution (3000 x g for 2 minutes), once with 50µL 80% ACN + 0.1% TFA (3000 x g for 2 minutes), once with 50µL 0.1% TFA (3000 x g for 2 minutes), then twice with 50µL H2O (3000 x g for 2 minutes). Samples were eluted using

50uL of 400mM ammonium hydroxide (NH4OH; pH 11-12; 1000 x g for 5 minutes), twice, and elutions combined. Samples were then spun at 3000 x g for 1 minute to clear out tips completely, and all eluates combined. All solutions were made in MS grade H2O.

After enrichment IMAC and TiO2, eluates were combined and dried using a speed vac before MS analysis.

3.1.4 LC-MS/MS analysis

Enriched phosphopeptides (as described in section 3.1.3) from 12 x 150mm plates worth of affinity purified FLAG-DHX38, HTATSF1, SUPT4H, CCDC6, CDC5L, PP4R2, or PP4R3A (treated with okadaic acid or vehicle alone) were re-suspended in 12µL of 5% formic acid and 5uL (5 x 150mm plates worth) loaded by autosampler onto fused silica capillary columns (0.75 m id) packed in-house with 10 cm Zorbax C18 (ZorbaxSB, 3.5 m) and pre-equilibrated with HPLC buffer A. The amount of enriched material loaded on column was equivalent to five 90% confluent 150 mm plates. The loaded column was placed in-line with an LTQ-Orbitrap Velos mass spectrometer equipped with an Eksigent Ultra HPLC. The HPLC gradient was delivered at 200 nl/min using nanoflow. Buffers A 93 and B and the HPLC gradient are as described in section 2.1.3. The parameters for data dependent acquisition on the LTQ-Orbitrap Velos were 1 high resolution MS scan followed by 10 low resolution MS/MS scans of the most abundant ions. Of note, an aliquot of the IMAC phosphopeptide enrichment flow through (equivalent to 2 x 150mm plates of affinity purified material) was analyzed by LC-MS/MS as described in section 2.1.3. This was done to ensure that all experimental steps upstream of phosphopeptide enrichment had worked as expected, the amount of starting material in both okadaic acid and vehicle treated samples was approximately equal, and that sample treatment had no effect on bait protein expression (e.g. the number of bait spectra observed by LC-MS/MS in each was similar).

3.1.5 Data Analysis

All ThermoFinnigan RAW files were saved in our local interaction proteomics LIMS, ProHits [253]. RAW files were uploaded into Proteome Discoverer software (V1.3.0.339; Thermo-Fisher Scientific), converted to MGF and searched using Mascot (Matrix Science, version 2.2) using the following parameters: one missed cleavage site, methionine oxidation, asparagine/glutamine deamidation, and serine/threonine/tyrosine phosphorylation as variable modifications. The fragment mass tolerance was 0.6 Da (monoisotopic mass), and the mass window for the precursor was 12 ppm (monoisotopic). Only +2 and +3 spectra were searched. .The searched database contained the human and adenovirus complement of the RefSeq protein database (version 45; 34602 entries searched). Proteome Discoverer false discovery rate (FDR) was calculated by searching against a decoy datatbase in which all protein sequences are reversed. Peptides which did not meet a FDR of 1% as assigned by Proteome Discoverer were omitted from further analysis. Phosphosites which were not assigned with ≥ 0.95 probability, as assigned by Proteome Discoverer PhosphoRS application, were also omitted from analysis. In brief, (as described in section 1.7.2 and Figure 1-4) the PhosphoRS algorithm works by first assuming that the peptide sequence generated by the search engine used (e.g. Mascot) is correct and that the MS/MS spectra contains no co- fragmenting isobaric peptides. The algorithm then filters the MS/MS spectra and a sequence probability is calculated for each possible peptide phospho-isoform based on 94 the probability the theoretical spectra for the phospho-isoform is a random match to the filtered MS/MS spectra, and the degree of difference between the best two matches. The probability that a given phosphosite is phosphorylated is then calculated as the sum of the sequence probabilities for each possible phosphor-isoform in which the site is phosphorylated [120]. Thermo-Fisher Scientific reports sites that are assigned a probability of ≥0.75 are truly phosphorylated. For Proteome Discoverer analyses, label free quantification was enabled (with peptide grouping) to assess the relative abundance of phosphopeptides in okadaic acid treated vs. untreated samples using parent ion signal intensity in MS1. Phosphopeptides that contained the same phosphorylated residue, but were quantified separately due to eluting separately (e.g. because of a missed cleavage or other post translational modification e.g. oxidation) had their areas summed together and are reported as a single phosphosite.

3.2 Results 3.2.1 PP4c interactor/subunit phosphosite identification

To investigate which PP4c interaction partners (and possibly regulatory subunits), may be substrates for the enzyme, I used mass spectrometry to identify serine and threonine residues that become phosphorylated upon treatment with the potent PP2A subfamily phosphatase inhibitor okadaic acid. Briefly, FLAG tagged versions of DHX38, HTATSF1, SUPT4H, CCDC6, CDC5L, PP4R2, and PP4R3A, were affinity purified from okadaic acid or vehicle treated cells, digested, and the subsequent phosphopeptides enriched and analyzed by LC-MS/MS (Figure 3-1). Phosphopeptides and phosphosites were identified using Proteome Discoverer software as described in section 1.7.2.

Figure 3-1. Experimental workflow for PP4c interacting protein or regulatory subunit phosphopeptide identification. (A) Twelve 150mm plates of HEK-293 Flp-In T-REx cells expressing either FLAG-DHX38, HTATSF1, SUPT4H, CCDC6, CDC5L, PP4R2, or PP4R3A were treated with 100nM okadaic acid or vehicle for 3h and (B) FLAG tagged proteins affinity purified in two 6 plate batches. (C) After purification and tryptic digest affinity purified material was pooled and then (D) phophopeptides enriched from 6 x 150mm plates using IMAC and 6 x 150mm plates using TiO2. (E) After enrichment, phosphopeptides were pooled and then 5 x 150mm plates worth of enriched material analyzed by LC-MS/MS on an LTQ-Orbitrap Velos. Phosphopeptides and sites were identified using Proteome Discoverer software as described in section 3-1. (F) Phosphopeptide abundance was quantified by monitoring the ion signal over time and calculating the area of the peak for the isotopic cluster of the peptide. (G) Phosphosite assignment was done using the Proteome Discoverer PhosphoRS algorithm and CID spectra.

Identified DHX38, HTATSF1, CCDC6, CDC5L, PP4R2, and PP4R3A phosphopeptides are listed in table 3-2. Of note, no phosphopeptides were identified for the DSIF component SUPT4H therefore I analyzed phosphopeptides identified on its interaction partner SUPT5H, a PP4c interacting protein of interest that I was unable to successfully clone. Additionally, while phosphopeptides are listed for CDC5L in table 3- 2, CDC5L will be left out of further analysis as CDC5L expression and recovery was very low.

Table 3-2. Summary table listing phosphosites identified for PP4c interacting proteins and regulatory subunits. Briefly, FLAG-DHX38, HTATSF1, SUPT4H, CCDC6, CDC5L, PP4R2, or PP4R3A were affinity purified from HEK-293 Flp-In T- REx cells treated with 100nM okadaic acid (Treated – Yes) or vehicle (Treated – No) for

3h; digested with trypsin, and phosphopeptides enriched using IMAC and TiO2. Five 150mm plates worth of enriched material was subjected to LC-MS/MS on an LTQ- Orbitrap Velos and phosphopetides and phosphosites identified using Proteome Discoverer software as described in section 3-1. Phosphosites assigned with ≥0.95 probability of being correct by Proteome Discoverer software are shown as lower case letters in the context of where they occur in the protein sequence associated with the listed GenInfo Identifier (GI) number. The probability of a correct phosphosite assignment is shown in brackets. Other posttranslational modifications that we also searched for (such as methionine oxidation, asparagine/glutamine deamidation) are omitted from the table for clarity. For proteins that were analyzed more than once (DHX38, HTATSF1, SUPT5H), all phosphosites are listed, not just those reproducibly detected. For phosphosites detected reproducibly the highest probability the residue is phosphorylated is shown. Phosphosites identified in bold are not currently listed in the PhosphoSitePlus database ([260]; 02/01/2012). */** Indicates the site has been demonstrated to be functionally significant in vivo. / indicates the site of trypsin missed cleavage. Peptides were selected corresponding to a Proteome Discoverer FDR of 1%.

Protein Treated Phosphopeptides Phosphosites GI:11067747 CDC5L Yes 110-DNEEET(2.5)t(97.5)DDPRK-123 T117 CDC5L Yes 381-GGLNt(100)PLHESDFSGVt(100)PQR-390 T385, T389 CDC5L Yes 414-TPs(100)NGAEGLt(100)PR-427 S417, T424 CDC5L No 414-T(0.1)Ps(99.9)NGAEGLTPR-427 S417

GI:46852390 CCDC6 Yes 45-SGGIVIs(100)PFR-56 S52 CCDC6 Yes 95-K/As(100)VT(0.1)IQAR-105 S98 CCDC6 Yes 104-AEQEEEFIs(100)NTLFK-119 S113 CCDC6 Yes 136-EEEFLt(100)NELSR-148 T142 CCDC6 Yes 179-KLENDt(100)ISK-189 T185 CCDC6 Yes 234-LDQPVs(100)APPs(100)PR-247 S240, S244* CCDC6 Yes 246-DIs(100)MEIDs(100)PENMMR-260 S249, S254 CCDC6 Yes 277-AAQLQHs(100)EK-287 S284 CCDC6 Yes 320-QLs(100)ES(3.7)ES(92.9)s(98)LEMDDER-336 S323, S328 CCDC6 Yes 335-YFNEMs(100)AQGLRPR-349 S341 99

CCDC6 Yes 415-RSNs(100)PDK-423 S419 CCDC6 No 45-SGGIVIs(100)PFR-56 S52 CCDC6 No 234-LDQPVs(100)APPs(100)PR-247 S240, S244* CCDC6 No 246-DISMEIDs(100)PENMMR-260 S254 CCDC6 No 320-QLs(100)ES(0.3)ES(3.8)s(99.8)LEMDDER-336 S323, S328 CCDC6 No 415-RSNs(100)PDK-423 S419

GI:28372531 PP4R2 Yes 141-NNSNs(100)LNR-150 S146 PP4R2 Yes 149-MNGVMFPGNs(99.9)PS(0.1)Y(0.1)TER-166 S159 PP4R2 Yes 165-SNINGPGt(100)PRPLNRPK-182 T173 PP4R2 Yes 181-Vs(99.8)LS(5.3)APMT(0.1)T(0.1)NGLPES(2.7)T(1)Ds(99.6)K/ S183, S189 EANLQQNEEK-211 PP4R2 Yes 210-NHSDS(2.9)S(94.1)T(2.9)S(0.1)ESEVS(0.2)S(4.1)Vs(100)P S226 LKNK-232 PP4R2 Yes 377-ETEELVGSNSs(100)K-390 S389 PP4R2 No 149-MNGVMFPGNs(99.9)PS(0.3)Y(0.3)TER-166 S159 PP4R2 No 200-EANLQQNEEK/NHSDSSTSESEVS(0.3)S(1.7)Vs(100)P S226 LK-230 PP4R2 No 377-ETEELVGS(1.6)Ns(98.4)SK-390 S389

GI:31657117 PP4R3A Yes 9-VYt(100)LNEDR-18 T12 PP4R3A Yes 22-GTGHVS(0.3)s(99.4)GY(0.3)VER-35 S29 PP4R3A Yes 43-AEs(100)DGSLLLESK-56 S46 PP4R3A Yes 103-DPSVDITQDLVDEs(100)EEER-122 S117 PP4R3A Yes 121-FDDMS(0.9)s(99.1)PGLELPSCELSR-140 S127 PP4R3A Yes 708-LKEs(100)EEK-716 S712 PP4R3A Yes 726-Qs(98.8)PS(1.4)FK/Ls(99.8)LS(1.2)S(0.2)GT(0.1)K-740 S728, S734 PP4R3A Yes 773-NT(0.1)s(99.9)QTAAITTK-785 S776 PP4R3A No 43-AEs(100)DGSLLLESK-56 S46 PP4R3A No 103-DPSVDITQDLVDEs(100)EEER-122 S117 PP4R3A No 121-FDDMS(0.4)s(99.6)PGLELPSCELSR-140 S127 PP4R3A No 740-TNLTSQSSTTNLPGS(1)PGS(94.8)PGS(7.3)PGs(97.2)P S764 GS(16.3)PGS(0.2)VPK-774 PP4R3A No 773-NT(0.2)s(99.6)QT(0.2)AAITTK-785 S776

GI:17999539 DHX38 Yes 0-MGDTs(100)EDAs(100)IHR-13 S5, S9 DHX38 Yes 28-S(0.1)K/s(100)AAs(100)EQHVFK-41 S31, S34 DHX38 Yes 75-VS(3.5)s(96.3)Y(0.2)KDWEESK/DDQK-91 S78 DHX38 Yes 80-DWEEs(100)KDDQK-91 S85 DHX38 Yes 114-VEt(100)PS(0.2)HPGGVSEEFWER-132 T117 DHX38 Yes 193-RNEPEs(100)PR-201 S199 DHX38 Yes 212-STWEEEDs(100)GYGs(100)S(0.2)R/R/ S220, S224, S232, SQWEs(99.8)Ps(99.2)Pt(99.7)PS(0.2)YR-241 S234, T236 DHX38 Yes 258-GK/Y(0.1)s(99.9)DDt(100)PLPt(100)PSYK-274 S262, T265, T269 DHX38 Yes 296-R/EEGEEGISFDt(100)EEER-313 T308 DHX38 Yes 361-RIs(100)AQR-368 S364 DHX38 Yes 381-MLT(0.1)s(99.9)GVVHR-391 S385 DHX38 Yes 434-DAt(95.5)S(4.5)DLAIIAR-446 T437 100

DHX38 Yes 489-AVt(100)EDGKVDYR-500 T492 DHX38 Yes 511-K/s(100)EAs(97.8)S(2.2)EFAK-522 S513, S516 DHX38 Yes 679-LIVT(0.1)s(99.9)ATMDAEK-692 S684 DHX38 Yes 717-t(100)PQEDYVEAAVK-730 T718 DHX38 Yes 1193-R/s(100)PLGs(100)VR-1200 S1195, S1199 DHX38 Yes 1209-KEQGEPMt(100)PR-1220 S1218 DHX38 No 212-STWEEEDSGYGSSR/R/SQWESPs(100)PTPSYR-241 S234 DHX38 No 296-R/EEGEEGIs(99.9)FDT(0.1)EEER-313 S305 DHX38 No 679-LIVT(0.1)s(99.8)AT(0.1)MDAEK-692 S684 DHX38 No 717-t(100)PQEDYVEAAVK-730 T718 DHX38 No 1193-R/s(100)PLGSVR-1200 S1195

GI:161169023 SUPT5H Yes 106-As(100)NIDNVVLDEDR-120 S108 SUPT5H Yes 641-HLVLAGGs(100)KPR-653 S649 SUPT5H Yes 652-DVTNFTVGGFAPMs(100)PR-669 S666 SUPT5H Yes 668-IS(2.9)s(97.4)PMHPSAGGQR-682 S671 SUPT5H Yes 681-GGFGs(99.8)PGGGS(0.2)GGMSR-697 S686 SUPT5H Yes 711-Is(100)QGPYK-719 S713 SUPT5H Yes 767-TPMY(0.1)Gs(99.7)QT(0.1)PMYGs(100)GSR-784 S773, S780 SUPT5H Yes 783-TPMYGs(99.9)Qt(99.9)PLQDGSR-799 S789, T791** SUPT5H Yes 798-T(0.3)PHY(4.5)Gs(100)Qt(99.7)PLHDGSR-814 S804, T806** SUPT5H Yes 813-t(100)PAQSGAWDPNNPNt(99.9)PS(4.8)R-832 T814**, T828 SUPT5H Yes 1021-VVSISSEHLEPIt(99.9)PT(0.2)K-1038 T1034 SUPT5H No 17-s(97.5)S(2.5)DGEEAEVDEER-31 S18 SUPT5H No 1021-VVSISSEHLEPIt(100)PT(0.2)K-1038 T1034

GI:21361437 HTATSF1 Yes 0-Ms(2.3)Gt(97.7)NLDGNDEFDEQLR-18 T4 HTATSF1 Yes 26-DGDTQTDAGGEPDs(100)LGQQPTDTPYEWDLDK/K-58 S40 HTATSF1 Yes 118-K/AEs(100)GWFHVEEDR-132 S122 HTATSF1 Yes 190-REs(100)VELALK-200 S193 HTATSF1 Yes 238-KLs(100)MQQK-246 S241 HTATSF1 Yes 309-HPDGVAs(97.1)Vs(100)FR-321 S316, S318 HTATSF1 Yes 383-R/S(4.4)Ds(100)Vs(100)As(100)ER-394 S387, S389, S391 HTATSF1 Yes 400-HFSEHPST(1.5)s(98.5)K-411 S409 HTATSF1 Yes 433-TEDGGEFEEGAs(100)ENNAK/Es(100)s(100)PEK-457 S445, S452, S453 HTATSF1 Yes 474-GFEGS(0.5)Cs(100)QK/ES(0.5)EEGNPVR/ S481, S494, S498 Gs(100)EEDs(100)PK/K-501 HTATSF1 Yes 519-Es(100)EDDLNK/Es(100)EEEVGPTK-538 S521, S529 HTATSF1 Yes 572-DLDEEGs(100)EK/ELHENVLDK-591 S579 HTATSF1 Yes 590-ELEENDSENs(100)EFEDDGs(100)EK-610 S600, S607 HTATSF1 Yes 609-VLDEEGs(100)ER-619 S616 HTATSF1 Yes 618-EFDEDs(100)DEKEEEEDt(99.6)Y(0.4)EK-637 S624, T633 HTATSF1 Yes 636-VFDDEs(100)DEKEDEEYADEK-655 S642 HTATSF1 Yes 671-LFEEs(100)DDKEDEDADGK-688 S676 HTATSF1 Yes 695-LFEDDDs(100)NEK/LFDEEEDS(0.1)s(99.9)EK-717 S702, S714 HTATSF1 No 383-R/SDs(99.9)VS(0.1)ASER-394 S387 HTATSF1 No 433-TEDGGEFEEGAs(100)ENNAK-451 S445 HTATSF1 No 474-GFEGS(0.1)Cs(99.9)QK/ESEEGNPVR/ S481, S494, S498 Gs(100)EEDs(100)PK-501 101

HTATSF1 No 519-Es(100)EDDLNK/Es(100)EEEVGPTK-538 S521, S529 HTATSF1 No 572-DLDEEGs(100)EK/ELHENVLDK-591 S579 HTATSF1 No 590-ELEENDSENs(99.7)EFEDDGs(100)EK-610 S600, S607 HTATSF1 No 609-VLDEEGs(100)ER-619 S616 HTATSF1 No 618-EFDEDs(100)DEKEEEEDTYEK-637 S624 HTATSF1 No 705-LFDEEEDS(0.1)s(99.9)EK-717 S714 *Involved in protein activation, apoptosis; **Involved in regulation of RNA transcription

102

Overall, an average of 50% (49 out of 97), of the phosphosites in the PhosphoSitePlus database ([260]; 02/01/2012) for the baits analyzed, were identified (when all identifications from both treated and untreated samples were considered). However, when just those baits which had been analyzed once (CCDC6, PP4R2, PP4R3A) were considered the percentage of phosphosites identified decreased to 36% overall (16 out of 44). In contrast, 62% (33 out of 53), of the phosphosites listed for the baits analyzed in duplicate (DHX38, HTATSF1, SUPT5H) were identified. Overall, this indicates that my approach for identifying PP4c interactor/substrate phosphosites is moderately robust for identifying known phosphosites (50% identified overall for 6 baits analyzed), but more so when more than one biological replicate is conducted (62% identified overall for 3 baits analyzed in duplicate vs. 36% for those baits analyzed only once; Table 3-2).

Of the PhosphoSitePlus sites identified, some sites were observed to be phosphorylated independently of okadaic acid treatment, indicating that in some cases these sites may be constitutively phosphorylated (Table 3-2). Analyzing samples in biological duplicates did not increase the number of PhosphoSitePlus sites identified to be phosphorylated independently of okadaic acid treatment (overall only 45% of the 33 PhosphoSitePlus sites identified for those baits analyzed in biological duplicates (DHX38, HTATSF1, SUPT5H) were observed to be phosphorylated independently of okadaic acid treatment, while 75% of the 16 PhosphoSitePlus sites identified for those baits analyzed only once (CCDC6, PP4R2, PP4R3A) were observed to be phosphorylated independently of okadaic acid treatment. This indicates that PhosphoSitePlus site detection (in the absence of okadaic acid treatment) may be bait specific (due to differences in the level of constitutive phosphorylation for each bait), or, that because replicate analysis allows for more PhosphoSitePlus sites to be identified overall (33 sites were identified for the baits analyzed in duplicate while only 16 sites were identified for the baits analyzed once), having more analyses of okadaic acid treated samples may lower the percentage of PhosphoSitePlus sites observed to be phosphorylated independently of okadaic acid treatment by increasing the total number of PhosphoSitePlus sites identified (4 of 16 PhosphoSitePlus sites identified in samples analyzed once were observed in okadaic acid treated samples only, while 18 of 33 103

PhosphoSitePlus sites identified in samples analyzed in duplicate were observed in okadaic acid treated samples only).

Overall, (56 of the 103 phosphosites identified (54%) were putative new sites for the 6 baits analyzed), as these sites have yet to be described in the PhosphoSitePlus database. This indicates that my method is moderately robust for identifying new phosphosites. Notably, for those baits analyzed in biological duplicates (DHX38, HTATSF1, SUPT5H), more putative new phosphosites were identified (41 sites) than were observed for those baits analyzed only once (15 sites); however, when compared to the total number of sites identified for samples analyzed in duplicate (72) or those samples analyzed only once (31) the percentage of putative novel phosphosites identified (57% vs. 48% respectively), did not differ by much, indicating that the identification of more putative novel phosphosites upon replicate analysis is due to the total number of phosphosite identifications increasing (both putative new and previously observed) and not due to a sampling of different novel phosphopeptides in each replicate analyses only. Perhaps more importantly, the majority of these putative new sites were detected exclusively upon treatment with okadaic acid (48 of the 56 putative novel phosphosites identified overall for the 6 baits analyzed), indicating that okadaic acid treatment itself is a very robust method for identifying novel phosphosites for the set of proteins analyzed (Table 3-2).

It must be noted however, that some of the phosphosites listed in the PhosphoSitePlus database are likely incorrectly assigned. This is due to the nature of the data curation, whereby the data is curated directly from a publication as described and not subjected to re-analysis [261]. This leads to inconsitiencies in data quality and likely to false site identification. However, PhosphosSitePlus, does list the number of times a given phosphosite has been detected in either low or high thoroughput experiments (a useful metric for assessing confidence in the site being real). A comparison of phosphosites identified by me to the number of instances they had been reported in the PhosphoSitePlus database by others did not reveal any distinct trend between the sites identified by me, or those missed by me, and the number of times they had been reported by others (i.e. the sites identified by me had been reported numerous times by others 104 while those missed were only reported once), indicating that the sites missed by me are just as likely to be real as not, and may be missed due to not being phosphorylated under the conditions used. An assessment of the putative new sites identified by me did not reveal any trend towards close proximity to known sites (indicating incorrect site assignment). Additionally, as PhosphoSitePlus is continually being updated, some of the phosphosites that I have listed as putative new phosphosites have been added to the database while this thesis was being prepared, increasing my confidence that some proportion of them are real.

3.2.2 Phosphosite detection reproducibility

When assessing the reproducibility of phosphosite identification for the baits analyzed in duplicate (DHX38, HTATSF1, SUPT5H), 53% of the phosphosites identified upon okadaic acid treatment were detected reproducibly (38 out of 72), as were 50% of the sites identified in the absence of okadaic acid treatment (10 out of 20; Figure 3-2).

105

Figure 3-2. Venn diagrams showing phosphosite identification overlap for DHX38, HTATSF1, and SUPT5H. Phosphosite identification overlap for A) DHX38 in okadaic acid treated biological replicates (DHX OK 1, DHX OK 2) and untreated samples (DHX NT 1, DHX NT 2). B) HTATSF1 in okadaic acid treated biological replicates (HTAT OK 1, HTAT OK 2) and untreated samples (HTAT NT 1, HTAT NT 2). C) SUPT5H in okadaic acid treated biological replicates (SPT5 OK 1, SPT5 OK 2) and untreated samples (SPT5 NT 1, SPT5 NT 2). Venn diagrams were created using the web application Venny [258].

106

This was interesting as overall substantially more phosphosites were detected upon okadaic acid treatment (72 vs. 20 respectively), yet the reproducibility of phosphosite detection did not differ upon okadaic acid treatment. Of note, the level of reproducibility did vary by bait, with most variation observed to occur with DHX38 (11 out of the 28 phosphosites identified on DHX38 upon okadaic acid treatment were only observed in one sample, (Figure 3-2; DHX OK 1); and only 1 of the 5 DHX38 phosphosites identified to be phosphorylated independently of okadaic acid treatment was detected reproducibly (Figure 3-2). Restricting this analysis to just those sites listed in the PhosphoSitePlus database (to increase the probability that they are real phosphosites) increased the percentage of sites detected reproducibly upon okadaic acid treatment to 67% overall for the 3 baits analyzed, and 60% overall in the absence of okadaic acid treatment (Figure 3-3), indicating that some of the lack of reproducibility in site detection observed by me may be due to false localization of the phosphosite by the PhosphoRS algorithm, further highlighting the need for manual validation. Again, as was observed above for all identified phosphosites (not just those listed in PhosphoSitePlus), more previously observed phosphosites were detected upon okadaic acid treatment than without (27 vs. 15), yet the reproducibility of phosphosite identification did not differ substantially between samples treated with okadaic acid (67% identified reproducibly) and those without (60% identified reproducibly). Phosphosites discussed in figures 3-2, 3-3 (and listed in Table 3-2), are mapped onto their respective proteins (for proteins analyzed in biological duplicate: DHX38, HTATSF1, SUPT5H), for ease of viewing (Figures 3-4 – 3-6).

107

Figure 3-3. Venn diagrams showing PhosphoSitePlus phosphosite identification overlap for DHX38, HTATSF1, and SUPT5H. Phosphosite identification overlap for A) DHX38 in okadaic acid treated biological replicates (DHX OK 1, DHX OK 2) and untreated samples (DHX NT 1, DHX NT 2). B) HTATSF1 in okadaic acid treated biological replicates (HTAT OK 1, HTAT OK 2) and untreated samples (HTAT NT 1, HTAT NT 2). C) SUPT5H in okadaic acid treated biological replicates (SPT5 OK 1, SPT5 OK 2) and untreated samples (SPT5 NT 1, SPT5 NT 2). Only phosphosites listed in the PhosphoSitePlus database ([260]; 02/01/2012) are considered. Venn diagrams were created using the web application Venny [258].

108

109

Figure 3-4. DHX38 protein diagram illustrating phosphosites identified in biological replicate analysis of FLAG-DHX38. Briefly, FLAG-DHX38 was affinity purified from HEK-293 Flp-In T-REx cells (A,C) treated with 100nM okadaic acid for 3h or (B,D) vehicle; digested with trypsin, and phophopeptides enriched using IMAC or TiO2. Five 150mm plates worth of enriched material was subjected to LC-MS/MS on an LTQ- Orbitrap Velos and phophopetides and sites identified using Proteome Discoverer software as described in section 3-1. Phosphosites listed in red are not currently listed in the PhosphoSitePlus database ([260]; 02/01/2012); phosphosites listed in black are, but were not identified in these analyses; phosphosites listed in blue are listed in PhosphoSitePlus and were detected in these analyses. Only sites assigned with ≥0.95 probability of being correct by Proteome Discoverer’s PhosphoRS algorithm are shown. DEAD – RNA helicase domain; Helicase – conserved helicase domain; HA2 – domain of unknown function (possibly nucleic acid binding); OB NTP – domain of unknown function.

110

111

Figure 3-5. HTATSF1 protein diagram illustrating phosphosites identified in biological replicate analysis of FLAG-HTATSF1. Briefly, FLAG-HTATSF1 was affinity purified from HEK-293 Flp-In T-REx cells (A,C) treated with 100nM okadaic acid for 3h or (B,D) vehicle; digested with trypsin, and phophopeptides enriched using

IMAC or TiO2. Five 150mm plates worth of enriched material was subjected to LC- MS/MS on an LTQ-Orbitrap Velos and phophopetides and sites identified using Proteome Discoverer software as described in section 3-1. Phosphosites listed in red are not currently listed in the PhosphoSitePlus database ([260]; 02/01/2012); phosphosites listed in black are, but were not identified in these analyses; phosphosites listed in blue are listed in PhosphoSitePlus and were detected in these analyses. Only sites assigned with ≥0.95 probability of being correct by Proteome Discoverer’s PhosphoRS algorithm are shown. RRM – RNA recognition motif (binds to single stranded RNA).

112

113

Figure 3-6. SUPT5H protein diagram illustrating phosphosites identified in biological replicate analysis of FLAG-SUPT4H. Briefly, FLAG-SUPT4H was affinity purified from HEK-293 Flp-In T-REx cells (A,C) treated with 100nM okadaic acid for 3h or (B,D) vehicle; digested with trypsin, and phophopeptides enriched using IMAC or

TiO2. Five 150mm plates worth of enriched material was subjected to LC-MS/MS on an LTQ-Orbitrap Velos and phophopetides and sites identified using Proteome Discoverer software as described in section 3-1. Phosphosites listed in red are not currently listed in the PhosphoSitePlus database ([260]; 02/01/2012); phosphosites listed in black are, but were not identified in these analyses; phosphosites listed in blue are listed in PhosphoSitePlus and were detected in these analyses. Only sites assigned with ≥0.95 probability of being correct by Proteome Discoverer’s PhosphoRS algorithm are shown. Supt5 – SUPT4H binding domain, needed for DSIF dimerization; KOW – Kyprides, Ouzounis, Woese motif (found in ribosomal proteins and bacterial transcription factor NusG).

114

3.2.3 Changes in PP4c interactor phosphorylation upon PP2A type phosphatase inhibition

To assess whether okadaic acid treatment led to an increase in protein phosphorylation at sites identified to be phosphorylated in both okadaic acid treated and untreated samples (Table 3-2), and to assess the reproducibility of phosphosite quantification across biological replicates (Figures 3-4 – 3-6), phosphopeptide abundance was quantified at MS1 (using the area of the peak for the isotopic cluster of a peptide of interest with Proteome Discoverer software; Tables 3-3 – 3-5), and phosphosite abundance compared between okadaic acid treated and untreated samples for those phosphosites which were reproducibly identified in both okadaic acid treated and untreated samples for the three baits analyzed in duplicate (Figures 3-7 – 3-9).

115

Table 3-3. Quantification of phosphopeptides identified in biological replicate analysis of FLAG-DHX38. Briefly, FLAG-DHX38 was affinity purified from HEK-293 Flp-In T-REx cells treated with 100nM okadaic acid for 3h or vehicle alone; digested with trypsin, and phophopeptides enriched using IMAC or TiO2. Five 150mm plates worth of enriched material was subjected to LC-MS/MS on an LTQ-Orbitrap Velos and phosphopetides and sites identified using Proteome Discoverer software as described in section 3-1. Peptides were selected corresponding to a Proteome Discoverer FDR of 1%. Phosphopeptide abundance was quantified by Proteome Discoverer software based on MS1 area. The relative abundance of the identified phosphopeptides in okadaic acid treated samples (Treated – Yes) is shown as compared to untreated samples (Treated – No). Phosphosite assignment was done by Proteome Discoverer’s PhosphoRS algorithm. Phosphosites assigned with ≥0.95 probability of being correct are shown as lowercase letters, the probability of a correct site assignment is shown in brackets. Other PTM’s are omitted for clarity. A) Biological Replicate 1. B) Biological Replicate 2. A Peptide Sequence (Probability of correct site assignment) MS1 Area Phosphosites Treated Phosphosites identified reproducibly in both treated and untreated samples 717-t(100)PQEDYVEAAVK-730 5.30E+06 T718 Yes 717-t(100)PQEDYVEAAVK-730 2.28E+06 T718 No Phosphosites identified reproducibly in treated samples only 0-MGDTSEDAs(100)IHR-13 6.44E+07 S9 Yes 28-S(0.1)Ks(99.8)AAS(0.1)EQHVFK-41 1.04E+07 S31 Yes 114-Vet(99.8)PS0.2HPGGVSEEFWER-132 4.07E+06 T117 Yes 193-RNEPEs(100)PR-201 3.50E+09 S199 Yes 212-STWEEEDSGYGs(100)SR-227 4.82E+08 S224 Yes 212-STWEEEDS(0.4)GYGs(98.7)S(0.4)RR-228 1.28E+08 S224 Yes 260-YSDDt(100)PLPTPSYK-274 2.52E+08 T265 Yes 296-REEGEEGISFDt(100)EEER-313 8.40E+06 T308 Yes 297-EEGEEGISFDt(100)EEER-313 1.48E+07 T308 Yes Phosphosites identified un-reproducibly in both treated and untreated samples 227-SQWES(3.9)Ps(95.9)PT(0.2)PSYR-241 5.13E+08 S234 Yes 227-SqWES(0.4)Ps(99.2)PT(0.4)PSYR-241 2.63E+06 S234 Yes 679-LIVT(0.1)s(99.9)ATMDAEK-692 1.64E+06 S684 Yes 679-LIVT(0.1)s(99.8)AT(0.1)MDAEK-692 3.86E+05 S684 No 1193-Rs(100)PLGSVR-1202 2.33E+08 S1195 Yes 1194-s(100)PLGSVR-1202 5.63E+07 S1195 No Phosphosites identified un-reproducibly in treated samples only 0-mGDTs(100)EDASIHR-13 1.64E+07 S5 Yes 30-SAAs(100)EQHVFK-41 1.08E+07 S34 Yes 75-VS(3.5)s(96.3)Y(0.2)KDWEESK-87 2.89E+06 S78 Yes 116

80-DWEEs(100)KDDQK-91 1.22E+07 S85 Yes 258-GKY(0.1)s(99.9)DDTPLPTPSYK-274 6.42E+06 S262 Yes 381-MLT(0.1)s(99.9)GVVHR-391 1.95E+06 S385 Yes 434-DAt(95.5)S(4.5)DLAIIAR-446 5.59E+04 T437 Yes 511-Ks(100)EASSEFAK-522 2.23E+07 S513 Yes 1193-SPLGs(100)VR-1202 8.83E+08 S1199 Yes 1209-KEQGEPMt(100)PR-1220 1.83E+07 T1218 Yes Phosphosites identified un-reproducibly in untreated samples only 297-EEGEEGIs(99.9)FDT(0.1)EEER-313 1.95E+05 S305 No Doubly phosphorylated peptides 212-STWEEEDs(100)GYGs(99.8)S(0.2)R-227 6.34E+06 S220, S224 Yes 227-SQWEs(99.8)PS(0.3)Pt(99.7)PS0.2)YR-241 1.33E+08 S232, T236 Yes 258-GKYSDDt(100)PLPt(100)PSYK-274 3.56E+06 T265, T269 Yes 260-YSDDt(100)PLPt(100)PSYK-274 3.08E+08 T265, T269 Yes

B Peptide Sequence (Probability of correct site assignment) MS1 Area Phosphosites Treated Phosphosites identified reproducibly in both treated and untreated samples 717-t(100)PQEDYVEAAVK-730 4.07E+06 T718 Yes 717-t(100)PQEDYVEAAVK-730 1.06E+06 T718 No Phosphosites identified reproducibly in treated samples only 0-MGDTSEDAs(100)IHR-13 7.25E+06 S9 Yes 0-mGDTSEDAs(100)IHR-13 1.18E+06 S9 Yes 28-S(0.1)Ks(99.8)AAS(0.1)EQHVFK-41 9.17E+06 S31 Yes 30-s(100)AASEQHVFK-41 2.23E+07 S31 Yes 114-Vet(100)PSHPGGVSEEFWER-132 1.73E+08 T117 Yes 193-RNEPEs(100)PR-201 5.09E+08 S199 Yes 212-STWEEEDSGYGs(100)SR-228 3.44E+08 S224 Yes 260-YSDDt(100)PLPTPSYK-274 2.17E+08 T265 Yes 258-GKYSDDt(100)PLPTPSYK-274 5.68E+07 T265 Yes 296-REEGEEGISFDt(100)EEER-313 3.51E+06 T308 Yes 297-EEGEEGISFDt(100)EEER-313 4.57E+06 T308 Yes Phosphosites identified un-reproducibly in both treated and untreated samples 227-SQWES(0.1)Ps(99.9)PTPSYR-241 6.69E+08 S234 Yes 226-RSQWES(0.1)Ps(99.7)PT(0.1)PSYR-241 1.14E+07 S234 Yes 227-SQWESPs(100)PTPSYR-241 2.81E+05 S234 No 679-LIVT(0.1)s(99.9)ATMDAEK-692 6.20E+06 S684 Yes 679-LIVT(0.1)s(99.9)ATmDAEK-692 3.41E+06 S684 Yes 1193-Rs(100)PLGSVR-1202 9.77E+07 S1195 Yes Phosphosites identified un-reproducibly in treated samples only 361-Ris(100)AQR-368 5.87E+08 S364 Yes 489-Avt(100)EDGKVDYR-500 1.86E+06 T492 Yes 512-SEAs(97.8)S(2.2)EFAK-522 6.74E+06 S516 Yes Doubly phosphorylated peptides 227-SQWEs(99.7)PS(0.5)Pt(99.7)PSYR-241 5.11E+05 S232, T236 Yes 258-GKYSDDt(100)PLPt(100)PSYK-274 4.28E+04 T265, T269 Yes 260-YSDDt(100)PLPt(100)PSYK-274 6.16E+05 T265, T269 Yes 117

118

Figure 3-7. Quantification of phosphopeptides reproducibly identified in biological replicate analysis of FLAG-DHX38. Briefly, FLAG-DHX38 was affinity purified from HEK-293 Flp-In T-REx cells treated with 100nM okadaic acid for 3h or vehicle alone; digested with trypsin, and phophopeptides enriched using IMAC or TiO2. Five 150mm plates worth of enriched material was subjected to LC-MS/MS on an LTQ-Orbitrap Velos and phophopetides and sites identified using Proteome Discoverer software as described in section 3-1. Peptides were selected corresponding to a Proteome Discoverer FDR of 1%. Phosphopeptide abundance was quantified by Proteome Discoverer software based on MS1 area. The relative abundance of the identified phosphopeptides in okadaic acid treated samples (blue) is shown as compared to untreated samples (red). Peptide areas were summed for peptides containing the same phosphosite but exhibiting different areas due to eluting separately (e.g. because they contained other posttranslational modifications (deamidation, oxidation) or missed cleavages). Doubly phosphorylated peptides are presented separately. Phosphosite assignment was done by Proteome Discoverer’s PhosphoRS algorithm. Phosphosites assigned with ≥0.95 probability of being correct are shown as lowercase letters, the probability of a correct site assignment is shown in brackets. Other PTM’s are omitted for clarity. A) Biological Replicate 1. B) Biological Replicate 2.

119

Table 3-4. Quantification of phosphopeptides identified in biological replicate analysis of FLAG-HTATSF1. Briefly, FLAG-HTATSF1 was affinity purified from HEK-293 Flp-In T-REx cells treated with 100nM okadaic acid for 3h or vehicle alone; digested with trypsin, and phophopeptides enriched using IMAC or TiO2. Five 150mm plates worth of enriched material was subjected to LC-MS/MS on an LTQ-Orbitrap Velos and phophopetides and sites identified using Proteome Discoverer software as described in section 3-1. Peptides were selected corresponding to a Proteome Discoverer FDR of 1%. Phosphopeptide abundance was quantified by Proteome Discoverer software based on MS1 area. The relative abundance of the identified phosphopeptides in okadaic acid treated samples (Treated – Yes) is shown as compared to untreated samples (Treated – No). Phosphosite assignment was done by Proteome Discoverer’s PhosphoRS algorithm. Phosphosites assigned with ≥0.95 probability of being correct are shown as lowercase letters, the probability of a correct site assignment is shown in brackets. Other PTM’s are omitted for clarity. A) Biological Replicate 1. B) Biological Replicate 2. A Peptide Sequence (Probability of correct site assignment) MS1 Area Phosphosites Treated Phosphosites identified reproducibly in both treated and untreated samples 433-TEDGGEFEEGAs(100)ENNAK-451 8.04E+07 S445 Yes 433-TEDGGEFEEGAs(100)ENNAK-451 1.68E+07 S445 No 483-ESEEGNPVRGs(100)EEDSPK-500 7.12E+06 S494 Yes 483-ESEEGNPVRGs(100)EEDSPK-500 2.23E+06 S494 No 492-GSEEDs(100)PK-501 3.70E+05 S498 Yes 492-GSEEDs(100)PKK-502 7.28E+07 S498 Yes 492-GSEEDs(100)PK-501 7.15E+05 S498 No 492-GSEEDs(100)PKK-502 2.93E+07 S498 No 519-ESEDDLNKEs(100)EEEVGPTK-538 4.87E+07 S529 Yes 519-ESEDDLNKEs(100)EEEVGPTK-538 3.75E+07 S529 No 572-DLDEEGs(100)EK-582 1.45E+08 S579 Yes 572-DLDEEGs(100)EKELHENVLDK-591 3.68E+07 S579 Yes 572-DLDEEGs(100)EK-582 6.46E+07 S579 No 572-DLDEEGs(100)EKELHENVLDK-591 6.16E+07 S579 No 609-VLDEEGs(100)ER-619 8.27E+08 S616 Yes 609-VLDEEGs(100)ER-619 4.21E+08 S616 No 618-EFDEDs(100)DEKEEEEDTYEK-637 4.56E+06 S624 Yes 618-EFDEDs(100)DEKEEEEDTYEK-637 3.77E+06 S624 No Phosphosites identified reproducibly in treated samples only 26-DGDTQTDAGGEPDs(100)LGQQPTDTPYEWDLDK-57 8.18E+04 S40 Yes 26-DGDTQT(0.1)DAGGEPDs(98.9)LGQQPT(0.3)D T(0.3)PY(0.3)EWDLDKK-58 2.97E+05 S40 Yes 238-KLs(100)MQQK-246 3.97E+06 S241 Yes Phosphosites identified un-reproducibly in both treated and 120 untreated samples 383-RSDs(99.9)VS(0.1)ASER-394 7.03E+05 S387 No 474-GFEGSCs(100)QKESEEGNPVR-490 1.68E+05 S481 Yes 474-GFEGS(0.1)Cs(99.9)QKESEEGNPVR-490 6.69E+04 S481 No 519-Es(100)EDDLNK-528 1.64E+06 S521 Yes 590-ELEENDSENSEFEDDGs(100)EK-610 1.85E+06 S607 Yes 590-ELEENDSENSEFEDDGs(100)EK-610 5.46E+06 S607 No Phosphosites identified un-reproducibly in treated samples only 0-Ms(2.3)Gt(97.7)NLDGNDEFDEQLR-18 1.08E+05 T4 Yes 119-Aes(100)GWFHVEEDR-132 1.02E+05 S122 Yes 190-Res(100)VELALK-200 5.81E+05 S193 Yes 309-HPDGVASVs(100)FR-321 9.53E+05 S318 Yes 384-SDS(1.2)Vs(98.8)ASER-394 3.65E+07 S389 Yes 400-HFSEHPST(1.5)s(98.5)K-411 6.48E+07 S409 Yes Doubly phosphorylated peptides 384-S(4.4)Ds(95.6)VS(4.4)As(95.6)ER-394 2.37E+06 S387, S391 Yes 383-RSDs(100)Vs(100)ASER-394 4.11E+06 S387, S389 Yes

B Peptide Sequence (Probability of correct site assignment) MS1 Area Phosphosites Treated Phosphosites identified reproducibly in both treated and untreated samples 433-TEDGGEFEEGAs(100)ENNAK-451 6.01E+08 S445 Yes 433-TEDGGEFEEGAs(100)EnNAK-451 1.73E+07 S445 Yes 433-TEDGGEFEEGAs(100)ENNAK-451 1.04E+08 S445 No 433-TEDGGEFEEGAs(100)EnNAK-451 5.51E+06 S445 No 483-ESEEGNPVRGs(100)EEDSPK-501 6.60E+06 S494 Yes 483-ESEEGNPVRGs(100)EEDSPK-501 3.00E+06 S494 No 492-GSEEDs(100)PK-501 5.67E+06 S498 Yes 492-GSEEDs(100)PKK-502 9.71E+07 S498 Yes 492-GSEEDs(100)PK-501 6.54E+06 S498 No 492-GSEEDs(100)PKK-502 1.15E+08 S498 No 519-ESEDDLNKEs(100)EEEVGPTK-538 4.42E+08 S529 Yes 519-ESEDDLNKEs(100)EEEVGPTK-538 2.38E+08 S529 No 572-DLDEEGs(100)EK-582 9.61E+08 S579 Yes 572-DLDEEGs(100)EKELHENVLDK-591 1.65E+09 S579 Yes 572-DLDEEGs(100)EK-582 1.20E+09 S579 No 572-DLDEEGs(100)EKELHENVLDK-591 2.81E+08 S579 No 609-VLDEEGs(100)ER-619 2.78E+09 S616 Yes 609-VLDEEGs(100)ER-619 1.71E+09 S616 No 618-EFDEDs(100)DEKEEEEDTYEK-637 4.00E+09 S624 Yes 618-EFDEDs(100)DEKEEEEDTYEK-637 6.59E+07 S624 No Phosphosites identified reproducibly in treated samples only 26-DGDTQTDAGGEPDs(100)LGQQPTDTPYEWDLDK-57 6.21E+07 S40 Yes 26-DGDTQTDAGGEPDs(99.9)LGQQPT(0.1)D TPYEWDLDKK-58 1.14E+08 S40 Yes 238-KLs(100)MQQK-246 3.21E+07 S241 Yes Phosphosites identified un-reproducibly in both treated and untreated samples 384-SDs(100)VSASER-394 9.23E+08 S387 Yes 121

383-RSDs(99.9)VS(0.1)ASER-394 2.60E+06 S387 No 474-GFEGS(0.5)Cs(99.1)QKES(0.5)EEGNPVR-490 2.77E+06 S481 Yes 519-Es(100)EDDLNK-528 7.39E+06 S521 Yes 519-Es(100)EDDLNK-528 8.40E+06 S521 No 590-ELEENDSENSEFEDDGs(100)EK-610 1.11E+08 S607 Yes 705-LFDEEEDS(0.1)s(99.9)EK-717 8.51E+05 S714 Yes 705-LFDEEEDS(0.1)s(99.9)EK-717 1.30E+05 S714 No Phosphosites identified un-reproducibly in treated samples only 309-HPDGVAs(97.1)VS(2.9)FR-321 2.14E+06 S316 Yes 383-RSDSVSAs(100)ER-394 8.29E+08 S391 Yes 636-VFDDEs(100)DEKEDEEYADEK-655 6.84E+06 S642 Yes 671-LFEEs(100)DDKEDEDADGK-688 3.26E+06 S676 Yes 695-LFEDDDs(100)NEK-706 4.29E+06 S702 Yes Phosphosites identified un-reproducibly in untreated samples only 590-ELEENDSENs(99.7)EFEDDGS(0.3)EK-610 1.58E+07 S600 No Doubly phosphorylated peptides 384-SDs(99.9)VS(3.2)As(96.9)ER-394 2.65E+08 S387, S391 Yes 590-ELEENDSENs(100)EFEDDGs(100)EK-610 5.80E+08 S600, S607 Yes 433-TEDGGEFEEGASENNAKEs(100)s(100)PEK-457 2.73E+08 S452, S453 Yes 483-ESEEGNPVRGs(100)EEDs(100)PK-501 2.20E+07 S494, S498 Yes 618-EFDEDs(100)DEKEEEEDt(99.6)Y(0.4)EK-637 2.34E+08 S624, T633 Yes

122

123

Figure 3-8. Quantification of phosphopeptides reproducibly identified in biological replicate analysis of FLAG-HTATSF1. Briefly, FLAG-HTATSF1 was affinity purified from HEK-293 Flp-In T-REx cells treated with 100nM okadaic acid for 3h or vehicle alone; digested with trypsin, and phophopeptides enriched using IMAC or TiO2. Five 150mm plates worth of enriched material was subjected to LC-MS/MS on an LTQ- Orbitrap Velos and phophopetides and sites identified using Proteome Discoverer software as described in section 3-1. Peptides were selected corresponding to a Proteome Discoverer FDR of 1%. Phosphopeptide abundance was quantified by Proteome Discoverer software based on MS1 area. The relative abundance of the identified phosphopeptides in okadaic acid treated samples (blue) is shown as compared to untreated samples (red). Peptide areas were summed for peptides containing the same phosphosite but exhibiting different areas due to eluting separately (e.g. because they contained other posttranslational modifications (deamidation, oxidation) or missed cleavages. Doubly phosphorylated peptides are presented separately. Phosphosite assignment was done by Proteome Discoverer’s PhosphoRS algorithm. Phosphosites assigned with ≥0.95 probability of being correct are shown as lowercase letters, the probability of a correct site assignment is shown in brackets. Other PTM’s are omitted for clarity. A) Biological Replicate 1. B) Biological Replicate 2.

124

Table 3-5. Quantification of SUPT5H phosphopeptides identified in biological replicate analysis of FLAG-SUPT4H. Briefly, FLAG-SUPT4H was affinity purified from HEK-293 Flp-In T-REx cells treated with 100nM okadaic acid for 3h or vehicle alone; digested with trypsin, and phophopeptides enriched using IMAC or TiO2. Five 150mm plates worth of enriched material was subjected to LC-MS/MS on an LTQ- Orbitrap Velos and phophopetides and sites identified using Proteome Discoverer software as described in section 3-1. Peptides were selected corresponding to a Proteome Discoverer FDR of 1%. Phosphopeptide abundance was quantified by Proteome Discoverer software based on MS1 area. The relative abundance of the identified phosphopeptides in okadaic acid treated samples (Treated – Yes) is shown as compared to untreated samples (Treated – No). Phosphosite assignment was done by Proteome Discoverer’s PhosphoRS algorithm. Phosphosites assigned with ≥0.95 probability of being correct are shown as lowercase letters, the probability of a correct site assignment is shown in brackets. Other PTM’s are omitted for clarity. A) Biological Replicate 1. B) Biological Replicate 2. A Peptide Sequence (Probability of correct site assignment) MS1 Area Phosphosites Treated Phosphosites identified reproducibly in both treated and untreated samples 1021-VVSISSEHLEPIt(99.9)PT(0.1)K-1038 6.03E+06 T1034 Yes 1021-VVSISSEHLEPIt(100)PTK-1038 4.21E+06 T1034 No Phosphosites identified reproducibly in treated samples only 106-As(100)NIDNVVLDEDR-120 1.54E+05 S108 Yes 652-DVTNFTVGGFAPMs(100)PR-669 1.13E+07 S666 Yes 652-DVTNFTVGGFAPms(100)PR-669 4.82E+07 S666 Yes 668-IS(0.2)s(99.8)PMHPSAGGQR-682 4.64E+06 S671 Yes 668-IS(2.9)s(97.1)PmHPSAGGQR-682 1.14E+07 S671 Yes 681-GGFGs(99.8)PGGGS(0.2)GGMSR-697 1.32E+05 S686 Yes 767-TPMYGSQTPMYGs(100)GSR-784 6.10E+06 S780 Yes 767-TPMYGSQTPmYGs(100)GSR-784 9.83E+06 S780 Yes 767-TPmYGSQTPmYGs(100)GSR-784 1.82E+07 S780 Yes 783-TPMYGs(99.9)QT(0.1)PLQDGSR-799 2.41E+07 S789 Yes 783-TPmYGs(99.9)QT(0.1)PLQDGSR-799 3.99E+07 S789 Yes 798-TPHYGs(100)QTPLHDGSR-814 4.10E+07 S804 Yes 813-TPAQSGAWDPNNPNt(99.9)PS(0.1)R-832 7.44E+07 T828 Yes Phosphosites identified un-reproducibly in treated samples only 641-HLVLAGGs(100)KPR-653 1.30E+05 S649 Yes 711-Is(100)QGPYK-719 7.25E+06 S713 Yes Phosphosites identified un-reproducibly in untreated samples only 17-s(97.5)S(2.5)DGEEAEVDEER-31 1.54E+05 S18 No 125

Doubly phosphorylated peptides 813-t(100)PAQSGAWDPNNPNt(95.2)PS(4.8)R-832 2.12E+06 T814, T828 Yes

B Peptide Sequence (Probability of correct site assignment) MS1 Area Phosphosites Treated Phosphosites identified reproducibly in both treated and untreated samples 1021-VVSISSEHLEPIt(99.8)PT(0.2)K-1038 1.12E+07 T1034 Yes 1021-VVSISSEHLEPIt(99.8)PT(0.2)K-1038 2.74E+06 T1034 No Phosphosites identified reproducibly in treated samples only 106-As(100)NIDNVVLDEDR-120 5.69E+05 S108 Yes 652-DVTNFTVGGFAPMs(100)PR-669 1.65E+07 S666 Yes 652-DVTNFTVGGFAPms(100)PR-669 4.51E+07 S666 Yes 668-IS(2.6)s(97.4)PmHPSAGGQR-682 1.13E+07 S671 Yes 681-GGFGs(99.8)PGGGS(0.2)GGMSR-669 6.60E+05 S686 Yes 767-TPMYGSQTPMY(0.2)Gs(95.7)GS(4.1)R-784 2.70E+07 S780 Yes 767-TPMYGSQTPmYGs(99.9)GSR-784 2.74E+07 S780 Yes 783-TPMYGs(99.9)QT(0.1)PLQDGSR-799 1.23E+08 S789 Yes 783-TPmYGs(99.7)QT(0.3)PLqDGSR-799 9.57E+05 S789 Yes 798-TPHYGs(100)QTPLHDGSR-814 1.24E+08 S804 Yes 813-TPAQSGAWDPNNPNt(99.8)PS(0.2)R-832 2.47E+08 T828 Yes Phosphosites identified un-reproducibly in treated samples only 767-TPmY(0.1)Gs(99.7)QT(0.1)PmYGSGSR-784 2.95E+07 S773 Yes 783-TPmYGS(0.1)Qt(99.9)PLQDGSR-799 1.05E+08 T791 Yes Doubly phosphorylated peptides 813-t(100)PAQSGAWDPNNPNt(99.8)PS(0.2)R-832 1.04E+06 T814, T828 Yes 798-T(0.3)PHY(4.5)Gs(95.5)Qt(99.7)PLHDGSR-814 1.16E+07 S804, T806 Yes

126

127

Figure 3-9. Quantification of SUPT5H phosphopeptides reproducibly identified in biological replicate analysis of FLAG-SUPT4H. Briefly, FLAG-SUPT4H was affinity purified from HEK-293 Flp-In T-REx cells treated with 100nM okadaic acid for 3h or vehicle alone; digested with trypsin, and phophopeptides enriched using IMAC or TiO2. Five 150mm plates worth of enriched material was subjected to LC-MS/MS on an LTQ- Orbitrap Velos and phophopetides and sites identified using Proteome Discoverer software as described in section 3-1. Peptides were selected corresponding to a Proteome Discoverer FDR of 1%. Phosphopeptide abundance was quantified by Proteome Discoverer software based on MS1 area. The relative abundance of the identified phosphopeptides in okadaic acid treated samples (blue) is shown as compared to untreated samples (red). Peptide areas were summed for peptides containing the same phosphosite but exhibiting different areas due to eluting separately (e.g. because they contained other posttranslational modifications (deamidation, oxidation) or missed cleavages. Doubly phosphorylated peptides are presented separately. Phosphosite assignment was done by Proteome Discoverer’s PhosphoRS algorithm. Phosphosites assigned with ≥0.95 probability of being correct are shown as lowercase letters, the probability of a correct site assignment is shown in brackets. Other PTM’s are omitted for clarity. A) Biological Replicate 1. B) Biological Replicate 2.

128

Upon analysis it was observed that the phosphosites identified reproducibly in both okadaic acid and vehicle treated cells demonstrated a range of -1.18 to 5.62 fold difference (Fd) in abundance upon okadaic acid treatment for the three baits analyzed (Tables 3-6 – 3-8; HTASF1 S624 (okadaic acid treated) 60.74 Fd outlier omitted because it fell outside of 2.25 standard deviations).

129

Table 3-6. Fold change in DHX38 phosphosite abundance upon okadaic acid treatment. Briefly, FLAG-DHX38 was affinity purified from HEK-293 Flp-In T-REx cells treated with 100nM okadaic acid for 3h or vehicle alone; digested with trypsin, and phophopeptides enriched using IMAC or TiO2. Five 150mm plates worth of enriched material was subjected to LC-MS/MS on an LTQ-Orbitrap Velos and phophopetides and sites identified using Proteome Discoverer software as described in section 3-1. Peptides were selected corresponding to a Proteome Discoverer FDR of 1%. Phosphopeptide abundance was quantified by Proteome Discoverer software based on MS1 area. Peptide areas were summed for peptides containing the same phosphosite but exhibiting different areas due to eluting separately (e.g. because they contained other posttranslational modifications (deamidation, oxidation) or missed cleavages). The relative abundance of the identified phosphosites in okadaic acid treated samples (Treated – Yes) is shown as compared to untreated samples (Treated – No), and the fold increase in phosphosite detection upon okadaic acid treatment is given in relation to the abundance of the phosphosite in an untreated sample. Phosphosite assignment was done by Proteome Discoverer’s PhosphoRS algorithm. Phosphosites assigned with ≥0.95 probability of being correct are shown as lowercase letters, the probability of a correct site assignment is shown in brackets. Other PTM’s are omitted for clarity. A) Biological Replicate 1. B) Biological Replicate 2.

130

A Peptide Sequence Fold Difference (Probability of correct site assignment) MS1 Area (OK/NT) Phosphosites Treated Phosphosites identified reproducibly in both treated and untreated samples 717-t(100)PQEDYVEAAVK-730 5.30E+06 2.33 T718 Yes 717-t(100)PQEDYVEAAVK-730 2.28E+06 T718 No Phosphosites identified un-reproducibly in both treated and untreated samples 679-LIVT(0.1)s(99.9)ATMDAEK-692 1.64E+06 4.25 S684 Yes 679-LIVT(0.1)s(99.8)AT(0.1)MDAEK-692 3.86E+05 S684 No 1193-Rs(100)PLGSVR-1202 2.33E+08 4.14 S1195 Yes 1194-s(100)PLGSVR-1202 5.63E+07 S1195 No

B Peptide Sequence Fold Difference (Probability of correct site assignment) MS1 Area (OK/NT) Phosphosites Treated Phosphosites identified reproducibly in both treated and untreated samples 717-t(100)PQEDYVEAAVK-730 4.07E+06 3.85 T718 Yes 717-t(100)PQEDYVEAAVK-730 1.06E+06 T718 No Phosphosites identified un-reproducibly in both treated and untreated samples 226-R/SQWES(0.1)Ps(99.9)PT(0.1)PSYR-241 6.80E+08 2418.21 S234 Yes 227-SQWESPs(100)PTPSYR-241 2.81E+05 S234 No

131

Table 3-7. Fold change in HTATSF1 phosphosite abundance upon okadaic acid treatment. Briefly, FLAG-HTATSF1 was affinity purified from HEK-293 Flp-In T-REx cells treated with 100nM okadaic acid for 3h or vehicle alone; digested with trypsin, and phophopeptides enriched using IMAC or TiO2. Five 150mm plates worth of enriched material was subjected to LC-MS/MS on an LTQ-Orbitrap Velos and phophopetides and sites identified using Proteome Discoverer software as described in section 3-1. Peptides were selected corresponding to a Proteome Discoverer FDR of 1%. Phosphopeptide abundance was quantified by Proteome Discoverer software based on MS1 area. Peptide areas were summed for peptides containing the same phosphosite but exhibiting different areas due to eluting separately (e.g. because they contained other posttranslational modifications (deamidation, oxidation) or missed cleavages. The relative abundance of the identified phosphosites in okadaic acid treated samples (Treated – Yes) is shown as compared to untreated samples (Treated – No), and the fold increase in phosphosite detection upon okadaic acid treatment is given in relation to the abundance of the phosphosite in an untreated sample. Phosphosite assignment was done by Proteome Discoverer’s PhosphoRS algorithm. Phosphosites assigned with ≥0.95 probability of being correct are shown as lowercase letters, the probability of a correct site assignment is shown in brackets. Other PTM’s are omitted for clarity. A) Biological Replicate 1. B) Biological Replicate 2.

132

A Peptide Sequence Fold Difference (Probability of correct site assignment) MS1 Area (OK/NT) Phosphosites Treated Phosphosites identified reproducibly in both treated and untreated samples 433-TEDGGEFEEGAs(100)ENNAK-451 8.04E+07 4.80 S445 Yes 433-TEDGGEFEEGAs(100)ENNAK-451 1.68E+07 S445 No 483-ESEEGNPVRGs(100)EEDSPK-500 7.12E+06 3.20 S494 Yes 483-ESEEGNPVRGs(100)EEDSPK-500 2.23E+06 S494 No 492-GSEEDs(100)PK/K-502 7.31E+07 2.44 S498 Yes 492-GSEEDs(100)PK/K-502 3.00E+07 S498 No 519-ESEDDLNKEs(100)EEEVGPTK-538 4.87E+07 1.30 S529 Yes 519-ESEDDLNKEs(100)EEEVGPTK-538 3.75E+07 S529 No 572-DLDEEGs(100)EK/ELHENVLDK-591 1.81E+08 1.44 S579 Yes 572-DLDEEGs(100)EK/ELHENVLDK-591 1.26E+08 S579 No 609-VLDEEGs(100)ER-619 8.27E+08 1.96 S616 Yes 609-VLDEEGs(100)ER-619 4.21E+08 S616 No 618-EFDEDs(100)DEKEEEEDTYEK-637 4.56E+06 1.21 S624 Yes 618-EFDEDs(100)DEKEEEEDTYEK-637 3.77E+06 S624 No Phosphosites identified un-reproducibly in both treated and untreated samples 474-GFEGSCs(100)QKESEEGNPVR-490 1.68E+05 2.51 S481 Yes 474-GFEGS(0.1)Cs(99.9)QKESEEGNPVR-490 6.69E+04 S481 No 590-ELEENDSENSEFEDDGs(100)EK-610 1.85E+06 -2.94 S607 Yes 590-ELEENDSENSEFEDDGs(100)EK-610 5.46E+06 S607 No

B Peptide Sequence Fold Difference (Probability of correct site assignment) MS1 Area (OK/NT) Phosphosites Treated Phosphosites identified reproducibly in both treated and untreated samples 433-TEDGGEFEEGAs(100)ENNAK-451 6.18E+08 5.62 S445 Yes 433-TEDGGEFEEGAs(100)ENNAK-451 1.10E+08 S445 No 483-ESEEGNPVRGs(100)EEDSPK-501 6.60E+06 2.20 S494 Yes 483-ESEEGNPVRGs(100)EEDSPK-501 3.00E+06 S494 No 492-GSEEDs(100)PK/K-502 1.03E+08 -1.18 S498 Yes 492-GSEEDs(100)PK/K-502 1.21E+08 S498 No 519-ESEDDLNKEs(100)EEEVGPTK-538 4.42E+08 1.85 S529 Yes 519-ESEDDLNKEs(100)EEEVGPTK-538 2.38E+08 S529 No 572-DLDEEGs(100)EK/ELHENVLDK-591 2.61E+09 1.76 S579 Yes 572-DLDEEGs(100)EK/ELHENVLDK-591 1.48E+09 S579 No 609-VLDEEGs(100)ER-619 2.78E+09 1.63 S616 Yes 609-VLDEEGs(100)ER-619 1.71E+09 S616 No 618-EFDEDs(100)DEKEEEEDTYEK-637 4.00E+09 60.74 S624 Yes 618-EFDEDs(100)DEKEEEEDTYEK-637 6.59E+07 S624 No Phosphosites identified un-reproducibly in both treated and untreated samples 384-SDs(100)VSASER-394 9.23E+08 354.59 S387 Yes 383-RSDs(99.9)VS(0.1)ASER-394 2.60E+06 S387 No 519-Es(100)EDDLNK-528 7.39E+06 -1.14 S521 Yes 519-Es(100)EDDLNK-528 8.40E+06 S521 No 133

705-LFDEEEDS(0.1)s(99.9)EK-717 8.51E+05 6.56 S714 Yes 705-LFDEEEDS(0.1)s(99.9)EK-717 1.30E+05 S714 No

134

Table 3-8. Fold change in SUPT5H phosphosite abundance upon okadaic acid treatment. Briefly, FLAG-SUPT4H was affinity purified from HEK-293 Flp-In T-REx cells treated with 100nM okadaic acid for 3h or vehicle alone; digested with trypsin, and phophopeptides enriched using IMAC or TiO2. Five 150mm plates worth of enriched material was subjected to LC-MS/MS on an LTQ-Orbitrap Velos and phophopetides and sites identified using Proteome Discoverer software as described in section 3-1. Peptides were selected corresponding to a Proteome Discoverer FDR of 1%. Phosphopeptide abundance was quantified by Proteome Discoverer software based on MS1 area. Peptide areas were summed for peptides containing the same phosphosite but exhibiting different areas due to eluting separately (e.g. because they contained other posttranslational modifications (deamidation, oxidation) or missed cleavages. The relative abundance of the identified phosphosites in okadaic acid treated samples (Treated – Yes) is shown as compared to untreated samples (Treated – No), and the fold increase in phosphosite detection upon okadaic acid treatment is given in relation to the abundance of the phosphosite in an untreated sample. Phosphosite assignment was done by Proteome Discoverer’s PhosphoRS algorithm. Phosphosites assigned with ≥0.95 probability of being correct are shown as lowercase letters, the probability of a correct site assignment is shown in brackets. Other PTM’s are omitted for clarity. A) Biological Replicate 1. B) Biological Replicate 2. A Peptide Sequence Fold Difference (Probability of correct site assignment) MS1 Area (OK/NT) Phosphosites Treated Phosphosites identified reproducibly in both treated and untreated samples 1021-VVSISSEHLEPIt(99.9)PT(0.1)K-1038 6.03E+06 1.43 T1034 Yes 1021-VVSISSEHLEPIt(100)PTK-1038 4.21E+06 T1034 No

B Peptide Sequence Fold Difference (Probability of correct site assignment) MS1 Area (OK/NT) Phosphosites Treated Phosphosites identified reproducibly in both treated and untreated samples 1021-VVSISSEHLEPIt(99.8)PT(0.2)K-1038 1.12E+07 4.09 T1034 Yes 1021-VVSISSEHLEPIt(99.8)PT(0.2)K-1038 2.74E+06 T1034 No

135

When the effect of okadaic acid treatment on phosphosite abundance (for those phosphosites identified reproducibly in both okadaic and non-treated samples) was assessed overall (all three baits analyzed together for both biological replicates), the average fold increase in phosphosite quantification was: 2.35 fold ± 1.59 (n = 17) (again HTATSF1 S624 omitted as outlier as it fell outside of 2.25 SDV’s). These results indicate that in most cases okadaic acid treatment results not only in the identification of more phosphosites (Table 3-2; Figures 3-2 – 3-6), but also in an enrichment of phosphosites that are identified to be phosphorylated in the absence of okadaic acid treatment (Tables 3-6 – 3-8; Figures 3-7 – 3-9). The reason for this could be two fold, 1 – these sites are targeted by PP2A subfamily phosphatases and okadaic acid treatment and PP2A subfamily inhibition (i.e. PP4c inhibition) results in their enrichment, or 2 – the increase in the total number of phosphopeptides present in the cell upon okadaic acid treatment creates a buffering effect whereby less of the phosphopeptides containing these sites are lost during sample preparation and they appear subsequently enriched. However which of the two scenarios is occuring remains to be addressed.

Analysis of phosphosite quantification for those sites which were not identified reproducibly in both okadaic acid treated and untreated samples, in biological duplicates, revealed a range of -2.94 – 2418 fold difference (Fd) in phosphosite abundance in okadaic acid treated samples as compared to an untreated samples for the three baits analyzed (Tables 3-6, 3-7). However, when the effect of okadaic acid treatment on phosphosite abundance was assessed for all phosphopeptides identified un-reproducibly in both okadaic and non-treated samples overall (all three baits analyzed together), the range and average fold difference in phosphosite quantification was: Range: -2.94 Fd – 6.56 Fd; Avg Fd = 2.23 ± 3.60 n = 6; after the outliers DHX38 S234 (2418 Fd) and HTATSF1 S387 (355 Fd) were removed. Notably, the new range and average fold difference in phosphosite abundance observed for phosphopeptides un-reproducibly identified in both okadaic acid treated/untreated samples in biological replicates (upon outlier removal) is now more in line with that observed for phosphosites reproducibly identified in biological replicates of okadaic acid treated/untreated samples (Range -1.18 Fd – 5.62 Fd; Avg. Fd = 2.35 ± 1.59 n = 17). This indicates that although phosphosites may be identified un-reproducibly across biological replicates, the data obtained in a 136 single replicate may still be useful for determining if okadaic acid treatment leads to phosphosite enrichment, so long as enough data is generated for statistical removal of outliers. With this in mind it is evident that multiple biological replicates be performed to properly assess phosphopeptide abundance. Additionally, the large discrepancy in phosphosite abundance observed in biological replicate analyses of HTATSF1 (HTATSF1 S624 Bio Rep1 Fd = 1.21; HTATSF1 S624 Bio Rep2 Fd = 60.74) indicates that multiple technical replicates are also needed to accurately quantify phosphosite abundance. The incorporation of additional technical replicates may also help in the detection of phosphopeptides observed to be present in one or more (but not all) of multiple biological replicates.

3.2.4 Reproducibility of phosphosite quantification across biological replicates

A comparison of phosphosite abundance in biological replicate 2 to phosphosite abundance in replicate 1, for those phosphosites identified reproducibly in biological replicates (with or without okadaic acid treatment), revealed a range of -7.62 Fd to 17.49 Fd for phosphosite abundance in biological replicate 2 as compared to replicate 1 (Rep2/Rep1), for the 3 baits analyzed, (Tables 3-9 – 3-11; DHX38 T117 (42.45 Fd); HTATSF1 S624 (okadaic acid treated, 879 Fd); and HTATSF1 S40 (464 Fd) removed as outliers).

137

Table 3-9. Quantification of DHX38 phosphopeptides reproducibly identified in biological replicate analysis of FLAG-DHX38. Briefly, FLAG-DHX38 was affinity purified from HEK-293 Flp-In T-REx cells treated with 100nM okadaic acid for 3h or vehicle alone; digested with trypsin, and phophopeptides enriched using IMAC or TiO2. Five 150mm plates worth of enriched material was subjected to LC-MS/MS on an LTQ- Orbitrap Velos and phophopetides and sites identified using Proteome Discoverer software as described in section 3-1. Peptides were selected corresponding to a Proteome Discoverer FDR of 1%. Phosphopeptide abundance was quantified by Proteome Discoverer software based on MS1 area. The relative abundance of the identified phosphopeptides in okadaic acid treated samples (Treated – Yes), and untreated samples (Treated – No), and the fold difference in phosphosite quantification between biological replicate analyses (Rep2/Rep1) is given. Where Rep2/Rep1 was < 1 the inverse was taken to get the negative fold difference in phosphosite abundance. Phosphosite assignment was done by Proteome Discoverer’s PhosphoRS algorithm. Phosphosites assigned with ≥0.95 probability of being correct are shown as lowercase letters, the probability of a correct site assignment is shown in brackets. Other PTM’s are omitted for clarity.

Peptide Sequence MS1 Area MS1 Area Fold Difference (Probability of correct site assignment) (Rep1) (Rep2) (Rep2/Rep1) Phosphosites Treated Phosphosites identified reproducibly in both treated and untreated samples 717-t(100)PQEDYVEAAVK-730 5.30E+06 4.07E+06 -1.30 T718 Yes 717-t(100)PQEDYVEAAVK-730 2.28E+06 1.06E+06 -2.15 T718 No Phosphosites identified reproducibly in treated samples only 0-MGDTSEDAs(100)IHR-13 6.44E+07 8.44E+06 -7.62 S9 Yes 28-S(0.1)K/s(100)AAS(0.1)EQHVFK-41 1.04E+07 3.15E+07 3.02 S31 Yes 114-Vet(100)PSHPGGVSEEFWER-132 4.07E+06 1.73E+08 42.45 T117 Yes 193-RNEPEs(100)PR-201 3.50E+09 5.09E+08 -6.88 S199 Yes 212-STWEEEDSGYGs(100)SR-228 6.10E+08 3.44E+08 -1.77 S224 Yes 258-GK/YSDDt(100)PLPTPSYK-274 2.52E+08 2.74E+08 1.09 T265 Yes 296-RE/EGEEGISFDt(100)EEER-313 2.32E+07 3.51E+06 -6.62 T308 Yes

138

Table 3-10. Quantification of HTATSF1 phosphopeptides reproducibly identified in biological replicate analysis of FLAG-HTATSF1. Briefly, FLAG-HTATSF1 was affinity purified from HEK-293 Flp-In T-REx cells treated with 100nM okadaic acid for 3h or vehicle alone; digested with trypsin, and phophopeptides enriched using IMAC or

TiO2. Five 150mm plates worth of enriched material was subjected to LC-MS/MS on an LTQ-Orbitrap Velos and phophopetides and sites identified using Proteome Discoverer software as described in section 3-1. Peptides were selected corresponding to a Proteome Discoverer FDR of 1%. Phosphopeptide abundance was quantified by Proteome Discoverer software based on MS1 area. The relative abundance of the identified phosphopeptides in okadaic acid treated samples (Treated – Yes), and untreated samples (Treated – No), and the fold difference in phosphosite quantification between biological replicate analyses (Rep2/Rep1) is given. Where Rep2/Rep1 was < 1 the inverse was taken to get the negative fold difference in phosphosite abundance. Phosphosite assignment was done by Proteome Discoverer’s PhosphoRS algorithm. Phosphosites assigned with ≥0.95 probability of being correct are shown as lowercase letters, the probability of a correct site assignment is shown in brackets. Other PTM’s are omitted for clarity.

Peptide Sequence MS1 Area MS1 Area Fold Difference (Probability of correct site assignment) (Rep1) (Rep2) (Rep2/Rep1) Phosphosites Treated Phosphosites identified reproducibly in both treated and untreated samples 433-TEDGGEFEEGAs(100)ENNAK-451 8.04E+07 6.18E+08 7.68 S445 Yes 433-TEDGGEFEEGAs(100)ENNAK-451 1.68E+07 1.10E+08 6.56 S445 No 483-ESEEGNPVRGs(100)EEDSPK-501 7.12E+06 6.60E+06 -1.08 S494 Yes 483-ESEEGNPVRGs(100)EEDSPK-501 2.23E+06 3.00E+06 1.35 S494 No 492-GSEEDs(100)PK/K-502 7.31E+07 1.03E+08 1.41 S498 Yes 492-GSEEDs(100)PK/K-502 3.00E+07 1.21E+08 4.03 S498 No 519-ESEDDLNKEs(100)EEEVGPTK-538 4.87E+07 4.42E+08 9.08 S529 Yes 519-ESEDDLNKEs(100)EEEVGPTK-538 3.75E+07 2.38E+08 6.36 S529 No 572-DLDEEGs(100)EK/ELHENVLDK-591 1.81E+08 2.61E+09 14.42 S579 Yes 572-DLDEEGs(100)EK/ELHENVLDK-591 1.26E+08 1.48E+09 11.75 S579 No 609-VLDEEGs(100)ER-619 8.27E+08 2.78E+09 3.37 S616 Yes 609-VLDEEGs(100)ER-619 4.21E+08 1.71E+09 4.06 S616 No 618-EFDEDs(100)DEKEEEEDTYEK-637 4.56E+06 4.00E+09 878.59 S624 Yes 618-EFDEDs(100)DEKEEEEDTYEK-637 3.77E+06 6.59E+07 17.49 S624 No Phosphosites identified reproducibly in treated samples only 26-DGDTQTDAGGEPDs(100)LGQQP 3.79E+05 1.76E+08 464.38 S40 Yes T(0.1)DTPYEWDLDK/K-58 238-KLs(100)MQQK-246 3.97E+06 3.21E+07 8.07 S241 Yes

139

Table 3-11. Quantification of SUPT5H phosphopeptides reproducibly identified in biological replicate analysis of FLAG-SUPT4H. Briefly, FLAG-SUPT4H was affinity purified from HEK-293 Flp-In T-REx cells treated with 100nM okadaic acid for 3h or vehicle alone; digested with trypsin, and phophopeptides enriched using IMAC or TiO2. Five 150mm plates worth of enriched material was subjected to LC-MS/MS on an LTQ- Orbitrap Velos and phophopetides and sites identified using Proteome Discoverer software as described in section 3-1. Peptides were selected corresponding to a Proteome Discoverer FDR of 1%. Phosphopeptide abundance was quantified by Proteome Discoverer software based on MS1 area. The relative abundance of the identified phosphopeptides in okadaic acid treated samples (Treated – Yes), and untreated samples (Treated – No), and the fold difference in phosphosite quantification between biological replicate analyses (Rep2/Rep1) is given. Where Rep2/Rep1 was < 1 the inverse was taken to get the negative fold difference in phosphosite abundance. Phosphosite assignment was done by Proteome Discoverer’s PhosphoRS algorithm. Phosphosites assigned with ≥0.95 probability of being correct are shown as lowercase letters, the probability of a correct site assignment is shown in brackets. Other PTM’s are omitted for clarity.

Peptide Sequence MS1 Area MS1 Area Fold Difference (Probability of correct site assignment) (Rep1) (Rep2) (Rep2/Rep1) Phosphosites Treated Phosphosites identified reproducibly in both treated and untreated samples 1021-VVSISSEHLEPIt(99.8)PT(0.2)K-1038 6.03E+06 1.12E+07 1.85 T1034 Yes 1021-VVSISSEHLEPIt(99.8)PT(0.2)K-1038 4.21E+06 2.74E+06 -1.54 T1034 No Phosphosites identified reproducibly in treated samples only 106-As(100)NIDNVVLDEDR-120 1.54E+05 5.69E+05 3.69 S108 Yes 652-DVTNFTVGGFAPMs(100)PR-669 5.94E+07 6.16E+07 1.04 S666 Yes 668-IS(2.6)s(97.4)PmHPSAGGQR-682 1.60E+07 1.13E+07 -1.41 S671 Yes 681-GGFGs(99.8)PGGGS(0.2)GGMSR-669 1.32E+05 6.60E+05 4.99 S686 Yes 767-TPMYGSQTPMY(0.2)Gs(99.9)GS(4.1)R-784 3.41E+07 5.44E+07 1.60 S780 Yes 783-TPMYGs(99.9)QT(0.3)PLQDGSR-799 6.40E+07 1.24E+08 1.94 S789 Yes 798-TPHYGs(100)QTPLHDGSR-814 4.10E+07 1.24E+08 3.02 S804 Yes 813-TPAQSGAWDPNNPNt(99.8)PS(0.2)R-832 7.44E+07 2.47E+08 3.32 T828 Yes

140

Overall, when all three baits were analyzed together there was a range of -7.62 Fd – 11.75 Fd; in phosphosite abundance between biological replicate 2 and replicate 1 (Rep2/Rep1) and an average fold difference of 1.96 ± 4.56 (n = 30) after outlier removal; (HTATSF1 S684 (okadaic acid treated, 879 Fd); HTATSF1 S40 (464 Fd); DHX38 T117 (42.45 Fd); HTATSF1 S684 (non-treated, 17.49 Fd); and HTATSF1 S579 (okadaic acid treated, 14.42 Fd) removed as outliers). These results demonstrate that substantial variation in phosphosite abundance exists between biological replicates, indicating that samples to be directly compared should be processed in parallel to ensure this variation does not lead to false identification of enriched phosphosites (when comparing the effect of okadaic acid treatment on phosphosite abundance, treated and untreated samples were processed in parallel).

A comparison of the variance in the ratios of phosphopeptide abundance between biological replicate 2 and replicate 1 (Rep2/Rep1), for phosphosites identified reproducibly in okadaic acid treated samples, and those identified reproducibly in untreated samples, revealed similar levels of variation occurred in untreated samples as did in okadaic acid treated samples (after outlier removal), when analyzed across all baits: (Untreated samples Rep2/Rep1: Range: -2.15 Fd – 17.49 Fd; Avg. Fd = 5.32 ± 6.26 n = 9; Treated samples Rep2/Rep1: Range: -7.62 Fd – 9.08 Fd; Avg. Fd = 1.3 ± 4.47 n = 22; (HTATSF1 S684 (okadaic acid treated, 879 Fd); HTATSF1 S40 (464 Fd); DHX38 T117 (42.45 Fd); and HTATSF1 S579 (okadaic acid treated, 14.42 Fd) removed as outliers). This indicates that sample processing and MS analysis, more likely than any effect of okadaic acid treatment, is responsible for the variation in phosphopeptide abundance observed between biological replicates. Of note, more outliers were present in okadaic acid treated samples (4), than in non-treated samples (0), indicating that okadaic acid treatment itself, and the subsequent increase in phosphosites observed (26 vs. 9 sites respectively), may be responsible for unusual measurements in phosphopeptide abundance to be recorded. 141

3.3 Discussion 3.3.1 Discerning whether PP4c interactors are possible substrates for the enzyme

When trying to determine which of the PP4c interacting proteins outlined in section 3.2.1 are possible PP4c substrates, the effect of intra sample variation in phosphosite abundance made confident identification of phosphosites enriched upon okadaic acid treatment problematic. While inter sample variation in phosphopeptide abundance was expected (due to variation introduced during sample processing), it was expected to affect all phosphopeptides within a sample equally (i.e. all phosphopeptides in biological replicate 2 are 10% more abundant than in replicate 1), however this was not observed to be the case (tables 3-9 – 3-11). In fact a substantial degree of intra sample variation was observed for both phosphosites detected in okadaic acid treated samples and those detected in untreated samples, when phosphosite abundance in replicate 2 was compared to replicate 1 (Fold difference in phosphosite abundance for phosphosites identified reproducibly in okadaic acid treated samples (after outlier removal), for 3 baits analyzed in duplicate: Range: -7.62 Fd – 9.08 Fd; Avg. Fd = 1.3 ± 4.47 n = 22; Fold difference in phosphosite abundance for phosphosites identified reproducibly in un-treated samples, for 3 baits analyzed in duplicate: Range: -2.15 Fd – 17.49 Fd; Avg. Fd = 5.32 ± 6.26 n = 9; Tables 3-9 – 3-11). In fact, this level of intra sample variation was more than was observed to occur between samples when phosphosite abundance after okadaic acid treatment was compared to a non-treated sample (for those phosphosites identified reproducibly in both okadaic and non-treated samples in both biological replicates only, all three baits analyzed together for both biological replicates: Range: -1.18 Fd – 5.62 Fd; Avg. Fd 2.35 ± 1.59, n = 17; HTATSF1 S624 omitted as outlier), indicating that substantial variation is introduced during sample prep and MS analysis, and further highlighting the need for samples that will be directly compared to each other to be harvested, processed, and analyzed in parallel to reduce variation as much as possible. Of note, these results are in stark contrast to those obtained by Choi et al., [129] where less than 16.2% relative standard deviation was detected between phosphopeptide peak areas 142 in four independent experiments (the standard deviations listed aboved range from 117% – 343% of the mean).

One plausible solution for identifying phosphosites enriched upon okadaic acid treatment, while taking the high amount of intra sample variation into account, was to look for phosphosites that display (upon okadaic acid treatment) an abundance that falls outside 2 standard deviations of the intra sample variation (for all bait peptides) when compared to its non-treated counterpart. These sites could then be followed up on specifically. In this case the average fold difference in phosphosite abundance between biological replicate 2 and replicate 1 (Rep2/Rep1) for both okadaic acid treated and untreated samples for all 3 baits analyzed was 1.96 ± 4.56 (n = 30) after outlier removal; (HTATSF1 S684 (okadaic acid treated, 879 Fd; and non-treated, 17.49 Fd); HTATSF1 S40 (464 Fd); DHX38 T117 (42.45 Fd); HTATSF1 S579 (okadaic acid treated, 14.42 Fd) removed as outliers as described above), indicating in order to be considered significantly enriched a phosphosite would have to reproducibly demonstrate an 11.08 fold increase in abundance upon okadaic acid treatment, of which none of the phosphosites I observed reproducibly in both okadaic treated and untreated sample did (Tables 3-6 – 3-8). Of the phosphosites identified reproducibly in both okadaic acid treated and untreated samples in both biological replicates, the HTATSF1 phosphopeptides containing S445 and S494 were identified to consistently exhibit a greater than 2 fold enrichment upon okadaic acid treatment (S445: Range: 5.62 Fd – 4.80 Fd; Avg. Fd = 5.21 n = 2; S494: Range: 3.20 Fd – 2.20 Fd; Avg. Fd = 2.7 n = 2); as did the DHX38 phosphopeptide containing T718 (T718: Range: 2.33 Fd – 3.85 Fd; Avg. Fd = 3.09 n = 2), however, the increase in phosphosite abundance was nowhere near 11 fold (maximum 5.62 Fd), making it hard to determine if these sites are targeted by PP2A subfamily phosphatases or just appearing to be enriched because of technical variation. In this case multiple technical replicates could be useful for reducing intra sample variation, making identification of phosphosites enriched upon okadaic acid treatment easier, however this was not assessed.

More simplistically, only the phosphosites that were reproducibly identified upon okadaic acid treatment could be selected for initial follow up (e.g. DHX38 S9, S31; HTATSF1 S40, S241; SUPT5H S108, S666 Tables 3-3 – 3-5; Figures 3-7 – 3-9). 143

However, the fact some phosphosites were identified only to be phosphorylated upon okadaic acid treatment in one biological replicate, but then identified to be phosphorylated in both okadaic and non treated samples in the other replicate (e.g. DHX38 S234, S684; HTATSF1 S387, S481; Tables 3-3, 3-4), indicates that more than two biological replicates, and possibly technical replicates, should be conducted to ensure that sites observed to be phosphorylated upon okadaic acid treatment are accurately identified before follow up experiments are conducted.

Of note, the SUPT5H phosphosites T791, T806, and T814, which evidence suggests are phosphorylated by P-TEFb, and involved in the switching of SUPT5H from a repressor to an activator of transcription and c-fos expression [262], were observed to be phosphorylated only upon okadaic acid treatment (T791 and T806 were identified un- reproducibly, with T806 occurring as a doubly phosphorylated peptide containing S804; while T814 was identified reproducibly in 2 biological replicates as a doubly phosphorylated peptide also containing T828; Table 3-5). This indicates these sites could be possible PP4c targeted sites and a means through which PP4c could regulate transcription, however for the reasons described above, more technical/biological replicates need to be performed first, in addition to further follow up experiments (to ensure that PP4c is targeting these sites and not other PP2A family phosphatases), before this can be determined concretely to be the case. However, in principle, the identification of phosphosites involved in transcriptional regulation does verify that okadaic acid treatment, followed by phosphosite identification, may be a viable way of uncovering PP4c substrates, targeted phosphosites, and understanding PP4c regulation of transcription (and splicing). 144

Chapter 4 Thesis Summary and Future Directions 4.1 Thesis Summary In this thesis I investigated the applicability of multidimensional fractionation approaches (MudPIT, RP/RP, GeLC) to affinity purified samples, to determine if AP-MS samples would benefit from sample fractionation, as do more complex samples. If successful, it was hoped that the application of these methodologies to the baits used by Ginny Chen to generate the PP4c interaction network (Figure 1-7), would allow for the identification of novel components of the transcription and splicing machineries that interact with PP4c, thereby expanding the network and broadening our understanding of PP4c regulation of transcription and splicing. Additionally, if useful, these methods could be applied to other AP-MS studies and the expansion of other interaction networks in the lab. Secondly, I sought to identify whether a subset of the PP4c interacting proteins identified (outlined in section 3.2.1) could be PP4c substrates in vivo, by using the potent PP2A phosphatase subfamily inhibitor okadaic acid, to inhibit PP4c (in addition to other phosphatases, e.g. PP2Ac, PP6c), and quantifying changes in phosphosite abundance upon phosphatase inhibition (note PP1c is also inhibited by okadaic acid albeit at much higher concentrations than PP2Ac [263-265]). Phosphosites that demonstrated enrichment after okadaic acid treatment would be flagged as possible PP4c targeted sites, and could be followed up on using additional experiments. Lastly, I investigated the functional consequences of PP4c depletion on mRNA transcription and splicing in EGF stimulated cells, as Ginny had previously demonstrated a role for PP4c in the regulation of transcription and splicing for certain genes (outlined in Appendix 1).

In summary, what I was able to demonstrate was that multidimensional fractionation of AP-MS samples resulted in a robust increase in the number of bait and prey spectra detected, but only a modest increase in number of unique peptide and protein identifications, and came at a cost of additional sample handing and instrument time. The limited benefit obtained through the use of these methods did not make it apparent that their application to PP4c interaction partners, would expand the PP4c network greatly, 145 and justify the additional workload involved. Traditional 1D LC-MS/MS appeared able to adequately identify all the core components of the protein complexes isolated and analyzed by AP-MS. Additionally, I observed that using the PP2A subfamily phosphates inhibitor okadaic acid to try and identify whether a subset of the PP4c interacting proteins identified could be PP4c substrates in vivo resulted in the identification of several interactor phosphosites that appeared to demonstrate enrichment upon okadaic acid treatment indicating that they could possibly be regulated by PP4c. However, the amount of intra sample variation observed when biological replicate MS analyses were compared made it hard to determine whether these sites were truly enriched upon okadaic acid treatment or just appeared to be enriched due to technical variation. Variation aside, however, a subset of the PP4c interactor phosphosites were reproducibly identified to be phosphorylated only upon okadaic acid treatment, indicating these sites are likely regulated by PP2A subfamily phosphatases and the best targets for initial follow up experiments (i.e. in vitro dephosphorylation assays of synthetic peptides or affinity purified phosphorylated interacting proteins). Of note, the identification of SUPT5H phosphosites suggested to regulate transcription (e.g. SUPT5H phosphosite T814), upon okadaic acid treatment exclusively, indicates that this project is headed in the right direction towards finding PP4c targeted sites, and a means through which PP4c could regulate transcription. Lastly, I was unable to validate the functional consequences of PP4c depletion on mRNA transcription and splicing in EGF stimulated cells (observed by Ginny), using multiple siRNAs targeting PP4c. At this time it is unclear what effects of siRNA treatment on mRNA transcription and splicing are due to PP4c depletion itself, or due to unidentified off target effects.

4.2 Future Directions 4.2.1 PP4c substrate identification

While Okadaic acid treatment has been demonstrated to be a potent PP2A subfamily phosphatase inhibitor, its inhibition of all PP2A subfamily phosphatases (i.e. PP2Ac, PP4c, and PP6c) limits it usefulness for the direct identification of PP4c substrates. As such, the phosphosites identified here must be subjected to further analysis to determine whether they are directly targeted by PP4c. This can be done using a more direct 146 approach (i.e. siRNA mediated depletion of PP4c) and identifying which phosphosites are enriched upon PP4c knockdown specifically (either on a bait protein of interest or globally), as compared to cells treated with a non-silencing siRNA, an siRNA targeting another phosphatase (i.e. PP2Ac), or cells expressing siRNA resistant PP4c. In any case, if a phosphosite observed to be phosphorylated upon okadaic acid treatment can be demonstrated to be enriched upon PP4c knockdown specifically, it will provide additional evidence that PP4c itself is responsible for dephosphorylation of the site and not another PP2A subfamily phosphatase (i.e. PP2Ac, PP6c). Additionally, as cellular perturbations such as phosphatase inhibiton or siRNA treatment can have global effects on the proteome [266] and phosphoproteome [267] any sites demonstrated to be enriched using both methods could be selected for initial follow up based on the assumption that the global perturbations of okadaic acid and siRNA treatment will be dissimilar and therefore the non-overlapping identifications made using these methods are not likely due to loss of PP4c function (in the experiments outlined in chapter 3, gene expression was driven by an exogenous (CMV) promoter therefore it is thought that the effect of phosphatase inhibition on bait expression would be minimal; however this was only assessed briefly by looking at the number of bait peptides in okadaic acid treated and untreated samples by MS).

The coupling of both inhibitor (okadaic acid) and siRNA directed approaches can further help to elucidate whether any cross talk occurs between PP4c, PP2Ac, PP6c (as occurs with kinases [261]), and what substrates may be targeted by multiple phosphatases, a process known to occur (e.g, evidence suggests FCP1, SCP1, and PP1c dephosphorylation of the CTD of RNA Pol II [268-273]; and dephosphorylation of γH2AX S139 can be mediated by PP4c , PP6c, PP2Ac and wild-type p53-induced phosphatase 1 (WIP1) [143, 222, 274-276]). This can be done by comparing phosphopeptides that are enriched upon inhibiton of all PP2A subfamily phosphatases to those that are enriched upon knockdown of each phosphatase and looking for overlapping identifications. Whether the interactions are mediated by a specific complex (i.e. PP4c- PP4R2-PP4R3A) or through the “sharing” of regulatory subunits (PP2Ac regulatory subunits were observed to co-purify with PP4c and vice versa – although the interaction was not confirmed to be direct (G. Chen Ph.D Thesis [157]) can then be determined by 147 targeting of regulatory subunits. Of further note, as okadaic acid has also been shown to inhibit PP1c (albeit weakly) and several PP1c binding motifs have been identified [277] these motifs could in theory be used to try to weed out any phosphopeptides that are enriched upon okadaic acid treatment due to regulation by PP1c, or, on a global scale, to discern probable PP1c substrates from PP4c substrates (although co-regulation by both phosphatases cannot be ruled out).

Direct evidence for PP4c targeting of a given phosphosite in vitro can be garnered using dephosphorylation assays. In this case a phosphorylated putative substrate can be isolated by affinity purification (i.e. after okadaic acid treatment) and incubated with the purified PP4c complex of interest (i.e. PP4c-PP4R2-PP4R3A), calf intestinal alkaline phosphatase (CIP), or a protein that has no demonstrated kinase or phosphatase activity and any change in protein phosphorylation assessed by MS, gel shift, or radioblot (if labeled with 32P). Alternatively, a synthetic peptide can be used. Phosphosites are no longer detected by MS after phosphoprotein incubation with PP4c can be inferred to be targeted by the PP4c complex used, whereas an increase in electrophoretic mobility or decrease in 32P signal can also be indicative of dephosphorylation by PP4c. Alternatively, a more global approach can be undertaken by adapting the methods presented by Knight et al., [278] for kinase substrate identification to phosphatases. In brief, S/T phosphatases can be irreversibly inhibited (e.g. with sodium fluoride), and the cellular lysate collected and desalted before a purified PP4c containing complex (or PP2Ac, PP6c) is added back in and the changes in global phosphorylation assessed by MS. This method was used for identifying both in vitro and in vivo substrates of p38 MAPK. The use of other cellular perturbations (e.g. UV, heat shock, osmotic stress) can provide information of PP4c substrates in vivo by monitoring the rate of substrate de-phosphorylation after cellular perturbation in the absence of PP4c (i.e. siRNA mediated; Figure 3-10). Proteins identified to be dephosphorylated quicker after stimuli removal in the presence of PP4c can be inferred to be dephosphorylated in vivo by PP4c, although direct evidence will still be needed. As PP4c has been implicated in such processes as DNA damage checkpoint recovery, mitosis, and TNF-α signaling, quantifying changes in PP4c interactor or global changes in phosphorylation under these conditions may help elucidate more meaningful phosphorylation events from than those caused by by PP2A subfamily inhibition alone. 148

Figure 3-10. Gel shift assay for identifying putative PP4c substrates by monitoring protein de-phosphorylation in the absence of PP4c. (A) Phosphorylation of the PP4c subunit PP4R3A was induced using 400mM sorbitol (osmotic stressor) and monitored by electrophoretic mobility (gel shift). Maximum phospho-PP4R3A was observed at 70 minutes. At 70 minutes sorbitol was removed and phospho-PP4R3A returned to normal levels at ~150 minutes. (B) Monitoring the rate of protein de-phosphoryaltion in the absence of PP4c (e.g. siRNA depleted) can help to identify putative PP4c substrates as those that retain their phosphorylation for a longer period of time. While sorbitol was used in the example above alternate methods of cellular perturbation can be used (e.g. heat shock). As an alternative to gel based assays harvested protein can be analyzed by MS to identify specific PP4c targeted phosphosites by MRM.

149

Even, in the case that a given phosphosite can be demonstrated to be directly targeted by PP4c, much will still remain to be learned about its function. Mutation of the site and functional readout (e.g. effect on mRNA transcription or splicing) is the most direct way of determining site function. In this case, mutation of a given site to a phosphomimetic residue (i.e. Aspartic or Glutamic acid) may result in a similar effect on mRNA transcription or splicing as is observed upon PP4c depletion (note that mutation to phosphomimetic residues does not always emulate protein phosphorylation). However, as multiple phosphosites may function together within a cell, mutation of one site may not result in any observable read out [279]. In this case, S/T sites that are in close proximity can all be mutated, as sites that are close by have been demonstrated to share interconnectivity [280, 281]. However, if the phosphosite functions in combination with other posttranslational modifications (PTMs) this could further complicate the analysis (an excellent example of which is the regulation of histones through PTM of their tails [282]).

Lastly, to further reduce inter sample variation, and to increase the accuracy of phosphosite quantification, a SILAC based approach can be undertaken to label PP4c interactors isolated from cells expressing PP4c with heavy isotopes, and PP4c interactors isolated from cells in which PP4c has been siRNA depleted with light isotopes. These samples can then be mixed after affinity purification of the interactor, effectively eliminating technical variation introduced during tryptic digest, phosphopeptide enrichment and MS analysis. Spiking in labeled peptides of known quantity in this approach can allow for absolute quantification of peptide abundance [283]. Alternatively, to SILAC based approaches, label free quantification of phosphopeptide abundance using a SRM approach could lead to a more accurate quantification of phosphopeptide abundance as a phosphopeptide of interest can be directly targeted during the MS analysis. However in this context the MS/MS fragment ions used for quantification must also provide positional information on the phosphosite location [261]. The use of this approach without phosphopeptide enrichment can additionally allow for both the phosphorylated and unphosphorylated verison of a peptide to be quantified, allowing for the stoichiometry between the two to be monitored and any effect of treatment on gene expression corrected. SRM, in combination with phosphopeptide fractionation 150 approaches (i.e. SCX [284]) could possibly lead to more accurate analysis of protein phosphorylation upon okadaic acid treatment or PP4c depletion. However, as AP-MS samples are already of a reduced complexity, it is unknown as to how much the limited phosphopeptides isolated will benefit from additional fractionation. Of note, the use of additional peptide fragmentation approaches (i.e. HCD, ETD, ECD) may also be complementary to these approaches as each of these methods has been demonstrated to identify a unique subset of phosphopeptides [110, 122] (using CID I was only able to identify ~50% of all known phosphosites listed in PhosphoSitePlus, however the proportion of the sites listed in the database that are incorrectly assigned is unknown and as such the increase that can be gained using these approaches is unknown). Further sample processing, such as the reduction and alkylation of samples, may also help to increase the number of phosphopeptides identified (i.e. by allowing for the identification of cysteine containing peptides).

4.3 Conclusions

Multidimensional fractionation of the AP-MS samples outlined herein did not result in a substantial increase in unique peptide and protein identifications, however it did increase the number of spectra observed and thus could be a useful tool when statistical methods based on spectral counting are used for data filtering or protein quantification (i.e. SAINT). It must be noted however, that baits bringing down an even more complex set of associating proteins may benefit from the multidimensional fractionation approaches outlined herein to a larger degree, allowing for more unique peptide and protein identifications to be than was observed in the current study.

Additionally, in this study it was demonstrated that okadaic acid treatment is a potent method for the induction of PP4c interactor phosphorylation, and a valuable tool for the identification of numerous previously uncharacterized phosphosites. The identification of phosphosites that are only detected upon okadaic acid treatment provides a good starting point from which more specific treatments (e.g. siRNA) can be used to whittle down which phosphosites appear targeted specifically by PP4c. Importantly, the identification of SUPT5H phosphosites involved in the regulation of transcription 151 indicates that through the use of these methods (and upon further experimentation) the identification of PP4c targeted phosphosites involved in transcriptional (or splicing) regulation is feasible.

In summary, it is hoped that by increasing our understanding of the cellular role PP4c plays we will be better equipped to understand the mechanism by which PP4c regulates the processes outlined in section 1.8, and how solid tumor resistance to the anticancer drug cisplatin develops [176].

152

References

1. Gingras, A.C., et al., A novel, evolutionarily conserved protein phosphatase complex involved in cisplatin sensitivity. Mol Cell Proteomics, 2005. 4(11): p. 1725-40.

2. Dunham, W.H., et al., A cost-benefit analysis of multidimensional fractionation of affinity purification-mass spectrometry samples. Proteomics, 2011. 11(13): p. 2603-12.

3. Gelpi, E., From large analogical instruments to small digital black boxes: 40 years of progress in mass spectrometry and its role in proteomics. Part I 1965- 1984. J Mass Spectrom, 2008. 43(4): p. 419-35.

4. Gelpi, E., From large analogical instruments to small digital black boxes: 40 years of progress in mass spectrometry and its role in proteomics. Part II 1985- 2000. J Mass Spectrom, 2009. 44(8): p. 1137-61.

5. Yates, J.R., A century of mass spectrometry: from atoms to proteomes. Nature Methods, 2011. 8(8): p. 633-37.

6. Carini, M., L. Regazzoni, and G. Aldini, Mass Spectrometric Strategies and their Applications for Molecular Mass Determination of Recombinant Therapeutic Proteins. Curr Pharm Biotechnol, 2011.

7. Ferguson, C.N., et al., Direct Ionization of Large Proteins and Protein Complexes by Desorption Electrospray Ionization-Mass Spectrometry. Anal Chem, 2011.

8. Pourhaghighi, M.R., J.M. Busnel, and H.H. Girault, High-sensitive protein analysis by FESI-CE-MALDI-MS. Electrophoresis, 2011. 32(14): p. 1795-803.

9. Steen, H. and M. Mann, The ABC's (and XYZ's) of peptide sequencing. Nat Rev Mol Cell Biol, 2004. 5(9): p. 699-711.

10. Bantscheff, M., et al., Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors. Nat Biotechnol, 2007. 25(9): p. 1035-44.

11. Breitkreutz, A., et al., A global protein kinase and phosphatase interaction network in yeast. Science, 2010. 328(5981): p. 1043-6.

12. Greco, T.M., et al., Nuclear import of histone deacetylase 5 by requisite nuclear localization signal phosphorylation. Mol Cell Proteomics, 2011. 10(2): p. M110 004317.

13. Kool, J., et al., Studying protein-protein affinity and immobilized ligand-protein affinity interactions using MS-based methods. Anal Bioanal Chem, 2011. 401(4): p. 1109-25. 153

14. Nesvizhskii, A.I. and R. Aebersold, Interpretation of shotgun proteomic data: the protein inference problem. Mol Cell Proteomics, 2005. 4(10): p. 1419-40.

15. Roque, A.C. and C.R. Lowe, Affinity chromatography: history, perspectives, limitations and prospects. Methods Mol Biol, 2008. 421: p. 1-21.

16. Raida, M., Drug target deconvolution by chemical proteomics. Curr Opin Chem Biol, 2011. 15(4): p. 570-5.

17. Jansen, G., et al., Drag&Drop cloning in yeast. Gene, 2005. 344: p. 43-51.

18. Muller, J. and A. Hemphill, Identification of a host cell target for the thiazolide class of broad-spectrum anti-parasitic drugs. Exp Parasitol, 2011. 128(2): p. 145- 50.

19. Slobodin, B. and J.E. Gerst, RaPID: an aptamer-based mRNA affinity purification technique for the identification of RNA and protein factors present in ribonucleoprotein complexes. Methods Mol Biol, 2011. 714: p. 387-406.

20. Tsai, B.P., et al., Quantitative profiling of in vivo-assembled RNA-protein complexes using a novel integrated proteomic approach. Mol Cell Proteomics, 2011. 10(4): p. M110 007385.

21. Wei, X., et al., A quantitative proteomic approach to prion disease biomarker research: delving into the glycoproteome. J Proteome Res, 2011. 10(6): p. 2687- 702.

22. Dekker, C., et al., The interaction network of the chaperonin CCT. EMBO J, 2008. 27(13): p. 1827-39.

23. Rodriguez, P., et al., Isolation of transcription factor complexes by in vivo biotinylation tagging and direct binding to streptavidin beads. Methods Mol Biol, 2006. 338: p. 305-23.

24. Brizzard, B., Epitope tagging. Biotechniques, 2008. 44(5): p. 693-5.

25. Jarvik, J.W. and C.A. Telmer, Epitope tagging. Annu Rev Genet, 1998. 32: p. 601-18.

26. Evan, G.I., et al., Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol, 1985. 5(12): p. 3610-6.

27. Seol, J.H., A. Shevchenko, and R.J. Deshaies, Skp1 forms multiple protein complexes, including RAVE, a regulator of V-ATPase assembly. Nat Cell Biol, 2001. 3(4): p. 384-91. 154

28. Hopp, T.P., et al., A short polypeptide marker sequence useful for recombinant protein identification and purification. Nature Biotechnology, 1988. 6: p. 1204- 10.

29. Olma, M.H., et al., An interaction network of the mammalian COP9 signalosome identifies Dda1 as a core subunit of multiple Cul4-based E3 ligases. J Cell Sci, 2009. 122(Pt 7): p. 1035-44.

30. Skarra, D.V., et al., Label-free quantitative proteomics and SAINT analysis enable interactome mapping for the human Ser/Thr protein phosphatase 5. Proteomics, 2011. 11(8): p. 1508-16.

31. Field, J., et al., Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol, 1988. 8(5): p. 2159-65.

32. Sowa, M.E., et al., Defining the human deubiquitinating enzyme interaction landscape. Cell, 2009. 138(2): p. 389-403.

33. Hochuli, E., et al., Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent. Nature Biotechnology, 1988. 6: p. 1321-25.

34. Li, Z., et al., Affinity purification of an archaeal DNA replication protein network. MBio, 2010. 1(5).

35. Schmidt, T.G. and A. Skerra, The random peptide library-assisted engineering of a C-terminal affinity peptide, useful for the detection and purification of a functional Ig Fv fragment. Protein Eng, 1993. 6(1): p. 109-22.

36. Chhabra, S.R., et al., Towards a Rigorous Network of Protein-Protein Interactions of the Model Sulfate Reducer Desulfovibrio vulgaris Hildenborough. PLoS One, 2011. 6(6): p. e21470.

37. Olinares, P.D., et al., Subunit Stoichiometry, Evolution, and Functional Implications of an Asymmetric Plant Plastid ClpP/R Protease Complex in Arabidopsis. Plant Cell, 2011. 23(6): p. 2348-61.

38. Smith, D.B. and K.S. Johnson, Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene, 1988. 67(1): p. 31-40.

39. Jones, J., et al., A targeted proteomic analysis of the ubiquitin-like modifier nedd8 and associated proteins. J Proteome Res, 2008. 7(3): p. 1274-87.

40. Tai, H.C., et al., Characterization of the Brain 26S Proteasome and its Interacting Proteins. Front Mol Neurosci, 2010. 3. 155

41. Chalfie, M., et al., Green fluorescent protein as a marker for gene expression. Science, 1994. 263(5148): p. 802-5.

42. Cristea, I.M., et al., Fluorescent proteins as proteomic probes. Mol Cell Proteomics, 2005. 4(12): p. 1933-41.

43. Galan, J.A., et al., Proteomic studies of Syk-interacting proteins using a novel amine-specific isotope tag and GFP nanotrap. J Am Soc Mass Spectrom, 2011. 22(2): p. 319-28.

44. Hubner, N.C., et al., Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions. J Cell Biol, 2010. 189(4): p. 739-54.

45. Hubner, N.C. and M. Mann, Extracting gene function from protein-protein interactions using Quantitative BAC InteraCtomics (QUBIC). Methods, 2011. 53(4): p. 453-9.

46. Uhlen, M., et al., Gene fusion vectors based on the gene for staphylococcal protein A. Gene, 1983. 23(3): p. 369-78.

47. Oeffinger, M., et al., Comprehensive analysis of diverse ribonucleoprotein complexes. Nat Methods, 2007. 4(11): p. 951-6.

48. Tackett, A.J., et al., Proteomic and genomic characterization of chromatin complexes at a boundary. J Cell Biol, 2005. 169(1): p. 35-47.

49. Rees, J.S., et al., In vivo analysis of proteomes and interactomes using Parallel Affinity Capture (iPAC) coupled to mass spectrometry. Mol Cell Proteomics, 2011. 10(6): p. M110 002386.

50. Butland, G., et al., Interaction network containing conserved and essential protein complexes in Escherichia coli. Nature, 2005. 433(7025): p. 531-7.

51. Zeghouf, M., et al., Sequential Peptide Affinity (SPA) system for the identification of mammalian and bacterial protein complexes. J Proteome Res, 2004. 3(3): p. 463-8.

52. Wang, J., et al., A protein interaction network for pluripotency of embryonic stem cells. Nature, 2006. 444(7117): p. 364-8.

53. Forget, D., et al., The protein interaction network of the human transcription machinery reveals a role for the conserved GTPase RPAP4/GPN1 and microtubule assembly in nuclear import and biogenesis of RNA polymerase II. Mol Cell Proteomics, 2010. 9(12): p. 2827-39.

54. Gavin, A.C., et al., Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature, 2002. 415(6868): p. 141-7. 156

55. Jeronimo, C., et al., Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme. Mol Cell, 2007. 27(2): p. 262-74.

56. Lambert, J.P., et al., Defining the budding yeast chromatin-associated interactome. Mol Syst Biol, 2010. 6: p. 448.

57. Rigaut, G., et al., A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol, 1999. 17(10): p. 1030-2.

58. Boldt, K., et al., Tandem affinity purification of ciliopathy-associated protein complexes. Methods Cell Biol, 2009. 91: p. 143-60.

59. Gloeckner, C.J., et al., A novel tandem affinity purification strategy for the efficient isolation and characterisation of native protein complexes. Proteomics, 2007. 7(23): p. 4228-34.

60. Gloeckner, C.J., et al., Tandem affinity purification of protein complexes from mammalian cells by the Strep/FLAG (SF)-TAP tag. Methods Mol Biol, 2009. 564: p. 359-72.

61. Burckstummer, T., et al., An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells. Nat Methods, 2006. 3(12): p. 1013-9.

62. Kyriakakis, P., et al., Tandem affinity purification in Drosophila: the advantages of the GS-TAP system. Fly (Austin), 2008. 2(4): p. 229-35.

63. Glatter, T., et al., An integrated workflow for charting the human interaction proteome: insights into the PP2A system. Mol Syst Biol, 2009. 5: p. 237.

64. Wepf, A., et al., Quantitative interaction proteomics using mass spectrometry. Nat Methods, 2009. 6(3): p. 203-5.

65. Kirkpatrick, D.S., et al., Proteomic identification of ubiquitinated proteins from human cells expressing His-tagged ubiquitin. Proteomics, 2005. 5(8): p. 2104-11.

66. Goel, A., et al., Relative position of the hexahistidine tag effects binding properties of a tumor-associated single-chain Fv construct. Biochim Biophys Acta, 2000. 1523(1): p. 13-20.

67. Hofemeister, H., et al., Recombineering, transfection, Western, IP and ChIP methods for protein tagging via gene targeting or BAC transgenesis. Methods, 2011. 53(4): p. 437-52.

68. Rumlova, M., et al., Comparison of classical and affinity purification techniques of Mason-Pfizer monkey virus capsid protein: the alteration of the product by an affinity tag. Protein Expr Purif, 2001. 23(1): p. 75-83. 157

69. Howson, R., et al., Construction, verification and experimental use of two epitope-tagged collections of budding yeast strains. Comp Funct Genomics, 2005. 6(1-2): p. 2-16.

70. Gavin, A.C., et al., Proteome survey reveals modularity of the yeast cell machinery. Nature, 2006. 440(7084): p. 631-6.

71. Krogan, N.J., et al., Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature, 2006. 440(7084): p. 637-43.

72. Fernandez, E., et al., Targeted tandem affinity purification of PSD-95 recovers core postsynaptic complexes and schizophrenia susceptibility proteins. Mol Syst Biol, 2009. 5: p. 269.

73. Allton, K., et al., Trim24 targets endogenous p53 for degradation. Proc Natl Acad Sci U S A, 2009. 106(28): p. 11612-6.

74. Goudreault, M., et al., A PP2A phosphatase high density interaction network identifies a novel striatin-interacting phosphatase and kinase complex linked to the cerebral cavernous malformation 3 (CCM3) protein. Mol Cell Proteomics, 2009. 8(1): p. 157-71.

75. Mak, A.B., et al., A lentiviral functional proteomics approach identifies chromatin remodeling complexes important for the induction of pluripotency. Mol Cell Proteomics, 2010. 9(5): p. 811-23.

76. Sardiu, M.E., et al., Probabilistic assembly of human protein interaction networks from label-free quantitative proteomics. Proc Natl Acad Sci U S A, 2008. 105(5): p. 1454-9.

77. Torres, J.Z., J.J. Miller, and P.K. Jackson, High-throughput generation of tagged stable cell lines for proteomic analysis. Proteomics, 2009. 9(10): p. 2888-91.

78. Strambio-de-Castillia, C., et al., A method for the rapid and efficient elution of native affinity-purified protein A tagged complexes. J Proteome Res, 2005. 4(6): p. 2250-6.

79. Chen, G.I., et al., PP4R4/KIAA1622 forms a novel stable cytosolic complex with phosphoprotein phosphatase 4. J Biol Chem, 2008. 283(43): p. 29273-84.

80. Tackett, A.J., et al., I-DIRT, a general method for distinguishing between specific and nonspecific protein interactions. J Proteome Res, 2005. 4(5): p. 1752-6.

81. Deeb, S.J., et al., Super-SILAC allows classification of diffuse large B-cell lymphoma subtypes by their protein expression profiles. Mol Cell Proteomics, 2012. 158

82. Campbell-Valois, F.X., et al., Quantitative proteomics reveals that only a subset of the endoplasmic reticulum contributes to the phagosome. Mol Cell Proteomics, 2012.

83. Emadali, A. and M. Gallagher-Gambarelli, [Quantitative proteomics by SILAC: practicalities and perspectives for an evolving approach]. Med Sci (Paris), 2009. 25(10): p. 835-42.

84. Ong, S.E., L.J. Foster, and M. Mann, Mass spectrometric-based approaches in quantitative proteomics. Methods, 2003. 29(2): p. 124-30.

85. Ong, S.E., et al., Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics, 2002. 1(5): p. 376-86.

86. Liu, H., R.G. Sadygov, and J.R. Yates, 3rd, A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal Chem, 2004. 76(14): p. 4193-201.

87. Paoletti, A.C., et al., Quantitative proteomic analysis of distinct mammalian Mediator complexes using normalized spectral abundance factors. Proc Natl Acad Sci U S A, 2006. 103(50): p. 18928-33.

88. Rinner, O., et al., An integrated mass spectrometric and computational framework for the analysis of protein interaction networks. Nat Biotechnol, 2007. 25(3): p. 345-52.

89. Behrends, C., et al., Network organization of the human autophagy system. Nature, 2010. 466(7302): p. 68-76.

90. Lavallee-Adam, M., et al., Modeling contaminants in AP-MS/MS experiments. J Proteome Res, 2011. 10(2): p. 886-95.

91. Choi, H., et al., SAINT: probabilistic scoring of affinity purification-mass spectrometry data. Nat Methods, 2011. 8(1): p. 70-3.

92. Choi, H., et al., SAINT-MS1: Protein-Protein Interaction Scoring Using Label- free Intensity Data in Affinity Purification-Mass Spectrometry Experiments. J Proteome Res, 2012.

93. Elschenbroich, S., et al., Peptide separations by on-line MudPIT compared to isoelectric focusing in an off-gel format: application to a membrane-enriched fraction from C2C12 mouse skeletal muscle cells. J Proteome Res, 2009. 8(10): p. 4860-9.

94. Gilar, M., et al., Comparison of 1-D and 2-D LC MS/MS methods for proteomic analysis of human serum. Electrophoresis, 2009. 30(7): p. 1157-67. 159

95. Horvatovich, P., et al., Multidimensional chromatography coupled to mass spectrometry in analysing complex proteomics samples. J Sep Sci, 2010. 33(10): p. 1421-37.

96. Schirle, M., M.A. Heurtier, and B. Kuster, Profiling core proteomes of human cell lines by one-dimensional PAGE and liquid chromatography-tandem mass spectrometry. Mol Cell Proteomics, 2003. 2(12): p. 1297-305.

97. Gilar, M., et al., Orthogonality of separation in two-dimensional liquid chromatography. Anal Chem, 2005. 77(19): p. 6426-34.

98. Miller, K.M. and S.P. Jackson, Histone marks: repairing DNA breaks within the context of chromatin. Biochem Soc Trans, 2012. 40(2): p. 370-6.

99. Populo, H., J.M. Lopes, and P. Soares, The mTOR Signalling Pathway in Human Cancer. Int J Mol Sci, 2012. 13(2): p. 1886-918.

100. Sun, H. and Y. Wang, Novel Ser/Thr protein phosphatases in cell death regulation. Physiology (Bethesda), 2012. 27(1): p. 43-52.

101. Moorhead, G.B., L. Trinkle-Mulcahy, and A. Ulke-Lemee, Emerging roles of nuclear protein phosphatases. Nat Rev Mol Cell Biol, 2007. 8(3): p. 234-44.

102. Olsen, J.V., et al., Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell, 2006. 127(3): p. 635-48.

103. Manning, G., et al., The protein kinase complement of the human genome. Science, 2002. 298(5600): p. 1912-34.

104. Yaffe, M.B. and A.E. Elia, Phosphoserine/threonine-binding domains. Curr Opin Cell Biol, 2001. 13(2): p. 131-8.

105. Hunter, T., Signaling--2000 and beyond. Cell, 2000. 100(1): p. 113-27.

106. Krebs, E.G., Historical perspectives on protein phosphorylation and a classification system for protein kinases. Philos Trans R Soc Lond B Biol Sci, 1983. 302(1108): p. 3-11.

107. Gerrits, B. and B. Bodenmiller, Mapping of phosphorylation sites by LC-MS/MS. Methods Mol Biol, 2010. 658: p. 127-36.

108. Wu, H.Y. and P.C. Liao, Analysis of protein phosphorylation using mass spectrometry. Chang Gung Med J, 2008. 31(3): p. 217-27.

109. D'Ambrosio, C., et al., Analytical methodologies for the detection and structural characterization of phosphorylated proteins. J Chromatogr B Analyt Technol Biomed Life Sci, 2007. 849(1-2): p. 163-80. 160

110. Paradela, A. and J.P. Albar, Advances in the analysis of protein phosphorylation. J Proteome Res, 2008. 7(5): p. 1809-18.

111. Kange, R., et al., Comparison of different IMAC techniques used for enrichment of phosphorylated peptides. J Biomol Tech, 2005. 16(2): p. 91-103.

112. Larsen, M.R., et al., Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics, 2005. 4(7): p. 873-86.

113. Bodenmiller, B., et al., Reproducible isolation of distinct, overlapping segments of the phosphoproteome. Nat Methods, 2007. 4(3): p. 231-7.

114. Zoumaro-Djayoon, A.D., A.J. Heck, and J. Munoz, Targeted analysis of tyrosine phosphorylation by immuno-affinity enrichment of tyrosine phosphorylated peptides prior to mass spectrometric analysis. Methods, 2012. 56(2): p. 268-74.

115. Bergstrom Lind, S., et al., Toward a comprehensive characterization of the phosphotyrosine proteome. Cell Signal, 2011. 23(8): p. 1387-95.

116. Matsuoka, S., et al., ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science, 2007. 316(5828): p. 1160-6.

117. Cortez, D., G. Glick, and S.J. Elledge, Minichromosome maintenance proteins are direct targets of the ATM and ATR checkpoint kinases. Proc Natl Acad Sci U S A, 2004. 101(27): p. 10078-83.

118. Boersema, P.J., S. Mohammed, and A.J. Heck, Phosphopeptide fragmentation and analysis by mass spectrometry. J Mass Spectrom, 2009. 44(6): p. 861-78.

119. Bodenmiller, B. and R. Aebersold, Quantitative analysis of protein phosphorylation on a system-wide scale by mass spectrometry-based proteomics. Methods Enzymol, 2010. 470: p. 317-34.

120. Taus, T., et al., Universal and confident phosphorylation site localization using phosphoRS. J Proteome Res, 2011. 10(12): p. 5354-62.

121. Sun, R.X., et al., Improved peptide identification for proteomic analysis based on comprehensive characterization of electron transfer dissociation spectra. J Proteome Res, 2010. 9(12): p. 6354-67.

122. Nagaraj, N., et al., Feasibility of large-scale phosphoproteomics with higher energy collisional dissociation fragmentation. J Proteome Res, 2010. 9(12): p. 6786-94.

123. Kelstrup, C.D., et al., Pinpointing phosphorylation sites: Quantitative filtering and a novel site-specific x-ion fragment. J Proteome Res, 2011. 10(7): p. 2937-48. 161

124. Palumbo, A.M. and G.E. Reid, Evaluation of gas-phase rearrangement and competing fragmentation reactions on protein phosphorylation site assignment using collision induced dissociation-MS/MS and MS3. Anal Chem, 2008. 80(24): p. 9735-47.

125. Mischerikow, N., et al., Comparative assessment of site assignments in CID and electron transfer dissociation spectra of phosphopeptides discloses limited relocation of phosphate groups. Mol Cell Proteomics, 2010. 9(10): p. 2140-8.

126. Neilson, K.A., et al., Less label, more free: approaches in label-free quantitative mass spectrometry. Proteomics, 2011. 11(4): p. 535-53.

127. Zhu, W., J.W. Smith, and C.M. Huang, Mass spectrometry-based label-free quantitative proteomics. J Biomed Biotechnol, 2010. 2010: p. 840518.

128. Chelius, D. and P.V. Bondarenko, Quantitative profiling of proteins in complex mixtures using liquid chromatography and mass spectrometry. J Proteome Res, 2002. 1(4): p. 317-23.

129. Choi, H., et al., Development of an off-line capillary column IMAC phosphopeptide enrichment method for label-free phosphorylation relative quantification. J Chromatogr B Analyt Technol Biomed Life Sci, 2011. 879(28): p. 2991-7.

130. Mueller, L.N., et al., An assessment of software solutions for the analysis of mass spectrometry based quantitative proteomics data. J Proteome Res, 2008. 7(1): p. 51-61.

131. Vandenbogaert, M., et al., Alignment of LC-MS images, with applications to biomarker discovery and protein identification. Proteomics, 2008. 8(4): p. 650-72.

132. America, A.H. and J.H. Cordewener, Comparative LC-MS: a landscape of peaks and valleys. Proteomics, 2008. 8(4): p. 731-49.

133. Gallien, S., E. Duriez, and B. Domon, Selected reaction monitoring applied to proteomics. J Mass Spectrom, 2011. 46(3): p. 298-312.

134. Elschenbroich, S. and T. Kislinger, Targeted proteomics by selected reaction monitoring mass spectrometry: applications to systems biology and biomarker discovery. Mol Biosyst, 2011. 7(2): p. 292-303.

135. Chen, Y., et al., Mascot-derived false positive peptide identifications revealed by manual analysis of tandem mass spectra. J Proteome Res, 2009. 8(6): p. 3141-7.

136. Goldstrohm, D.A., et al., Importance of manual validation for the identification of phosphopeptides using a linear ion trap mass spectrometer. J Biomol Tech, 2011. 22(1): p. 10-20. 162

137. Nichols, A.M. and F.M. White, Manual validation of peptide sequence and sites of tyrosine phosphorylation from MS/MS spectra. Methods Mol Biol, 2009. 492: p. 143-60.

138. Beausoleil, S.A., et al., A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat Biotechnol, 2006. 24(10): p. 1285-92.

139. Lemeer, S., et al., Phosphorylation site localization in peptides by MALDI MS/MS and the Mascot Delta Score. Anal Bioanal Chem, 2012. 402(1): p. 249-60.

140. Ruttenberg, B.E., et al., PhosphoScore: an open-source phosphorylation site assignment tool for MSn data. J Proteome Res, 2008. 7(7): p. 3054-9.

141. Lahesmaa-Korpinen, A.M., et al., Integrated data management and validation platform for phosphorylated tandem mass spectrometry data. Proteomics, 2010. 10(19): p. 3515-24.

142. Li, Y., et al., Relationship between ATM and Ribosomal Protein S6 Revealed by the Chemical Inhibition of Ser/Thr Protein Phosphatase Type 1. Biosci Biotechnol Biochem, 2012. 76(3): p. 486-94.

143. Nakada, S., et al., PP4 is a gamma H2AX phosphatase required for recovery from the DNA damage checkpoint. EMBO Rep, 2008. 9(10): p. 1019-26.

144. Kurimchak, A. and X. Grana, PP2A holoenzymes negatively and positively regulate cell cycle progression by dephosphorylating pocket proteins and multiple CDK substrates. Gene, 2012.

145. Wurzenberger, C. and D.W. Gerlich, Phosphatases: providing safe passage through mitotic exit. Nat Rev Mol Cell Biol, 2011. 12(8): p. 469-82.

146. O'Neill, A.K., M.J. Niederst, and A.C. Newton, Suppression of survival signalling pathways by the phosphatase PHLPP. FEBS J, 2012.

147. Bai, Y., et al., Phosphoproteomics identifies driver tyrosine kinases in sarcoma cell lines and tumors. Cancer Res, 2012.

148. Janssens, V. and A. Rebollo, The Role and Therapeutic Potential of Ser/Thr Phosphatase PP2A in Apoptotic Signalling Networks in Human Cancer Cells. Curr Mol Med, 2012. 12(3): p. 268-87.

149. Zhang, Q. and F.X. Claret, Phosphatases: the new brakes for cancer development? Enzyme Res, 2012. 2012: p. 659649.

150. Harsha, H.C. and A. Pandey, Phosphoproteomics in cancer. Mol Oncol, 2010. 4(6): p. 482-95. 163

151. Tiganis, T., PTP1B and TCPTP: Non-redundant phosphatases in insulin signaling and glucose homeostasis. FEBS J, 2012.

152. Braithwaite, S.P., et al., Targeting phosphatases as the next generation of disease modifying therapeutics for Parkinson's disease. Neurochem Int, 2012.

153. Doddareddy, M.R., T. Rawling, and A.J. Ammit, Targeting mitogen-activated protein kinase phosphatase-1 (MKP-1): structure-based design of MKP-1 inhibitors and upregulators. Curr Med Chem, 2012. 19(2): p. 163-73.

154. Voronkov, M., S.P. Braithwaite, and J.B. Stock, Phosphoprotein phosphatase 2A: a novel druggable target for Alzheimer's disease. Future Med Chem, 2011. 3(7): p. 821-33.

155. Moran, M.F., et al., Emerging applications for phospho-proteomics in cancer molecular therapeutics. Biochim Biophys Acta, 2006. 1766(2): p. 230-41.

156. Honkanen, R.E. and T. Golden, Regulators of serine/threonine protein phosphatases at the dawn of a clinical era? Curr Med Chem, 2002. 9(22): p. 2055-75.

157. Chen, G.I., Identification and Characterization of Novel Interactors of Human Protein Phosphatase 4 using Mass Spectrometry Technology, in Molecular Genetics2010, University of Toronto. p. 244.

158. Kloeker, S., et al., Parallel purification of three catalytic subunits of the protein serine/threonine phosphatase 2A family (PP2A(C), PP4(C), and PP6(C)) and analysis of the interaction of PP2A(C) with alpha4 protein. Protein Expr Purif, 2003. 31(1): p. 19-33.

159. Huang, X., A. Cheng, and R.E. Honkanen, Genomic organization of the human PP4 gene encoding a serine/threonine protein phosphatase (PP4) suggests a common ancestry with PP2A. Genomics, 1997. 44(3): p. 336-43.

160. Cohen, P.T., Novel protein serine/threonine phosphatases: variety is the spice of life. Trends Biochem Sci, 1997. 22(7): p. 245-51.

161. Brewis, N.D. and P.T. Cohen, Protein phosphatase X has been highly conserved during mammalian evolution. Biochim Biophys Acta, 1992. 1171(2): p. 231-3.

162. Hu, M.C., et al., Genomic structure of the mouse PP4 gene: a developmentally regulated protein phosphatase. Gene, 2001. 278(1-2): p. 89-99.

163. Hastie, C.J., et al., A novel 50 kDa protein forms complexes with protein phosphatase 4 and is located at centrosomal microtubule organizing centres. Biochem J, 2000. 347 Pt 3: p. 845-55. 164

164. Lyu, J., E.H. Jho, and W. Lu, Smek promotes histone deacetylation to suppress transcription of Wnt target gene brachyury in pluripotent embryonic stem cells. Cell Res, 2011. 21(6): p. 911-21.

165. Jia, H., et al., PP4 and PP2A regulate Hedgehog signaling by controlling Smo and Ci phosphorylation. Development, 2009. 136(2): p. 307-16.

166. Zhou, G., J.S. Boomer, and T.H. Tan, Protein phosphatase 4 is a positive regulator of hematopoietic progenitor kinase 1. The Journal of biological chemistry, 2004. 279(47): p. 49551-61.

167. Zhou, G., et al., Protein phosphatase 4 is involved in tumor necrosis factor-alpha- induced activation of c-Jun N-terminal kinase. The Journal of biological chemistry, 2002. 277(8): p. 6391-8.

168. Zhang, X., et al., Histone deacetylase 3 (HDAC3) activity is regulated by interaction with protein serine/threonine phosphatase 4. Genes Dev, 2005. 19(7): p. 827-39.

169. Yeh, P.Y., et al., Suppression of MEK/ERK signaling pathway enhances cisplatin- induced NF-kappaB activation by protein phosphatase 4-mediated NF-kappaB p65 Thr dephosphorylation. The Journal of biological chemistry, 2004. 279(25): p. 26143-8.

170. Hu, M.C., et al., Protein phosphatase X interacts with c-Rel and stimulates c- Rel/nuclear factor kappaB activity. The Journal of biological chemistry, 1998. 273(50): p. 33561-5.

171. Kim, J.A., et al., Protein phosphatases pph3, ptc2, and ptc3 play redundant roles in DNA double-strand break repair by homologous recombination. Mol Cell Biol, 2011. 31(3): p. 507-16.

172. Lee, D.H., et al., A PP4 phosphatase complex dephosphorylates RPA2 to facilitate DNA repair via homologous recombination. Nat Struct Mol Biol, 2010. 17(3): p. 365-72.

173. Chowdhury, D., et al., A PP4-phosphatase complex dephosphorylates gamma- H2AX generated during DNA replication. Molecular cell, 2008. 31(1): p. 33-46.

174. O'Neill, B.M., et al., Pph3-Psy2 is a phosphatase complex required for Rad53 dephosphorylation and replication fork restart during recovery from DNA damage. Proceedings of the National Academy of Sciences of the United States of America, 2007. 104(22): p. 9290-5.

175. Keogh, M.C., et al., A phosphatase complex that dephosphorylates gammaH2AX regulates DNA damage checkpoint recovery. Nature, 2006. 439(7075): p. 497- 501. 165

176. Gingras, A.C., et al., A novel, evolutionarily conserved protein phosphatase complex involved in cisplatin sensitivity. Molecular & cellular proteomics : MCP, 2005. 4(11): p. 1725-40.

177. Wang, B., et al., Protein phosphatase PP4 is overexpressed in human breast and lung tumors. Cell Res, 2008. 18(9): p. 974-7.

178. Martin-Granados, C., et al., Depletion of protein phosphatase 4 in human cells reveals essential roles in centrosome maturation, cell migration and the regulation of Rho GTPases. Int J Biochem Cell Biol, 2008. 40(10): p. 2315-32.

179. Toyo-oka, K., et al., Protein phosphatase 4 catalytic subunit regulates Cdk1 activity and microtubule organization via NDEL1 dephosphorylation. J Cell Biol, 2008. 180(6): p. 1133-47.

180. Sumiyoshi, E., A. Sugimoto, and M. Yamamoto, Protein phosphatase 4 is required for centrosome maturation in mitosis and sperm meiosis in C. elegans. J Cell Sci, 2002. 115(Pt 7): p. 1403-10.

181. Helps, N.R., et al., Protein phosphatase 4 is an essential enzyme required for organisation of microtubules at centrosomes in Drosophila embryos. J Cell Sci, 1998. 111 ( Pt 10): p. 1331-40.

182. Mourtada-Maarabouni, M. and G.T. Williams, Protein phosphatase 4 regulates apoptosis in leukemic and primary human T-cells. Leuk Res, 2009. 33(11): p. 1539-51.

183. Mourtada-Maarabouni, M. and G.T. Williams, Protein phosphatase 4 regulates apoptosis, proliferation and mutation rate of human cells. Biochim Biophys Acta, 2008. 1783(8): p. 1490-502.

184. Kloeker, S. and B.E. Wadzinski, Purification and identification of a novel subunit of protein serine/threonine phosphatase 4. The Journal of biological chemistry, 1999. 274(9): p. 5339-47.

185. Chen, G.I., et al., PP4R4/KIAA1622 forms a novel stable cytosolic complex with phosphoprotein phosphatase 4. The Journal of biological chemistry, 2008. 283(43): p. 29273-84.

186. Carnegie, G.K., et al., Protein phosphatase 4 interacts with the Survival of Motor Neurons complex and enhances the temporal localisation of snRNPs. J Cell Sci, 2003. 116(Pt 10): p. 1905-13.

187. Virshup, D.M. and S. Shenolikar, From promiscuity to precision: protein phosphatases get a makeover. Molecular cell, 2009. 33(5): p. 537-45.

188. Hubbard, M.J. and P. Cohen, On target with a new mechanism for the regulation of protein phosphorylation. Trends Biochem Sci, 1993. 18(5): p. 172-7. 166

189. Bilsland, E. and J.A. Downs, Tails of histones in DNA double-strand break repair. Mutagenesis, 2005. 20(3): p. 153-63.

190. Usui, H., et al., Three distinct forms of type 2A protein phosphatase in human erythrocyte cytosol. J Biol Chem, 1988. 263(8): p. 3752-61.

191. Chen, J., R.T. Peterson, and S.L. Schreiber, Alpha 4 associates with protein phosphatases 2A, 4, and 6. Biochem Biophys Res Commun, 1998. 247(3): p. 827- 32.

192. Prickett, T.D. and D.L. Brautigan, The alpha4 regulatory subunit exerts opposing allosteric effects on protein phosphatases PP6 and PP2A. J Biol Chem, 2006. 281(41): p. 30503-11.

193. Nanahoshi, M., et al., Alpha4 protein as a common regulator of type 2A-related serine/threonine protein phosphatases. FEBS letters, 1999. 446(1): p. 108-12.

194. LeNoue-Newton, M., et al., The E3 ubiquitin ligase- and protein phosphatase 2A (PP2A)-binding domains of the Alpha4 protein are both required for Alpha4 to inhibit PP2A degradation. J Biol Chem, 2011. 286(20): p. 17665-71.

195. McConnell, J.L., et al., Alpha4 is a ubiquitin-binding protein that regulates protein serine/threonine phosphatase 2A ubiquitination. Biochemistry, 2010. 49(8): p. 1713-8.

196. Kong, M., et al., Alpha4 is an essential regulator of PP2A phosphatase activity. Mol Cell, 2009. 36(1): p. 51-60.

197. Wada, T., et al., DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs. Genes Dev, 1998. 12(3): p. 343-56.

198. Wen, Y. and A.J. Shatkin, Transcription elongation factor hSPT5 stimulates mRNA capping. Genes Dev, 1999. 13(14): p. 1774-9.

199. Schwer, B. and C. Guthrie, PRP16 is an RNA-dependent ATPase that interacts transiently with the spliceosome. Nature, 1991. 349(6309): p. 494-9.

200. Fong, Y.W. and Q. Zhou, Stimulatory effect of splicing factors on transcriptional elongation. Nature, 2001. 414(6866): p. 929-33.

201. Yan, D., et al., CUS2, a yeast homolog of human Tat-SF1, rescues function of misfolded U2 through an unusual RNA recognition motif. Mol Cell Biol, 1998. 18(9): p. 5000-9.

202. Breitkreutz, B.J., et al., The BioGRID Interaction Database: 2008 update. Nucleic Acids Res, 2008. 36(Database issue): p. D637-40. 167

203. Spector, D.L. and A.I. Lamond, Nuclear speckles. Cold Spring Harb Perspect Biol, 2011. 3(2).

204. Lamond, A.I. and D.L. Spector, Nuclear speckles: a model for nuclear organelles. Nat Rev Mol Cell Biol, 2003. 4(8): p. 605-12.

205. Grillari, J., et al., SNEV is an evolutionarily conserved splicing factor whose oligomerization is necessary for spliceosome assembly. Nucleic Acids Res, 2005. 33(21): p. 6868-83.

206. Aksaas, A.K., et al., Protein kinase a-dependent phosphorylation of serine 119 in the proto-oncogenic serine/arginine-rich splicing factor 1 modulates its activity as a splicing enhancer protein. Genes Cancer, 2011. 2(8): p. 841-51.

207. Sumanasekera, C., et al., C6 pyridinium ceramide influences alternative pre- mRNA splicing by inhibiting protein phosphatase-1. Nucleic Acids Res, 2011.

208. Buratowski, S., Progression through the RNA polymerase II CTD cycle. Mol Cell, 2009. 36(4): p. 541-6.

209. Wahl, M.C., C.L. Will, and R. Luhrmann, The spliceosome: design principles of a dynamic RNP machine. Cell, 2009. 136(4): p. 701-18.

210. Egloff, S., et al., Serine-7 of the RNA polymerase II CTD is specifically required for snRNA gene expression. Science, 2007. 318(5857): p. 1777-9.

211. Misteli, T. and D.L. Spector, The cellular organization of gene expression. Curr Opin Cell Biol, 1998. 10(3): p. 323-31.

212. Misteli, T. and D.L. Spector, Protein phosphorylation and the nuclear organization of pre-mRNA splicing. Trends Cell Biol, 1997. 7(4): p. 135-8.

213. Misteli, T. and D.L. Spector, Serine/threonine phosphatase 1 modulates the subnuclear distribution of pre-mRNA splicing factors. Mol Biol Cell, 1996. 7(10): p. 1559-72.

214. Xiao, Y., et al., Analysis of a splice array experiment elucidates roles of chromatin elongation factor Spt4-5 in splicing. PLoS Comput Biol, 2005. 1(4): p. e39.

215. Lindstrom, D.L., et al., Dual roles for Spt5 in pre-mRNA processing and transcription elongation revealed by identification of Spt5-associated proteins. Mol Cell Biol, 2003. 23(4): p. 1368-78.

216. Mermoud, J.E., P.T. Cohen, and A.I. Lamond, Regulation of mammalian spliceosome assembly by a protein phosphorylation mechanism. EMBO J, 1994. 13(23): p. 5679-88. 168

217. Mermoud, J.E., P. Cohen, and A.I. Lamond, Ser/Thr-specific protein phosphatases are required for both catalytic steps of pre-mRNA splicing. Nucleic Acids Res, 1992. 20(20): p. 5263-9.

218. Ortlepp, D., et al., The mammalian homologue of Prp16p is overexpressed in a cell line tolerant to Leflunomide, a new immunoregulatory drug effective against rheumatoid arthritis. RNA, 1998. 4(8): p. 1007-18.

219. Zhou, Z. and R. Reed, Human homologs of yeast prp16 and prp17 reveal conservation of the mechanism for catalytic step II of pre-mRNA splicing. EMBO J, 1998. 17(7): p. 2095-106.

220. Brewis, N.D., et al., PPX, a novel protein serine/threonine phosphatase localized to centrosomes. EMBO J, 1993. 12(3): p. 987-96.

221. O'Neill, B.M., et al., Pph3-Psy2 is a phosphatase complex required for Rad53 dephosphorylation and replication fork restart during recovery from DNA damage. Proc Natl Acad Sci U S A, 2007. 104(22): p. 9290-5.

222. Chowdhury, D., et al., A PP4-phosphatase complex dephosphorylates gamma- H2AX generated during DNA replication. Mol Cell, 2008. 31(1): p. 33-46.

223. Fujita, T., et al., Up-regulation of P-TEFb by the MEK1-extracellular signal- regulated kinase signaling pathway contributes to stimulated transcription elongation of immediate early genes in neuroendocrine cells. Mol Cell Biol, 2008. 28(5): p. 1630-43.

224. Aida, M., et al., Transcriptional pausing caused by NELF plays a dual role in regulating immediate-early expression of the junB gene. Mol Cell Biol, 2006. 26(16): p. 6094-104.

225. Yamada, T., et al., P-TEFb-mediated phosphorylation of hSpt5 C-terminal repeats is critical for processive transcription elongation. Mol Cell, 2006. 21(2): p. 227-37.

226. Core, L.J., J.J. Waterfall, and J.T. Lis, Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science, 2008. 322(5909): p. 1845-8.

227. Baumli, S., et al., The structure of P-TEFb (CDK9/cyclin T1), its complex with flavopiridol and regulation by phosphorylation. EMBO J, 2008. 27(13): p. 1907- 18.

228. Fujita, T., et al., Gene-specific recruitment of positive and negative elongation factors during stimulated transcription of the MKP-1 gene in neuroendocrine cells. Nucleic Acids Res, 2007. 35(3): p. 1007-17. 169

229. Peterlin, B.M. and D.H. Price, Controlling the elongation phase of transcription with P-TEFb. Mol Cell, 2006. 23(3): p. 297-305.

230. Ping, Y.H. and T.M. Rana, DSIF and NELF interact with RNA polymerase II elongation complex and HIV-1 Tat stimulates P-TEFb-mediated phosphorylation of RNA polymerase II and DSIF during transcription elongation. J Biol Chem, 2001. 276(16): p. 12951-8.

231. Chen, Y., et al., DSIF, the Paf1 complex, and Tat-SF1 have nonredundant, cooperative roles in RNA polymerase II elongation. Genes Dev, 2009. 23(23): p. 2765-77.

232. Schroeder, S.C., et al., Dynamic association of capping enzymes with transcribing RNA polymerase II. Genes Dev, 2000. 14(19): p. 2435-40.

233. Ho, C.K. and S. Shuman, Distinct roles for CTD Ser-2 and Ser-5 phosphorylation in the recruitment and allosteric activation of mammalian mRNA capping enzyme. Mol Cell, 1999. 3(3): p. 405-11.

234. Krueger, B.J., et al., LARP7 is a stable component of the 7SK snRNP while P- TEFb, HEXIM1 and hnRNP A1 are reversibly associated. Nucleic Acids Res, 2008. 36(7): p. 2219-29.

235. Byers, S.A., et al., HEXIM2, a HEXIM1-related protein, regulates positive transcription elongation factor b through association with 7SK. J Biol Chem, 2005. 280(16): p. 16360-7.

236. Yik, J.H., et al., Compensatory contributions of HEXIM1 and HEXIM2 in maintaining the balance of active and inactive positive transcription elongation factor b complexes for control of transcription. J Biol Chem, 2005. 280(16): p. 16368-76.

237. Yik, J.H., et al., A human immunodeficiency virus type 1 Tat-like arginine-rich RNA-binding domain is essential for HEXIM1 to inhibit RNA polymerase II transcription through 7SK snRNA-mediated inactivation of P-TEFb. Mol Cell Biol, 2004. 24(12): p. 5094-105.

238. Hastie, C.J. and P.T. Cohen, Purification of protein phosphatase 4 catalytic subunit: inhibition by the antitumour drug fostriecin and other tumour suppressors and promoters. FEBS letters, 1998. 431(3): p. 357-61.

239. Horvatovich, P., et al., Multidimensional chromatography coupled to mass spectrometry in analysing complex proteomics samples. Journal of separation science, 2010. 33(10): p. 1421-37.

240. Elschenbroich, S., et al., Peptide separations by on-line MudPIT compared to isoelectric focusing in an off-gel format: application to a membrane-enriched 170

fraction from C2C12 mouse skeletal muscle cells. Journal of proteome research, 2009. 8(10): p. 4860-9.

241. Schirle, M., M.A. Heurtier, and B. Kuster, Profiling core proteomes of human cell lines by one-dimensional PAGE and liquid chromatography-tandem mass spectrometry. Molecular & cellular proteomics : MCP, 2003. 2(12): p. 1297-305.

242. Lawo, S., et al., HAUS, the 8-subunit human Augmin complex, regulates centrosome and spindle integrity. Curr Biol, 2009. 19(10): p. 816-26.

243. Nakada, S., et al., Non-canonical inhibition of DNA damage-dependent ubiquitination by OTUB1. Nature, 2010. 466(7309): p. 941-6.

244. Ewing, R.M., et al., Large-scale mapping of human protein-protein interactions by mass spectrometry. Mol Syst Biol, 2007. 3: p. 89.

245. Washburn, M.P., D. Wolters, and J.R. Yates, 3rd, Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol, 2001. 19(3): p. 242-7.

246. Wei, N., G. Serino, and X.W. Deng, The COP9 signalosome: more than a protease. Trends Biochem Sci, 2008. 33(12): p. 592-600.

247. Stancato, L.F., et al., The native v-Raf.hsp90.p50 heterocomplex contains a novel immunophilin of the FK506 binding class. J Biol Chem, 1994. 269(35): p. 22157- 61.

248. Tzivion, G., Z. Luo, and J. Avruch, A dimeric 14-3-3 protein is an essential cofactor for Raf kinase activity. Nature, 1998. 394(6688): p. 88-92.

249. Gingras, A.C., B. Raught, and N. Sonenberg, eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu Rev Biochem, 1999. 68: p. 913-63.

250. Chen, G.I. and A.C. Gingras, Affinity-purification mass spectrometry (AP-MS) of serine/threonine phosphatases. Methods, 2007. 42(3): p. 298-305.

251. Taylor, P., et al., Automated 2D peptide separation on a 1D nano-LC-MS system. J Proteome Res, 2009. 8(3): p. 1610-6.

252. Shevchenko, A., et al., In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc, 2006. 1(6): p. 2856-60.

253. Liu, G., et al., ProHits: integrated software for mass spectrometry-based interaction proteomics. Nature biotechnology, 2010. 28(10): p. 1015-7.

254. Kessner, D., et al., ProteoWizard: open source software for rapid proteomics tools development. Bioinformatics, 2008. 24(21): p. 2534-6. 171

255. Keller, A., et al., Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem, 2002. 74(20): p. 5383-92.

256. Nesvizhskii, A.I., et al., A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem, 2003. 75(17): p. 4646-58.

257. Choi, H., D. Fermin, and A.I. Nesvizhskii, Significance analysis of spectral count data in label-free shotgun proteomics. Mol Cell Proteomics, 2008. 7(12): p. 2373- 85.

258. Oliveros, J.C., VENNY. An interactive tool for comparing lists with Venn Diagrams. http://bioinfogp.cnb.csic.es/tools/venny/index.html, 2007.

259. Olhovsky, M., et al., OpenFreezer: a reagent information management software system. Nat Methods, 2011. 8(8): p. 612-3.

260. Hornbeck, P.V., et al., PhosphoSite: A bioinformatics resource dedicated to physiological protein phosphorylation. Proteomics, 2004. 4(6): p. 1551-61.

261. St-Denis, N. and A.C. Gingras, Mass spectrometric tools for systematic analysis of protein phosphorylation. Prog Mol Biol Transl Sci, 2012. 106: p. 3-32.

262. Yamada, T., et al., P-TEFb-mediated phosphorylation of hSpt5 C-terminal repeats is critical for processive transcription elongation. Molecular cell, 2006. 21(2): p. 227-37.

263. Fagerholm, A.E., D. Habrant, and A.M. Koskinen, Calyculins and related marine natural products as serine-threonine protein phosphatase PP1 and PP2A inhibitors and total syntheses of calyculin A, B, and C. Mar Drugs, 2010. 8(1): p. 122-72.

264. Gupta, V., et al., A model for binding of structurally diverse natural product inhibitors of protein phosphatases PP1 and PP2A. J Med Chem, 1997. 40(20): p. 3199-206.

265. Honkanen, R.E., et al., Characterization of natural toxins with inhibitory activity against serine/threonine protein phosphatases. Toxicon, 1994. 32(3): p. 339-50.

266. Wu, R., et al., Correct interpretation of comprehensive phosphorylation dynamics requires normalization by protein expression changes. Mol Cell Proteomics, 2011. 10(8): p. M111 009654.

267. Bodenmiller, B., et al., Phosphoproteomic analysis reveals interconnected system-wide responses to perturbations of kinases and phosphatases in yeast. Sci Signal, 2010. 3(153): p. rs4. 172

268. Tombacz, I., et al., The RNA Pol II CTD phosphatase Fcp1 is essential for normal development in Drosophila melanogaster. Gene, 2009. 446(2): p. 58-67.

269. Suh, M.H., et al., Fcp1 directly recognizes the C-terminal domain (CTD) and interacts with a site on RNA polymerase II distinct from the CTD. Proc Natl Acad Sci U S A, 2005. 102(48): p. 17314-9.

270. Kamenski, T., et al., Structure and mechanism of RNA polymerase II CTD phosphatases. Mol Cell, 2004. 15(3): p. 399-407.

271. Washington, K., et al., Protein phosphatase-1 dephosphorylates the C-terminal domain of RNA polymerase-II. J Biol Chem, 2002. 277(43): p. 40442-8.

272. Hausmann, S. and S. Shuman, Characterization of the CTD phosphatase Fcp1 from fission yeast. Preferential dephosphorylation of serine 2 versus serine 5. J Biol Chem, 2002. 277(24): p. 21213-20.

273. Dubois, M.F., et al., Phosphorylation of the RNA polymerase II largest subunit during heat shock and inhibition of transcription in HeLa cells. J Cell Physiol, 1994. 158(3): p. 417-26.

274. Moon, S.H., et al., Wild-type p53-induced phosphatase 1 dephosphorylates histone variant gamma-H2AX and suppresses DNA double strand break repair. J Biol Chem, 2010. 285(17): p. 12935-47.

275. Douglas, P., et al., Protein phosphatase 6 interacts with the DNA-dependent protein kinase catalytic subunit and dephosphorylates gamma-H2AX. Mol Cell Biol, 2010. 30(6): p. 1368-81.

276. Chowdhury, D., et al., gamma-H2AX dephosphorylation by protein phosphatase 2A facilitates DNA double-strand break repair. Mol Cell, 2005. 20(5): p. 801-9.

277. Roy, J. and M.S. Cyert, Cracking the phosphatase code: docking interactions determine substrate specificity. Sci Signal, 2009. 2(100): p. re9.

278. Knight, J.D., et al., A novel whole-cell lysate kinase assay identifies substrates of the p38 MAPK in differentiating myoblasts. Skelet Muscle, 2012. 2(1): p. 5.

279. Nash, P., et al., Multisite phosphorylation of a CDK inhibitor sets a threshold for the onset of DNA replication. Nature, 2001. 414(6863): p. 514-21.

280. Schweiger, R. and M. Linial, Cooperativity within proximal phosphorylation sites is revealed from large-scale proteomics data. Biol Direct, 2010. 5: p. 6.

281. Yachie, N., et al., In silico analysis of phosphoproteome data suggests a rich-get- richer process of phosphosite accumulation over evolution. Mol Cell Proteomics, 2009. 8(5): p. 1061-71. 173

282. Lee, J.S., E. Smith, and A. Shilatifard, The language of histone crosstalk. Cell, 2010. 142(5): p. 682-5.

283. Gerber, S.A., et al., Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc Natl Acad Sci U S A, 2003. 100(12): p. 6940-5.

284. Villen, J. and S.P. Gygi, The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry. Nat Protoc, 2008. 3(10): p. 1630- 8.

285. Kent, W.J., BLAT--the BLAST-like alignment tool. Genome Res, 2002. 12(4): p. 656-64.

286. Langmead, B., et al., Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol, 2009. 10(3): p. R25.

287. Vandesompele, J., et al., Accurate normalization of real-time quantitative RT- PCR data by geometric averaging of multiple internal control genes. Genome Biol, 2002. 3(7): p. RESEARCH0034.

288. Rasband, W.S., ImageJ, 1997-2008.

289. GraphPad Software, I., GraphPad Software, QuickCalcs, t test calculator., 2009.

290. Semizarov, D., et al., Specificity of short interfering RNA determined through gene expression signatures. Proc Natl Acad Sci U S A, 2003. 100(11): p. 6347- 52.

291. Sigoillot, F.D., et al., A bioinformatics method identifies prominent off-targeted transcripts in RNAi screens. Nat Methods, 2012. 9(4): p. 363-6.

292. Singh, S., A.S. Narang, and R.I. Mahato, Subcellular fate and off-target effects of siRNA, shRNA, and miRNA. Pharm Res, 2011. 28(12): p. 2996-3015.

293. Schultz, N., et al., Off-target effects dominate a large-scale RNAi screen for modulators of the TGF-beta pathway and reveal microRNA regulation of TGFBR2. Silence, 2011. 2: p. 3.

294. Petri, S., et al., Increased siRNA duplex stability correlates with reduced off- target and elevated on-target effects. RNA, 2011. 17(4): p. 737-49.

295. Sigoillot, F.D. and R.W. King, Vigilance and validation: Keys to success in RNAi screening. ACS Chem Biol, 2011. 6(1): p. 47-60.

296. Jackson, A.L., et al., Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol, 2003. 21(6): p. 635-7. 174

297. Sioud, M., Promises and challenges in developing RNAi as a research tool and therapy. Methods Mol Biol, 2011. 703: p. 173-87.

298. Kanasty, R.L., et al., Action and reaction: the biological response to sirna and its delivery vehicles. Mol Ther, 2012. 20(3): p. 513-24.

299. Naito, Y., et al., siDirect 2.0: updated software for designing functional siRNA with reduced seed-dependent off-target effect. BMC Bioinformatics, 2009. 10: p. 392.

300. Bramsen, J.B., et al., Improved silencing properties using small internally segmented interfering RNAs. Nucleic Acids Res, 2007. 35(17): p. 5886-97.

301. Jackson, A.L., et al., Position-specific chemical modification of siRNAs reduces "off-target" transcript silencing. RNA, 2006. 12(7): p. 1197-205.

302. Zhou, H., et al., Genome-scale RNAi screen for host factors required for HIV replication. Cell Host Microbe, 2008. 4(5): p. 495-504.

175

Appendices Appendix 1: Effect of siRNA directed depletion of PP4c on gene expression and RNA splicing

Work by Ginny Chen implicated PP4c in the regulation of transcription and splicing by identifying changes in mRNA transcription and alternate exon inclusion upon siRNA depletion of PP4c with or without epidermal growth factor (EGF) stimulation. To reiterate, Ginny observed a significant decrease in the expression of the AP-1 transcription factors JUNB and FOSB upon PP4c depletion in EGF stimulated cells by qRT-PCR (Figure 1-10), something she hypothesized could be mediated through interactions between PP4c-PP4R2-PP4R3A and the components of the DSIF complex SUPT4H and SUPT5H (Figure 1-7). Knockdown of SUPT5H produced a similar decrease in both JUNB and FOSB expression, indicating that it functions in the same pathway as PP4c to regulate EGF induction of gene expression. Prompted by these results, Ginny collaborated with the Blencowe lab to perform RNAseq analysis of four samples (siPP4c ± EGF and Control siRNA ± EGF). This approach was undertaken to generate unbiased lists of genes which demonstrate a change in either transcription or splicing in the absence of a functional PP4c holoenzyme. siRNA directed depletion of PP4c resulted in a significant alteration in the alternate exon inclusion of 95 genes, and the expression of several EGF inducible genes (as observed by RNAseq). Interestingly, Ginny observed an increase in 7SK RNA expression, a component of the 7SK snRNP inhibitor of P-TEFb (a regulator of the DSIF complex) [234-237] upon PP4c depletion, another possible mechanism by which PP4c could be mediating JUNB/FOSB expression. However, she was unable to discern whether PP4c regulation of these or other components of the transcription and splicing machineries (i.e. HTATSF-1, RNGTT, and DHX38) provided the mechanism whereby PP4c regulates mRNA transcription and splicing.

Here I set out to validate whether siRNA depletion of PP4c results in a consistent effect on mRNA transcription and alternate exon inclusion using several different siRNAs targeting PP4c (Ginny’s results were observed using only one siRNA – 176 explained below), a requisite before further investigation into what components of the mRNA transcription and splicing machineries PP4c regulates could be conducted. Once validated, the role of various components of the transcription and splicing machineries observed to interact with PP4c-PP4R2-PP4R3A would next be investigated by siRNA directed depletion (as was done for the DSIF component SUPT5H) to assess whether PP4c regulation of mRNA transcription and splicing is mediated through its interactions with these proteins.

A1.1 Materials and Methods A1.1.1 Cell culture and siRNA directed PP4c depletion

Hela cells were plated at 8 x 104 cells/well in a 6 well format (Greiner bio-one; Cat# 657160) in 2.5 mL of Dulbecco’s Modified Eagles Medium (high glucose) supplemented with 5% FBS, and 5% calf serum (no antibiotics). Twenty four hours later cells were transfected with 27nM siRNA targeting PP4c (Dharmacon; D-008486-01 (siPP4c01): 5´- GCACUGAGAUCUUUGACUA-3´; D-008486-03 (siPP4c03): 5´- GGAGCCGGCUACCUAUUUG-3´; D-008486-04 (siPP4c04): 5´- GACAAUCGACCGAAAGCAA-3´; D-008486-05 (siPP4c05): 5´- GCACUUAAGGUUCGCUAUC-3´; or non-targeting siRNAs Control38: 5´- AAGCAUCUGAGCUAAGUAGUA-3´; Control53: 5´- AAGCACUCACAUGGAGUCGUC-3´), using 3 µL Lipofectamine RNAiMAX transfection reagent (Invitrogen; Cat.# 13778), and 500 µL OptiMEM serum free media (Invitrogen; Cat# 31985) per well, as per the manufacturer’s directions. Twenty six hours post transfection media was aspirated and replaced with 3 mL Dulbecco’s Modified Eagles Medium (high glucose; no FBS, no calf serum, no antibiotics). Fifty hours post transfection cells were stimulated (or left un-stimulated) with 0.1µg/mL epidermal growth factor (PeproTech; Cat# AF-100-15), for 30 minutes. Cells harvested for RNA extraction were harvested by media aspiration followed by on plate lysis using 1mL TRI Reagent (Applied Biosystems; Cat# AM9738) and frozen at -80°C. Cells harvested for western blot were harvested after media aspiration and washing with PBS, by scraping with a rubber spatula. Cells were pelleted by centrifugation, and frozen at -80°C. 177

A1.1.2 Western blotting

Cell lysis buffer was 50 mM Hepes-KOH pH 8.0, 100 mM KCl, 2 mM EDTA, 0.1% NP40, 10% glycerol, 1 mM PMSF, 1 mM DTT and protease inhibitor cocktail (Sigma; P8340; 1:500). Cells were lysed on ice, subjected to one freeze-thaw cycle, and lysate cleared by centrifugation (20,800 x g, 20 minutes, 4°C). Lysate protein concentration was determined by Bradford assay. 20 µg of protein was resolved by SDS-PAGE, and transferred to nitrocellulose. Antibodies for PP4c were obtained from Bethyl Laboratories (A300-893A). Antibodies for α-4 (used as a loading control) were raised in rabbit (rabbit 2972) against a bacterially expressed GST- α-4 protein (IGBP1) and a kind gift from Dr. N. Sonenberg. Secondary antibody was ECL Rabbit IgG, HRP-Linked (GE Healthcare; Cat# NA934). Protein detection was facilitated using LumiGLO (Cell Signaling Technology; Cat# 7003).

A1.1.3 RNA isolation, reverse transcription, PCR, and qRT- PCR

Cellular RNA was extracted from TRI Reagent as per the manufacturer’s instructions using chloroform instead of bromochloropropane. After extraction, RNA was re- suspended to 100ng/µL in nuclease free H2O and assessed for quality by agarose gel electrophoresis. Reverse transcription was done using 650 ng of total RNA and SuperScript III Reverse transcriptase (Invitrogen; Cat# 18080) as per the kit directions, using 500ng of oligo dT12-18 (Invitrogen; Cat# 18418) and the following parameters: during step 2 samples were placed on ice for 10 minutes. In step three 1µL of RNasin Plus RNase inhibitor (Promega; Cat# N2615) was substituted for RNaseOUT (Invitrogen; Cat# 10777-019) to prevent RNA degradation. During step 5, samples were incubated at

50ºC for 1h. After reverse transcription cDNA was diluted 1:30 in nuclease free H2O for PCR assays. PCR was conducted using Expand High Fidelity PCR System (Roche; Cat# 11732650001) as per the kit instructions. PP4c primers were 5´- GATCTTTGACTACCTCAGCCTG-3´ and 5´-ACCACGTCACTGCCAAATAG-3´. PCR sample composition was as directed by the manufacturer; and included 1µL of each forward and reverse primers (from 5µM stocks), and 30µL of diluted (1:30) cDNA, for a final reaction volume of 50µL. PCR conditions were 94ºC for 2 min; followed by 30 178 cycles of 94ºC 15 sec, 52ºC 1 min, 72ºC 30 sec; after PCR cycling samples were held at 72ºC for 7 min. Ten microliters of PCR product was resolved using agarose gel electrophoresis and visualized using ethidium bromide staining to assess PP4c knockdown efficiency at the mRNA level. qRT-PCR was conducted using SYBR Green PCR Master Mix (Applied Biosystems; Cat#4367659) as per the kit instructions. Primers are described in table A1-1. qRT-PCR sample composition was as directed by the manufacturer; and included 0.45µL of each forward and reverse primers (from 5µM stocks), and 5µL of diluted (1:30) cDNA, for a final reaction volume of 20µL. qRT-PCR was conducted on Applied Biosystems 7500 Fast Real-Time PCR System. qRT-PCR conditions were 95ºC for 10 min; followed by 40 cycles of 95ºC 15 sec, 60ºC 1 min; after PCR cycling samples were heated to 95ºC 15 sec, then cooled to 60ºC 1 min, before heating to 95ºC for generation of the amplicon melting curve. Sample fluorescence was measured for each of the 40 cycles during the 60ºC 1 min step. The temperature ramp rate between all steps (including for generation of the PCR amplicon melting curve) was set at 100%. The presence of only one PCR product was ensured by the presence of only one major peak in the amplicon melting curve; in addition 10L of PCR product was resolved using agarose gel electrophoresis and visualized using ethidium bromide staining.

179

Table A1-1. PCR primers used for gene expression analyses by qRT-PCR. PCR Primers Gene Forward Reverse

JUNB 5´-CCGGATGTGCACTAAAATGGA-3´ 5´-TCAGGAGTTTGTAGTCGTGTAGAGAGA-3´ FOSB 5´-CTGCCAACCACAATTCAATGA-3´ 5´-AGATGCAAAATACAAAAAGAGAACCA-3´ NR4A1 5´-GGCATGGTGAAGGAAGTTGT-3´ 5´-CGGAGAGCAGGTCGTAGAAC-3´ ATF3 5´-CGCCTTTCATCTGGATTCTACAA-3´ 5´-GACACTGCTGCCTGAATCCTAA-3´ NRAS 5´-AAATACGCCAGTACCGAATGAAA-3´ 5´-GCAATCCCATACAACCCTGAGT-3´ NOTCH2 5´-GTGCAGGAATTGGAAAGTTGGA-3´ 5´-GGCCGCTTCAGAGGAAAAG-3´ 7SK 5´-CTTCGGTCAAGGGTATACGAGT-3´ 5´-ATGCAGCGCCTCATTTGGATGT-3´ EGFR 5´-GGAGAACTGCCAGAAACTGACC-3´ 5´-GCCTGCAGCACACTGGTTG-3´ SPRY4 5´-GGTATTTTGTTTTCCCCTCTAATGAGA-3´ 5´-CCTCATGCACTTCCAGTTTCAC-3´ KAT2B 5´-CATGAGTGAACGCCTCAAGA-3´ 5´-TAGGCACACTGCTTGGTGAG-3´ CDK6 5´-TGAACCAAAATGCCACATACACT-3´ 5´-TTCGGCCTTTCGCATAGG-3´ MLLT1 5´-CCCCAGTGCAAATCTCTGTGT-3´ 5´-TGGGCAAGAAAGGTATTCGATAC-3´ SOX18 5´-CTCTGCTCTCTCATACGCGTGTA-3´ 5´-GCCCAGAAGCCCAGGAA-3´ KLF6 5´-TGGGAGCTGGAGAGGATGTC-3´ 5´-TGTCAGTCCTTGGAGAAGAGTATTTG-3´ TAF13 5´-GGCAATGTCAATTGGAAGACAA-3´ 5´-CCTTGGGTCCTTTCGAATCA-3´ HPRT1 5´-GACCAGTCAACAGGGGACATA-3´ 5´-CCAAGGAAAGCAAAGTCTGC-3´

180

A1.1.4 RNAseq analysis of alternate exon inclusion

RNA sequencing was performed by Illumina Inc. using polyA+ RNA isolated from total RNA extracts. Briefly, RNA sequencing reads from siPP4c ± EGF and Control53 ± EGF were computationally aligned to a database of expressed sequence tags and cDNA- derived alternate splicing junctions using the BLAST like alignment tool (BLAT; [285]). Reads were considered properly aligned if at least 48 of 50 nucleotides matched a splicing junction and at least 5 nucleotides mapped to each of the two exons forming the splice junction. Alternate exon inclusion was calculated as the percentage of RNA sequence reads that mapped alternate exon inclusion events out of the total number of alternate exon inclusion or exclusion events. At least 20 reads were needed for each splice junction for the results to be considered reliable. Sequenced reads were computationally aligned to the human genome (hg19) using Bowtie v0.12.7 [286]. Exons that are differentially spliced upon PP4c knockdown were identified using statistical measures and genes were selected for follow up analyses if the alternate splicing event encompassed a defined or conserved domain, allowing functional consequences to be inferred. RNAseq analyses were conducted by Xinchen Wang in collaboration with the Blencowe lab. RNA samples and genes selected for follow up analysis were prepared/identified by Ginny Chen.

A1.1.5 Splicing Assay

Aliquots from RNA stocks (100ng/µL) were diluted to a final concentration of 2ng/µL in nuclease free H2O. NUMA1 and WBSCR1 alternative exon inclusion were assessed using QIAGEN OneStep RT-PCR kit (Cat# 210212). PCR primers were designed to span the alternate exon of interest such that mRNAs in which the exon had been included would generate a larger PCR product than mRNAs in which it had been excluded. Primers were: NUMA1 5´-CTGCGGGCAGAGAAGGCCA-3´ and 5´- CCAGTTTCTTACTCAGTTCTTCCAC-3´; WBSCR1 5´- CTGTTGGGCGATCGGTCACT-3´ and 5´-TCTGTGGGTTCTCTGAAATCCATG-3´.

RT-PCR sample composition was: 0.8µL nuclease free H2O; 2µL QIAGEN OneStep RT- PCR 5x Buffer; 0.4µL 10mM dNTP mix; 0.6µL of each forward and reverse primers (from 10µM stocks); 0.5µL QIAGEN OneStep RT-PCR Enzyme Mix; 0.1µL of 181

RiboLock RNase inhibitor (Fermentas; Cat# EO0381), and 5µL of diluted (2ng/µL) RNA per sample, for a final reaction volume of 10µL. RT-PCR conditions were 50ºC 30 min, 95ºC 15 min; followed by 27 cycles of 94ºC 40 sec, 52ºC 50 sec (for NUMA1) or 55ºC for 30 sec (for WBSCR1), 72ºC 60 sec; after PCR cycling samples were held at 72ºC for 10 minutes. Ten microliters of PCR product was resolved using agarose gel electrophoresis and visualized using ethidium bromide staining to evaluate alternate exon inclusion upon cellular treatment. Digital images of gels were captured using a BIO - RAD Gel Doc XR+ system, with exposure time optimized for intense bands, and ensuring that no image pixels were saturated.

A1.1.6 Data analysis qRT-PCR data was analyzed using Gene X-2 Gene Expression Analysis for iCycler iQ Real-Time PCR Detection System software [287]. JUNB and FOSB expression was normalized to Hypoxanthine phosphoribosyltransferase 1 (HPRT1) expression before inter sample comparison [287]. Alternate exon inclusion upon siRNA and EGF treatment was analyzed using ImageJ software ([288]; 12/15/2011). Briefly, the intensities of the top (alternate exon included; IntTop), and bottom (alternate exon excluded; IntBot), PCR amplicon bands were measured from digital images and the background intensity (Bac) subtracted from each of the measurements (IntTop – Bac = IntTopc; IntBot – Bac =

IntBotc). The percentage alternate exon inclusion was then calculated as: Percentage alternate exon inclusion = (IntTopc/(IntTopc+ IntBotc)) x 100%.

A1.2 Results A1.2.1 Effect of PP4c depletion on gene expression

As a first step into assessing whether PP4c is involved in the regulation of RNA transcription and splicing, I sought to reproduce Ginny Chen’s results showing that PP4c depletion with siPP4c-N3 (identical sequence to siPP4c04) results in a decrease in JUNB and FOSB expression upon EGF stimulation as compared to a non-targeting siRNA Control53 (Figure 1-10). Additionally, I sought to further validate her results by using three other siRNAs targeting PP4c (siPP4c01, siPP4c03, and siPP4c05) and another non- 182 targeting siRNA (Control38), to reduce the probability that what she was observing was due to siRNA off target effects and not PP4c depletion itself. This was important as Ginny’s results were garnered using what were believed to be two unique siRNAs targeting PP4c, however upon closer inspection they were identified to contain a majority of the same seed sequence (nucleotides 2-8 from the 5´ end, shown in bold), and in fact were almost entirely the same (sequence overlap shown in italics; siPP4c-N1: 5´- AAGACAAUCGACCGAAAGCAA-3´ and siPP4c-N3: 5´- GACAAUCGACCGAAAGCAA-3´), indicating that any off target effects generated by the two would likely be similar. Upon inspection, all four siRNAs were shown to deplete PP4c protein and mRNA levels similarly (Figure A1-1); however, siPP4c03 was observed to result in additional cell death and therefore was not used for gene expression or splicing analyses. 183

184

Figure A1-1. PP4c protein and mRNA levels after siRNA and EGF treatment. Hela cells were treated with one of four siRNAs targeting PP4c (siPP4c01, 03, 04, 05) or one of two non-targeting siRNAs (Control38-53) and the effect on (A) PP4c protein and (B) mRNA levels assessed in the presence or absence of EGF stimulation. (C) Ribosomal RNA was used to assess RNA quality. PP4c protein levels were assessed by western blot using 20µg of cleared cell lysate (PP4c is the bottom band of the doublet). PP4c mRNA levels were assessed by reverse transcription, PCR assay, and agarose gel electrophoresis; the PP4c amplicon is 231 bp. Ribosomal RNA was visualized by agarose gel electrophoresis and ethidium bromide staining. All three siRNAs were shown to deplete PP4c protein and mRNA levels similarly. RT(-) is a negative control lacking the reverse transcriptase enzyme and is used to check for DNA carryover during the RNA extraction procedure. RT H2O is a negative control lacking RNA template and is used to look for sample contamination during the reverse transcription and PCR reactions. siPP4c03 resulted in a low protein yield (12µg), due to cell death, and was not used further.

185

However, an analysis of the effect of each of the three PP4c targeting siRNAs and two non-targeting controls on JUNB and FOSB expression in the presence or absence of EGF stimulation, led to the conclusion that while all three PP4c siRNAs resulted in similar levels of PP4c protein and mRNA depletion their effect on JUNB and FOSB expression was markedly different when assessed by qRT-PCR assay (Figures A1-2, A1- 3; siPP4c03 omitted due to toxicity).

186

Figure A1-2. FOSB expression upon siRNA and epidermal growth factor (EGF) treatment. Hela cells were treated with one of three siRNAs targeting PP4c (siPP4c01, 04, 05) or one of two non-targeting siRNAs (Control38, 53) and the effect on FOSB expression assayed by qRT-PCR in the presence or absence of EGF stimulation. FOSB expression was normalized to HPRT1 expression. Error bars are 1 standard deviation of the mean. A) Biological Replicate 1. B) Biological Replicate 2. The effect of each of the three PP4c siRNAs on FOSB expression was markedly different, while the effect of the two non-targeting controls was observed to be similar. siPP4c04 resulted in the greatest decrease in FOSB expression as compared to either non-targeting control. 187

Figure A1-3. JUNB expression upon siRNA and epidermal growth factor (EGF) treatment. Hela cells were treated with one of three siRNAs targeting PP4c (siPP4c01, 04, 05) or one of two non-targeting siRNAs (Control38, 53) and the effect on JUNB expression assayed by qRT-PCR in the presence or absence of EGF stimulation. JUNB expression was normalized to HPRT1 expression. Error bars are 1 standard deviation of the mean. A) Biological Replicate 1. B) Biological Replicate 2. The effect of each of the three PP4c siRNAs on JUNB expression was markedly different and the effect of the two non-targeting controls was not reproducibly observed to be similar. siPP4c04 resulted in the greatest decrease in JUNB expression as compared to either non-targeting control. 188

Although the 3 siRNAs used (outlined in Figures A1-2, A1-3) did not lead to a consistent effect on either JUNB or FOSB expression (as assessed by qRT-PCR assay), the effect of each PP4c targeting siRNA was consistent across biological replicates, leading to the conclusion that the observed differences between each siRNA were real (possibly due to off target effects) and not the result of technical variation. Of note, while the level of PP4c knockdown across siPP4c01, 04 and 05 treated samples appears to be equivalent as monitored by both qRT-PCR (not shown) and western blot assays (Figure A1-1), the fact that PP4c is an enzyme does allow for the possibility that subtle differences in the level of PP4c knockdown (that cannot be discerned visually) could account for the observed difference in JUNB and FOSB expression (Figures A1-2, A1-3). Notably however, the effect of siPP4c04 on FOSB expression remained consistent with what had been previously observed by Ginny Chen by qRT-PCR assay, (significant down regulation of FOSB expression upon PP4c depletion and EGF stimulation as compared to Control53; Unpaired t-test: Bio Rep 1: p≤ 0.001, Average FOSB expression: siPP4c04+EGF = 33.31 ± 5.10, n = 3; Control53+EGF = 202.73 ± 12.21, n = 3; Bio Rep 2: p≤ 0.001, Average FOSB expression: siPP4c04+EGF = 42.26 ± 10.75, n = 3; Control53+EGF = 195.19 ± 9.47, n = 3). In addition, the level of FOSB expression (upon PP4c depletion and EGF stimulation) was observed to be significantly decreased compared to an additional non-targeting siRNA Control38 (Unpaired t-test: Bio Rep 1: p≤ 0.001, Average FOSB expression: siPP4c04+EGF = 33.31 ± 5.10, n = 3; Control38+EGF = 187.23 ± 13.59, n = 3; Bio Rep 2; p≤ 0.001, Average FOSB expression: siPP4c04+EGF = 42.26 ± 10.75, n = 3; Control38+EGF = 154.66 ± 15.47, n = 3). The effect of PP4c depletion on JUNB expression however, was not so clear cut.

A qRT-PCR assessment of PP4c depletion using siPP4c04 ± EGF treatment, on the expression of genes revealed to be differentially expressed upon PP4c depletion by RNAseq, (Table A1-2; RNAseq was performed by Xinchen Wang of the Blencowe lab), revealed that 7/15 genes exhibit expression profiles consistent with what was observed by RNAseq (± EGF in both biological replicates), 3/15 genes match the RNAseq data ± EGF in at least one biological replicate, 4/15 match the RNAseq data only + or – EGF, and only 1 gene does not match the RNAseq data in either case (Table A1-3). Raw data can been found in Table A1-4. However, as the effect of PP4c depletion using siPP4c04 on 189

JUNB and FOSB expression could not be validated using other PP4c targeting siRNAs, it remained questionable whether depletion of PP4c using these siRNAs would result in a reproducible effect on the expression of the genes presented in table A1-2. To investigate this possibility, the expression of ATF3 and NR4A1, two genes observed to be significantly down regulated upon PP4c depletion in an EGF dependant manner (similar to what was observed for JUNB and FOSB; Table A1-2) was assayed using siPP4c01, siPP4c05, and Control38 (data not shown). In this case, as was observed for both JUNB and FOSB, depletion of PP4c using different siRNAs targeting PP4c resulted in a wide ranging effect on ATF3 and NR4A1 expression (siPP4c01 resulted in a significant up- regulation in ATF3 expression upon EGF stimulation while siPP4c05 resulted in no effect; siPP4c01 resulted in a significant up-regulation in NR4A1 expression upon EGF stimulation while siPP4c05 resulted a significant down regulation). Due to these inconsistencies the effect of these siRNAs on the expression of other genes outlined in table A1-2 was not assessed.

190

Table A1-2. RNAseq genes demonstrated to be differentially expressed upon siPP4c ± EGF treatment selected for validation by qRT-PCR. Hela cells were treated with siPP4c04 or Control53 and the effect on gene expression in the presence or absence of EGF stimulation measured by RNAseq. A subset of genes from the total RNAseq gene lists were selected for validation based on having a high confidence of being differentially expressed and either involved in EGF signaling or exhibiting expression profiles similar to JUNB or FOSB upon siRNA ± EGF treatment. RNAseq analyses were conducted by Xinchen Wang in collaboration with the Blencowe lab.

Fold change in gene expression (siRNA/Control) Gene siPP4c04/Control53 P-Value siPP4c04 + EGF/ Control53 + EGF P-Value

JUNB ND NA -1.70 2.70E-04 FOSB ND NA -2.31 1.30E-06 NR4A1 ND NA -1.78 1.40E-04 ATF3 ND NA -1.91 3.50E-05 NRAS -1.98 8.00E-06 -1.90 4.80E-05 NOTCH2 -2.01 9.70E-07 -2.08 8.30E-06 7SK 1.94 8.00E-04 2.13 5.90E-04 EGFR -1.91 7.00E-06 -1.83 7.60E-05 SPRY4 2.18 6.70E-04 ND NA KAT2B -2.60 4.20E-07 -2.53 1.60E-06 CDK6 -2.26 4.20E-08 -2.18 2.40E-06 MLLT1 -1.92 2.60E-05 -1.86 9.10E-05 SOX18 -2.87 1.60E-04 ND NA KLF6 ND NA -1.64 3.60E-04 TAF13 -2.46 1.10E-06 -2.50 9.20E-07 *ND - No significant difference detected **NA - Not applicable

191

Table A1-3. qRT-PCR validation of genes observed to be differentially expressed upon PP4c depletion (by RNAseq) in the presence or absence of epidermal growth factor (EGF) treatment. Briefly, Hela cells were treated with siPP4c04 or non-targeting siRNA Control53 and the effect on gene expression for those genes identified to be differentially expressed by RNAseq assessed by qRT-PCR in the presence or absence of EGF stimulation. Gene expression was normalized to HPRT1 expression. The fold change in gene expression (siPP4c ± EGF/Control53 ± EGF) is listed for Biological Replicate1; Biological Replicate2. Where siRNA/Control was < 1 the inverse was taken to get the negative fold difference in gene expression. Genes identified to be expressed in fashion consistent to what was observed by RNAseq in both biological replicates are shown in green; genes where data from only 1 biological replicate matches what was observed by RNAseq are shown in orange; genes which do not match what was observed by RNAseq are shown in red. Overall, 7/15 genes match the RNAseq data in the presence or absence of EGF treatment in both biological replicates (green in both ± EGF), 3/15 genes match the RNAseq data in the presence or absence of EGF treatment in at least one biological replicate (orange in + or – EGF, green in the other), 4/15 genes match the RNAseq data only in the presence or absence of EGF treatment (red in + or – EGF, green in the other), and only 1 gene does not match the RNAseq data in either case (red in both ± EGF).

192

Fold change in gene expression (siRNA/Control) Gene siPP4c04/Control53 siPP4c04 + EGF/ Control53 + EGF

JUNB 1.55; 1.66 -1.14; -1.64 FOSB 5.50; 7.63 -6.08; -4.61 NR4A1 1.35; 1.73 -2.41; -2.21 ATF3 1.42; 3.29 -3.17; -2.98 NRAS -2.43; -2.60 -2.81; -2.52 NOTCH2 -3.56; -3.94 -3.74; -3.22 7SK 2.91; 5.35 2.58; 8.02 EGFR -2.05; -1.99 -2.41; -1.59 SPRY4 5.33; 17.78 2.09; 8.96 KAT2B -3.91; -4.39 -4.76; -5.34 CDK6 -2.92; -3.42 -3.25; -3.22 MLLT1 -2.45; -2.82 -2.50; -2.62 SOX18 -4.07; -6.06 -9.58; -6.82 KLF6 1.10; 1.40 -2.87; -1.37 TAF13 -1.16; 1.01 -1.70; -1.21

193

Table A1-4. Raw data from qRT-PCR validation of genes observed to be differentially expressed upon PP4c depletion (by RNAseq) in the presence or absence of epidermal growth factor (EGF) treatment. Briefly, Hela cells were treated with siPP4c04 or non-targeting siRNA Control53 and the effect on gene expression for those genes identified to be differentially expressed by RNAseq assessed by qRT-PCR in the presence or absence of EGF stimulation. Gene expression was normalized to HPRT1 expression. (A) The average gene expression and standard deviation of the mean (stdev) in the absence of EGF treatment is listed for Biological Replicate1; Biological Replicate2 (BioRep1; BioRep2). (B) The average gene expression and stdev, in the presence of EGF treatment is listed for Biological Replicate1; Biological Replicate2. In all cases the number of technical replicates per biological replicate was 3. P-values from an unpaired t-test comparing gene expression upon PP4c depletion using siPP4c04 to gene expression upon Control53 treatment in the presence or absence of EGF treatment is also listed for Biological Replicate1; Biological Replicate2. T- tests were conducted using GraphPad software [289]. P > 0.05 is not statistically significant; 0.01 ≤ P ≤ 0.05 is statistically significant; 0.001 ≤ P < 0.01 is very statistically significant; 0.0001 ≤ P < 0.001 is extremely statistically significant.

194

A Average gene expression ± stdev (BioRep1; BioRep2) Gene siPP4c04 Control53 P-Value (BioRep1; BioRep2)

JUNB 1.55±0.25; 1.66±0.46 1.00±0.06; 1.00±0.09 0.0215; 0.0713 FOSB 5.50±0.98; 7.71±2.32 1.00±0.08; 1.01±0.12 0.0014; 0.0075 NR4A1 1.35±0.06; 1.73±0.09 1.00±0.04; 1.00±0.04 0.0016; 0.0003 ATF3 1.42±0.08; 3.29±0.45 1.00±0.08; 1.00±0.05 0.0037; 0.0010 NRAS 1.07±0.05; 1.00±0.03 2.61±0.27; 2.60±0.14 0.0007; 0.0001 NOTCH2 1.02±0.03; 1.00±0.07 3.64±0.08; 3.94±0.30 0.0001; 0.0001 7SK 2.91±0.17; 9.69±2.44 1.00±0.06; 1.81±0.09 0.0001; 0.0050 EGFR 1.17±0.11; 1.00±0.28 2.40±0.22; 1.99±0.13 0.0010; 0.0057 SPRY4 5.33±0.54; 17.78±4.33 1.00±0.15; 1.00±0.54 0.0002; 0.0026 KAT2B 1.01±0.12; 1.42±0.38 3.95±0.28; 6.24±0.73 0.0001; 0.0005 CDK6 1.02±0.04; 1.00±0.04 2.98±0.14; 3.42±0.19 0.0001; 0.0001 MLLT1 1.00±0.05; 1.05±0.04 2.45±0.30; 2.97±0.36 0.0013; 0.0008 SOX18 1.27±0.35; 1.31±0.14 5.18±0.90; 7.95±1.63 0.0022; 0.0022 KLF6 1.10±0.05; 1.40±0.21 1.00±0.22; 1.00±0.09 0.4855; 0.0410 TAF13 1.00±0.11; 1.12±0.13 1.67±0.27; 1.11±0.13 0.0179; 0.9295

B Average gene expression ± stdev (BioRep1; BioRep2) Gene siPP4c04 + EGF Control53 + EGF P-Value (BioRep1; BioRep2)

JUNB 6.75±0.51; 5.93±0.92 7.73±0.42; 9.75±1.71 0.0620; 0.0275 FOSB 33.31±5.10; 42.26±10.75 202.73±12.21; 195.19±9.47 0.0001; 0.0001 NR4A1 20.09±1.45; 19.78±1.31 48.41±2.14; 43.87±3.93 0.0001; 0.0005 ATF3 7.48±0.62; 17.02±0.93 23.73±0.1.94; 50.86±3.48 0.0002; 0.0001 NRAS 1.00±0.10; 1.08±0.09 2.81±0.29; 2.73±0.09 0.0006; 0.0001 NOTCH2 1.00±0.15; 1.13±0.33 3.74±0.34; 3.64±0.09 0.0002; 0.0002 7SK 3.23±0.31; 8.02±0.90 1.25±0.10; 1.00±0.07 0.0005; 0.0002 EGFR 1.00±0.11; 1.08±0.16 2.41±0.41; 1.72±0.26 0.0047; 0.0250 SPRY4 6.46±0.71; 18.46±3.68 3.09±0.44; 2.06±0.55 0.0022; 0.0016 KAT2B 1.00±0.11; 1.00±0.13 4.76±0.27; 5.24±0.67 0.0001; 0.0004 CDK6 1.00±0.05; 1.11±0.17 3.25±0.15; 3.04±0.40 0.0001; 0.0016 MLLT1 1.08±0.04; 1.00±0.10 2.71±0.32; 2.62±0.07 0.0010; 0.0001 SOX18 1.00±0.24; 1.00±0.18 9.58±3.43; 6.82±1.59 0.0125; 0.0033 KLF6 1.15±0.39; 3.52±0.60 3.31±0.15; 4.83±0.59 0.0009; 0.0543 TAF13 1.17±0.12; 1.00±0.27 1.99±0.37; 1.21±0.17 0.0228; 0.3179

195

In summary, while I was able to reproduce the decrease in FOSB expression (observed by Ginny Chen using siPP4c04 + EGF, both by RNAseq and qRT-PCR; Table A1-2, Figure 1-10) I was unable to achieve as significant a decrease in JUNB expression upon siPP4c04 + EGF treatment as was observed by her (Figures 1-10, A1-2, A1-3).. Additionally, I was unable to reproduce these results using several other siRNAs targeting PP4c (siPP4c01, siPP4c05), nor could I reproduce the effect of siPP4c04 on ATF3 or NR4A1 expression using these alternate siRNAs. This raises important questions as to whether the effect of siPP4c04 on JUNB and FOSB expression is due to PP4c depletion or some unknown siRNA off target effect.

While it appears that the RNAseq data generated by Xinchen Wang can be validated by qRT-PCR if the same siRNA that was used to generate it is used in these analyses, it seems unlikely that the alternate siRNAs (siPP4c01, siPP4c05) will be useful as they could not reproduce the effect of PP4c depletion using siPP4c04 on JUNB, FOSB, ATF3, or NR4A1 expression; therefore other methods such as a rescue experiment will likely need be performed to validate if the observed effect of PP4c depletion using siPP4c04 on gene expression is due to PP4c depletion itself or some unknown siRNA off target effect. Indeed, even the alternate control (Control38) did not produce the same effect on 7SK RNA expression as was observed with Control53 (PP4c depletion with siPP4c04 resulted in a significant up-regulation in 7SK RNA as compared to Control53, while no significant difference existed between siPP4c04 and Control38; data not shown), indicating that additional follow up experiments are needed before any affect on gene transcription can be attributed to PP4c (i.e. through regulation of P-TEFb).

A1.2.2 Effect of PP4c depletion on RNA splicing

While the effect of PP4c depletion on JUNB, FOSB (Figures A1-2, A1-3), ATF3, or NR4A1 (data not shown) expression using siPP4c01 and siPP4c05 was, in most cases, not consistent with what was observed upon PP4c depletion using siPP4c04, I was still interested in determining whether the effect of PP4c depletion using these alternate siRNAs would be consistent when RNA splicing was evaluated. To this effect, I used RT-PCR to examined the outcome of siPP4c01, 04, 05, and Control38 and 53 treatment on alternate exon inclusion in the genes NUMA1 (nuclear mitotic apparatus protein 1; 196

Figures A1-4, A1-5) and WBSCR1 (Williams-Beuren syndrome chromosome region1; Figures A1-4, A1-6) two of the 17 genes selected for follow up analysis from the 95 total genes identified by Xinchen and Ginny to demonstrate differential alternate exon inclusion upon siPP4c04 and EGF treatment by RNAseq analysis.

197

Table A1-5. RNAseq genes demonstrated to be differentially spliced upon siPP4c ± EGF selected for validation by RT-PCR assay. Hela cells were treated with siPP4c04 or Control53 and the effect on alternate exon inclusion in the presence or absence of EGF stimulation measured by RNAseq. A subset of genes from the total RNAseq gene lists were selected for validation based on having a high confidence of being differentially spliced, and if the alternate splicing event encompassed a defined or conserved domain so that functional consequences could be inferred. Only the two of these genes analyzed by RT-PCR (out of the 17 selected for follow up validation), are listed. Absolute Percent Difference in Alternate Exon Inclusion upon siRNA depletion of PP4c Gene |Control53 - siPP4c04| P-Value |Control53 + EGF - siPP4c04 + EGF| P-Value

NUMA1 33.56% 7.80E-13 27.80% 1.54E-08 WBSCR1 10.54% 1.70E-08 ND NA

*ND - No significant difference detected (<10% difference in alternate exon inclusion upon PP4c depletion) **NA - Not applicable

198

199

Figure A1-4. NUMA1 and WBSCR1 alternate exon inclusion after siRNA and EGF treatment. Hela cells were treated with one of three siRNAs targeting PP4c (siPP4c01, 04, 05) or one of two non-targeting siRNAs (Control38-53) and the effect on (A) NUMA1 and (B) WBSCR1 alternate exon inclusion assessed by RT-PCR assay in the presence or absence of EGF stimulation. NUMA1 alternate exon inclusion results in a 400 base pair (bp) PCR product, alternate exon exclusion results in a 358bp PCR product. WBSCR1 alternate exon inclusion results in a 293bp PCR product, alternate exon exclusion results in a 233bp product. PCR amplicons were visualized by agarose gel electrophoresis and ethidium bromide staining. Alternate exon inclusion upon siRNA and EGF treatment was analyzed using ImageJ software [288]. Briefly, as shown for WBSCR1, (red boxes) the intensities of the top (alternate exon included (1); IntTop), and bottom (alternate exon excluded (2); IntBot), PCR amplicon bands were measured from digital images, and the background intensity (3) subtracted from each of the measurements (IntTopc, IntBotc). The percentage alternate exon inclusion was then calculated as: Percentage alternate exon inclusion = (IntTopc/(IntTopc+ IntBotc)) x 100%. RT(-) is a negative control lacking the reverse transcriptase enzyme and is used to check for DNA carryover during the RNA extraction procedure. RT H2O is a negative control lacking RNA template and is used to look for sample contamination during reverse transcription and PCR analysis. In this biological replicate no PCR product was observed upon Control53 + EGF treatment. 200

201

Figure A1-5. NUMA1 alternate exon inclusion after siRNA and EGF treatment. Hela cells were treated with one of three siRNAs targeting PP4c (siPP4c01, 04, 05) or one of two non-targeting siRNAs (Control38-53) and the effect on NUMA1 alternate exon inclusion assessed by RT-PCR assay in the presence or absence of EGF stimulation. PCR amplicons were visualized by agarose gel electrophoresis and ethidium bromide staining. Alternate exon inclusion upon siRNA and EGF treatment was analyzed using ImageJ software [288]. Percentage alternate exon inclusion = (IntTopc/(IntTopc+ IntBotc)) x 100%. A) Biological Replicate 1. B) Biological Replicate 2. Error bars are 1 standard deviation of the mean. In biological replicate 1, samples are only analyzed in technical duplicates so error bars are not shown. In biological replicate 2, no PCR product was observed upon Control53 + EGF treatment. The effect of each of the three PP4c siRNAs on NUMA1 alternate exon inclusion was markedly different, as was the effect of the two non-targeting controls. A comparison of siPP4c04 and Control53 revealed results consistent with that observed by RNAseq (BioRep1: |Control53 – siPP4c04| = 26.83% difference; |Control53+EGF – siPP4c04+EGF| = 29.29% difference; BioRep2: |Control53 – siPP4c04| = 32.66% difference).

202

203

Figure A1-6. WBSCR1 alternate exon inclusion after siRNA and EGF treatment. Hela cells were treated with one of three siRNAs targeting PP4c (siPP4c01, 04, 05) or one of two non-targeting siRNAs (Control38-53) and the effect on WBSCR1 alternate exon inclusion assessed by RT-PCR assay in the presence or absence of EGF stimulation. PCR amplicons were visualized by agarose gel electrophoresis and ethidium bromide staining. Alternate exon inclusion upon siRNA and EGF treatment was analyzed using

ImageJ software [288]. Percentage alternate exon inclusion = (IntTopc/(IntTopc+

IntBotc)) x 100%. A) Biological Replicate 1. B) Biological Replicate 2. Error bars are 1 standard deviation of the mean. In biological replicate 1, samples are only analyzed in technical duplicates so no error bars are shown. In biological replicate 2, no PCR product was observed upon Control53 + EGF treatment. The effect of siPP4c01 and 04 on WBSCR1 alternate exon inclusion were markedly different from that observed using siPP4c05. A comparison of siPP4c04 and Control53 revealed results consistent with that observed by RNAseq (BioRep1: |Control53 – siPP4c04| = 12.25% difference; |Control53+EGF – siPP4c04+EGF| = 8.66% difference; BioRep2: |Control53 – siPP4c04| = 10.43% difference).

204

Again, as was observed for JUNB and FOSB expression (Figures A1-2, A1-3), siRNA directed depletion of PP4c using each of the 3 siRNA outlined above did not lead to a consistent effect on either NUMA1 or WBSCR1 alternate exon inclusion (as assessed by RT-PCR assay), however the effect of each siRNA on NUMA1 or WBSCR1 alternate exon inclusion was consistent across biological replicates (Figures A1-5, A1-6), leading to the conclusion that the observed differences in NUMA1 or WBSCR1 alternate exon inclusion with each siRNA are real and likely due to off target effects, not technical variation. In this case too, it is interesting to note that the siRNAs themselves also appear to have an effect not only on NUMA1 splicing but NUMA1 expression (Figure A1-4), with siPP4c04 exhibiting the least amount of gene expression (faintest PCR bands) and siPP4c05 the most, further evidence that the effect of siRNA on gene expression may be attributable to off target effects and not PP4c depletion itself. However, in this case it must be noted that gene expression was not normalized to a reference gene as was done in the qRT-PCR assays so the apparent differences in gene expression with each of the siRNAs targeting PP4c could be due to sample loading errors. However, as the loading of technical replicates was consistent I would argue against this. Lastly, consistent with the results obtained for FOSB (Figure A1-2) and the majority of the RNAseq genes (Table A1-3), the effect of siPP4c04 on NUMA1 and WBSCR1 alternate exon inclusion as compared to Control53 (Figures A1-5, A1-6), remained consistent with what was observed by RNAseq analysis (Table A1-5).

In summary, while I was able to reproduce the effect of siPP4c04 directed PP4c depletion on FOSB expression observed by Ginny Chen (Figure A1-2), and the majority of the genes analyzed by RNAseq (Table A1-3), I was unable to validate these results using several other siRNAs targeting PP4c (Figures A1-2, A1-3; data from ATF3 and NR4A1 not shown). Additionally, I was unable to reproduce the effect of siPP4c04 on NUMA1 and WBSCR1 alternate exon inclusion using these siRNAs (Figures A1-5, A1-6). This raises important questions as to whether the effect of siPP4c04 on gene expression and alternate exon inclusion is due to PP4c depletion itself or siRNA off target effects. Of note, even Control38 and 53 were observed to induce a striking difference in NUMA1 alternate exon inclusion (Figures A1-4, A1-5) indicating that even non-targeting siRNAs can regulate alternate exon inclusion, although having no significant homology to any 205 known mRNA sequence, indicating off target effects are responsible. At this point it appears unlikely that the genes identified by RNAseq to be differentially expressed or spliced upon PP4c depletion can be validated by qRT-PCR/RT-PCR using these siRNAs therefore other methods such as a rescue experiment will likely need be performed.

A1.3 Discussion A1.3.1 siRNA off target effects and proper siRNA controls

Small interfering RNAs (siRNAs), while initially thought to be a highly specific for knocking down mRNA [290], have since become recognized as having, in most cases, a host of off target effects. These off target effects, which manifest as changes in the expression of genes not regulated by the target of interest, are thought to be facilitated in part through a misdirection of the RNA induced silencing complex (RISC), and primarily, through RISC entering, or the siRNA itself perturbing, the microRNA (miRNA) pathway [291-296].

In brief, when siRNA enters the cell, it is taken up by the RISC complex and processed such that the sense strand (passenger strand), is cleaved by the nuclease AGO2 allowing for it to be separated from the antisense strand (guide strand); the guide strand is then used as a template, directing RISC to complementary mRNAs which are bound and cleaved by AGO2, then further degraded by cellular nucleases resulting in gene silencing [294, 295, 297]. Problematically, guide strand targeting of RISC to mRNAs which have only partial sequence complementary can result in the cleavage of unintended targets [290, 297], or, alternatively, RISC uptake of the passenger strand in place of the guide strand can result in the misdirection of the RISC complex and the targeting of erroneous mRNAs leading to unintended silencing of non-targeted genes and “off target” effects [295, 298]. Additionally, the introduction of exogenous siRNA into the cell can result in sequestration of components of the RNA interference pathway, most notably the RISC complex, resulting in mis-regulation of endogenous miRNAs [294, 295]. In brief, miRNAs are 22-24 nucleotide RNAs that when taken up by RISC direct RISC (in most cases) to the 3´ UTR of genes where they facilitate RISC binding resulting in translation inhibition or the targeting of the mRNA for degradation [293, 295, 297]. Exogenous 206 siRNA displacement of endogenous miRNA from RISC is thought to result in a loss of gene repression and off target effects [292, 295, 298]. Not only this, but nucleotides 2-8 from the 5´ end of the siRNA “the seed sequence” have been shown to function through RISC in fashion similar to miRNAs. Matching 6 or 7 of these nucleotides to the 3´ UTR of an mRNA can result in gene suppression and “miRNA like” off target effects [291- 293, 295]. Because 3´ UTR sequences are shared by many mRNAs, siRNAs which enter the miRNA pathway can affect the expression of hundreds of genes resulting in numerous off target effects. In fact this is thought to be the predominant method by which most siRNA off target effects occur [298], as a single miRNA is though to be able to regulate ~300 genes [295], and even the most infrequently occurring 7 nucleotide seed sequence was bioinformatically shown to have complementary matches in the 3´ UTR of 17 different genes [299]. Studies have indicated that siRNAs functioning as miRNAs may nonspecifically reduce the expression of 30 – 100 genes by ≥ 2 fold and even the top hits of an RNAi screen were observed to be due to miRNA like off target effects [295].

The prevalence of miRNA like off target effects from siRNA was effectively demonstrated in a large scale siRNA screen where 6000 genes were targeted using 21,000 distinct siRNAs to identify novel components of the TGF-β pathway [293]. In this case the investigators found no novel components of the TGF-β pathway; however they did identify 176 siRNAs that gave false positive results through miRNA like depletion of TGF-β receptors type I and II. In the experiments outlined by Schultz et al., [293], TGF-β signaling was measured through GFP-SMAD2 nuclear localization (upon TGF-β stimulation, TGF-β binds the type II TGF- β receptor which then phosphorylates and forms a heterodimer with the type I receptor leading to SMAD2 phosphorylation and its shuttling from the cytoplasm to the nucleus), because the TGF-β signaling read out (GFP- SMAD2 nuclear localization) was directly dependant on all components of the TGF-β pathway (not targeted by siRNA) functioning accordingly, the authors point out that it is important to ensure that siRNAs used to investigate signaling pathways do not contain potential seed matches to components of the pathway. This raises important concerns as to whether siPP4c04, shown to down regulate FOSB expression upon EGF stimulation (in addition to EGF signaling components, e.x. EGFR, NRAS) is actually mediating this 207 action through PP4c depletion or through miRNA like off target effects facilitated through the matching of its seed sequence to regions in the 3´ UTR of these genes.

Methods to eliminate off target effects include the biochemical modification of the siRNA to reduce the probability of passenger strand incorporation into RISC [295, 298, 300], or to increase the specificity of the guide strand [292, 297, 301]. Additionally, bioinformatic analyses of mRNA 3´ UTR sequences can be conducted to identify whether a given siRNA seed sequence may complement a region of the 3´ UTR and possibly result in miRNA like off target effects [291]. However, not all siRNAs found to induce miRNA like off target effects in TGF-β receptors I and II had seed sequence matches to these receptors indicating this approach is not all encompassing [293]. Another popular approach, and one that was used here, is to use multiple siRNAs targeting different regions of an mRNA of interest to separate siRNA off target effects from the effect of target depletion. While Semizarov et al., [290] demonstrated that the use of this methodology resulted in similar gene expression profiles for each siRNA, and concluded that the siRNAs were highly specific, others have found [296], as did I, that each siRNA resulted in a unique expression pattern that appeared to be dependent on siRNA sequence rather than the intended target, indicating that only a small proportion of the observed effects on gene expression were target specific (those that were consistently observed for each of the siRNAs used) and the others likely due to off target effects. Notably, Jackson et al., [296] observed that even the use of siRNA targeting luciferase resulted in reproducible gene regulation for several genes despite a lack of homology, an effect similar to what I observed for NUMA1 alternate exon inclusion, where my two non- targeting siRNAs (Control38 and Control53) each resulted in different levels of NUMA1 alternate exon inclusion, indicating that at least one of them must be regulating NUMA1 alternate exon inclusion through some as yet unidentified off target effect (Figures A1-4, A1-5).

My observed lack of consistency in JUNB or FOSB expression, or NUMA1 alternate exon inclusion, when 3 distinct siRNAs targeting PP4c were used, indicates that the observed effect on gene expression and alternate exon inclusion is likely due to off target effects and not PP4c depletion (Figures A1-2, A1-3, A1-5). In contrast, the 208 consistent effect of siPP4c04, and siPP4c01 on WBSCR1 alternate exon inclusion indicates that WBSCR1 alternate exon inclusion may in fact be regulated by PP4c (Figure A1-6).

Of additional interest when trying to discern off target effects from target dependent effects is the fact that [296] observed that a subset of the transcripts down regulated upon siRNA treatment were down regulated before the siRNA target protein (MAPK14) was even depleted, indicating that these transcripts were silenced by siRNA off target effects and not a loss of MAPK14 activity. Additionally, the presence of another set of transcripts that were differentially expressed only after MAPK14 protein depletion provided them with a list of genes more likely to be regulated by MAPK14. Interestingly, these genes shared less sequence homology to the siRNAs used than the genes that were down-regulated quickly, indicating that the preceding transcripts may have been subjected to off target effects. Based on this observation, it may be of interest to observe the expression or alternate splicing of the genes outlined in tables 2-2, 2-5, at both early and late time points after siRNA treatment, to identify those genes that exhibit differential expression/splicing only after PP4c protein depletion, indicating that their expression or splicing is regulated by PP4c. Invariably however, it appears the best method for identification of siRNA off target effects at this time is the rescue experiment, in which a non-targetable form of the target gene is introduced into the cell/organism of interest and a reversal of the effect of siRNA generated if knockdown of the target gene is indeed responsible for the observed effect [295]. This is important as it directly demonstrates that depletion of the target protein itself is responsible for the observed effect. Even in cases where the use of multiple siRNAs to target a protein results in a consistent effect on gene expression the percentage of rescue can be low (4/9 genes; [302]), indicating that this is an important follow up experiment for the validation of all siRNA experiments and one that needs to be done in the context of the experiments outlined here. 209

A1.4 Conclusions and Future Directions A1.4.1PP4c regulation of Transcription and Splicing

In summary, I have shown that siRNA directed PP4c depletion using multiple siRNAs targeting PP4c leads to inconsistent effects on gene expression and alternate exon inclusion (Figures A1-2, A1-3, A1-5, A1-6), and that the effect of siRNA appears to be dependant on the siRNA sequence itself rather than the intended target, an effect that has been observed by others [296].

Going forward it appears that the best way to identify effects on gene expression and alternate exon inclusion induced by PP4c depletion is through a rescue experiment, as described above. To this effect a rescue plasmid expressing siPP4c04 resistant PP4c [143] has been secured which upon co-transfection, or the generation of stable cell lines, should allow for differential gene expression or alternate exon inclusion dependant on PP4c to be distinguished from siPP4c04 off target effects. In addition to this, a catalytically dead form of this PP4c vector can be generated to assess whether any observed rescue is directly dependant on PP4c phosphatase activity.

On another note, modified ON-TARGETplus siRNA (Dharmacon), which has been modified to reduce passenger stand selection by RISC, enhance guide strand specificity, and been bioinformatically analyzed to reduce the probability guide strand seed regions will produce miRNA like off target effects, can be used in future experiments to help to reduce the probability that any observed effect on gene expression or splicing is due to off target effects (siGENOME, a less specific siRNA, Dharmacon; was used in the experiments outlined above). Initially, these siRNAs can be used to recreate the RNAseq experiments (described briefly above) and the resulting perturbed genes surveyed for those genes that reproducibly display a consistent level of expression or alternate exon inclusion upon PP4c depletion for each of the siRNAs used; or, alternatively, these siRNAs can be used to validate the RNAseq data already generated if their use is shown to yield results consistent to those generated using siPP4c04. Additionally, by monitoring the expression or alternate exon inclusion of the transcripts identified to be differentially expressed or spliced upon siPP4c treatment over time, I can 210 identify those transcripts that are differentially expressed or spliced only after PP4c depletion, as was done by [296], to ensure that changes in expression or alternate exon inclusion are due to PP4c protein depletion and not siRNA off target effects. Any genes identified to be consistently down regulated upon siRNA treatment can be analyzed further, to assess whether they contain 3´ UTR sequences complimentary to the siRNAs used, which would allow for the possibility they are being regulated through miRNA like off target effects [295].

Lastly, the use of AGO2-/- mouse embryonic fibroblasts can allow for siPP4c miRNA like off target effects to be distinguished from the effects of PP4c depletion, as AGO2 is the only one of the four mammalian argonaute proteins (AGO1, AGO2, AGO3, AGO4) with nuclease activity and been shown to mediate siRNA directed mRNA cleavage and degradation. The others, while they can still bind to and take up siRNA are believed to only facilitate miRNA like silencing or degradation of the mRNA. If the use of an siRNA targeting PP4c in this cell line (i.e. siPP4c04) results in a similar effect on gene expression and alternate exon inclusion as was observed in the experiments outlined above (using Hela cells) one can likely conclude that the observed effects are not directed by AGO2 and not due to siRNA directed depletion of PP4c, but rather facilitated by one of the other AGO proteins and likely due to miRNA like off target effects [294].

If indeed the effects of siRNA directed PP4c depletion can be attributed to a loss of PP4c itself and not some unknown off-target or “miRNA like” effect, the components of the transcription and splicing machineries observed to interact with PP4c can next be perturbed (using siRNA) to identify which components (i.e. HTATSF1, DHX38) when depleted affect mRNA transcription or splicing in a manner consistent with that observed with PP4c depletion, indicating regulation by PP4c (as was done for SUPT5H). These proteins can then be narrowed down on to identify whether they are bona fide PP4c substrates using the methods outlined in chapter 3.

In regards to PP4c regulating mRNA transcription through regulation of 7SK RNA, exogenous expression of CDK9 (PTEF-b is comprised of CDK9 and Cyclin T1) can be used to elucidate whether PTEF-b inhibition through 7SK RNA up-regulation 211

(upon PP4c depletion) is the mechanism whereby PP4c regulates mRNA transcription (i.e. JUN/FOS). If exogenous expression of CDK9 can revert the effect of PP4c depletion on mRNA transcription it will provide additional evidence that PP4c regulation of 7SK RNA may be a mechanism whereby PP4c regulates gene transcription.

212

Appendix 2: A cost-benefit analysis of multidimensional fractionation of affinity purification- mass spectrometry samples A2.1 Supplementary Tables

Table S2-1. A) List of the proteins removed because they are listed as "frequent fliers" in our internal FLAG AP-MS HEK293 cell database (contains >1000 AP-MS analyses). HUGO Gene Symbols and Entrez Gene ID are listed. B) Proteins removed from subsequent analysis because they were detected in FLAG alone negative controls. Briefly, HEK293 cells expressing the FLAG epitope alone were harvested, lysed, and prepared as described. AP samples were analyzed using RP, RP/RP, GeLC, MudPIT, or the TripleTOF 5600. The maximal values for each parameter across the seven negative controls are listed. COPS5, and COPS4 were detected at low levels in FLAG alone controls but these proteins were not removed from the data set as they are either bait proteins or known interactors and quantitatively enriched in the analyses.

A. Gene Gene Gene Gene GeneName ID GeneName ID GeneName ID GeneName ID ACTA1 58 HNRNPA3 220988 NONO 4841 RPS16 6217 ACTB 60 HNRNPAB 3182 NPM1 4869 RPS17 6218 ACTBL2 345651 HNRNPC 3183 PABPC1 26986 RPS18 6222 ATP5A1 498 HNRNPD 3184 PABPC4 8761 RPS19 6223 ATP5B 506 HNRNPH1 3187 PARP1 142 RPS2 6187 C11orf84 144097 HNRNPH2 3188 PDHB 5162 RPS20 6224 C6orf170 221322 HNRNPK 3190 POTEE 445582 RPS25 6230 CALM2 805 HNRNPL 3191 PPM1B 5495 RPS26 6231 CAPZA1 829 HNRNPM 4670 PRDX1 5052 RPS28 6234 CAPZA2 830 HSPA1B 3304 PRDX4 10549 RPS3 6188 CAPZB 832 HSPA1L 3305 PRMT5 10419 RPS3A 6189 CBR1 873 HSPA5 3309 PRPF31 26121 RPS3AP49 400652 CBR3 874 HSPA6 3310 PRPF8 10594 RPS4X 6191 CD53 963 HSPA8 3312 PRPS1 5631 RPS6 6194 CFL1 1072 HSPA9 3313 PRPSAP1 5635 RPS8 6202 CLASP2 23122 HSPD1 3329 PRPSAP2 5636 RPS9 6203 CLNS1A 1207 IGF2BP1 10642 PRSS1 5644 RUVBL2 10856 IGHV1OR15- CLTC 1213 1 388077 PRSS12 8492 SF3B1 23451 IGHV1OR15- CMBL 134147 5 390530 PRSS2 5645 SF3B3 23450 IGHV3OR16- CORO1C 23603 13 28303 PRSS3 5646 SFPQ 6421 CTRB1 1504 IGJ 3512 QPCT 25797 SLC25A5 292 213

IGKV1OR2- CTRB2 440387 118 339562 RBM10 8241 SLC25A6 293 IGKV1ORY- CTRC 11330 1 439957 RCN2 5955 SNRNP70 6625 CTRL 1506 ILF2 3608 RIOK1 83732 SNRPD1 6632 RP11- DARS 1615 ILF3 3609 631M21.2 347688 SNRPD3 6634 RP5- DBN1 1627 IVNS1ABP 10625 1033B10.18 730754 SNRPN 6638 DBT 1629 KARS 3735 RPL10 6134 SPIN1 10927 DCD 117159 KIF11 3832 RPL10A 4736 SPTAN1 6709 DDX17 10521 KRT10 3858 RPL11 6135 SPTBN1 6711 DDX5 1655 LOC389950 389950 RPL12 6136 STK38 11329 DHX15 1665 LOC390712 390712 RPL13 6137 STK38L 23012 DHX9 1660 LOC390714 390714 RPL13A 23521 SUN2 25777 EEF1A1 1915 LOC391405 391405 RPL15 6138 SYNCRIP 10492 EEF1B2 1933 LOC391427 391427 RPL17 6139 THRAP3 9967 EEF1D 1936 LOC392217 392217 RPL18 6141 TMOD3 29766 EEF1G 1937 LOC440361 440361 RPL19 6143 TMPRSS11D 9407 EIF2S1 1965 LOC440370 440370 RPL21 6144 TRIM21 6737 EIF4B 1975 LOC440557 440557 RPL23 9349 TRIM28 10155 FLNA 2316 LOC440577 440577 RPL23A 6147 TRYX3 136541 GSTM1 2944 LOC440786 440786 RPL27 6155 TTN 7273 GSTM2 2946 LOC440871 440871 RPL3 6122 TUBA1A 7846 GSTM3 2947 LOC440891 440891 RPL30 6156 TUBA1B 10376 GSTP1 2950 LOC442031 442031 RPL31 6160 TUBA1C 84790 H1FX 8971 LOC442032 442032 RPL36 25873 TUBB 203068 hCG_2023776 402562 LOC442033 442033 RPL38 6169 TUBB2A 7280 HIST1H1C 3006 LOC442034 442034 RPL4 6124 TUBB2B 347733 HIST1H1E 3008 LOC643386 643386 RPL5 6125 TUBB2C 10383 HIST1H2AI 8329 LOC646821 646821 RPL6 6128 TUBB3 10381 HIST1H4A 8359 LTBP1 4052 RPL7 6129 TUBB4 10382 HNRNPA0 10949 MYH10 4628 RPL7A 6130 TUBB6 84617 HNRNPA1 3178 MYH11 4629 RPL8 6132 TXN 7295 HNRNPA1P4 389674 MYH14 79784 RPL9 6133 TYSND1 219743 HNRNPA2B1 3181 MYH9 4627 RPLP0 6175 VIM 7431 HNRNPR 10236 MYL12A 10627 RPLP1 6176 VSIG6 388078 HNRNPU 3192 MYL6 4637 RPLP2 6181 VSIG7 390531 HSP90AA1 3320 MYO18A 399687 RPS11 6205 WDR77 79084 HSP90AB1 3326 MYO1C 4641 RPS14 6208 XRCC5 7520 HSPA1A 3303 NCL 4691 RPS15A 6210 XRCC6 2547

B. HUGO Gene NCBI Entrez spectral unique % sequence Name Gene ID ProteinProphet counts peptides coverage S100A9 6280 1 39 11 68.4 ACTA2 59 1 21 5 26.9 HIST1H2AB 8335 1 19 4 27.3 DSP 1832 1 17 7 4.3 EPRS 2058 1 17 15 10.3 S100A8 6279 1 17 6 35.5 STATH 6779 1 16 3 48.4 PEX12 5193 0.9702 16 2 5.3 HIST1H1D 3007 1 15 4 18.6 214

SUB1 10923 1 15 5 22.8 DDX21 9188 1 15 14 19.4 ABHD3 171586 0.9984 14 2 3.2 LOC730839 730839 1 13 10 13.8 ERC1 23085 1 12 12 12.7 FLG2 388698 0.9999 12 2 1.4 LIMA1 51474 1 11 9 13.4 CALM3 808 1 11 6 32.9 ALB 213 1 10 4 3.6 MAP1B 4131 1 10 10 4.9 LOC100294459 100294459 1 9 4 1.8 MYBBP1A 10514 1 9 8 7.3 HIST1H2BD 3017 1 8 4 34.9 AKAP5 9495 1 8 6 23.9 HRNR 388697 1 8 7 11.3 E2F7 144455 1 8 8 11.9 RARS 5917 1 7 7 11.4 HIST1H2BB 3018 1 7 4 35.7 LARS 51520 1 7 6 6 SF3B2 10992 1 7 6 5.9 KIAA1967 57805 1 7 6 8.9 BCLAF1 9774 1 7 6 8.6 LYZ 4069 1 7 3 18.2 LSM14A 26065 1 6 5 11.4 LUC7L2 51631 1 6 5 12.2 MARS 4141 1 6 5 5.8 SR140 23350 1 6 6 6.6 RBM17 84991 1 6 6 16.2 TXNDC12 51060 1 5 3 22.7 PRPF6 24148 1 5 5 6.3 MYO5B 4645 1 5 5 6.3 CST1 1469 0.9999 5 2 7.8 ANXA1 301 1 5 4 17.6 COPS5 10987 1 5 3 8.7 SERPINB4 6318 1 5 3 7.9 COPS4 51138 1 5 4 15.8 GIGYF2 26058 1 5 5 4.9 RPS23 6228 0.9998 5 4 21.7 MYO1B 4430 1 5 5 4.7 YBX1 4904 1 5 5 34 RPL28 6158 1 5 5 32.8 AFF4 27125 0.9964 5 2 2.3 AIFM1 9131 1 4 4 8.2 PGK1 5230 0.9555 4 2 4.3 DYNC1H1 1778 0.9994 4 4 1.1 RPL29 6159 0.9983 4 2 14.5 HIST2H4A 8370 1 4 3 33 RPL34 6164 0.9944 4 3 20.5 PLEC 5339 0.983 4 3 1.4 RPL35 11224 0.9796 4 2 13.8 RPL35A 6165 0.9951 4 4 23.6 SFRS4 6429 0.9962 4 4 8.3 TAF4 6874 1 4 4 4.1 RPL26 6154 0.9965 4 4 16.6 215

IRS4 8471 1 4 4 2.7 PTS 5805 1 4 3 20 C17orf89 284184 0.9632 4 2 14.9 RNF219 79596 1 4 4 5.8 NOP2 4839 1 4 4 5.9 IARS 3376 1 4 4 3.4 SFRS7 6432 0.9999 4 4 14.7 RPL7AP66 388474 1 4 3 12 SFN 2810 0.9999 4 3 11.7 LRPPRC 10128 1 4 4 2.8 CSTB 1476 1 4 4 53.1 CDSN 1041 0.9807 4 2 5.1 RPL24 6152 1 4 4 19.1 SFRS6 6431 1 4 4 10.8 SF3A1 10291 1 3 3 4.7 IPO8 10526 1 3 3 3.7 FABP5L7 728641 0.9996 3 2 11.9 RPL14 9045 1 3 2 5.6 SFRS1 6426 1 3 3 11.7 PUF60 22827 0.996 3 3 6.8 RPS16P1 441876 1 3 2 14.4 RPL18A 6142 0.9987 3 2 9.1 SFRS3 6428 0.996 3 2 20.7 MYO1D 4642 0.9988 3 3 2.8 MATR3 9782 0.9999 3 3 4.5 PGAM5 192111 0.9999 3 3 12.2 DHX35 60625 0.9996 3 3 3 CHERP 10523 0.9997 3 3 4.7 RRBP1 6238 1 3 3 4.3 OAT 4942 1 3 3 6.6 ADD1 118 1 3 3 5.1 TMPO 7112 1 3 3 11.9 GSN 2934 1 3 3 4.1 RBBP4 5928 0.9795 2 2 5.4 RPL27AP6 389435 0.9906 2 2 14.2 UBC 7316 0.9985 2 2 23.6 C1QBP 708 0.9983 2 2 12.1 RPS5 6193 0.9941 2 2 12.7 LOC653881 653881 0.9967 2 2 15.1 ARG1 383 1 2 2 6.8 MAPKAPK3 7867 0.9852 2 2 6.5 RPS13 6207 0.9976 2 2 14.6 LRRC59 55379 0.9982 2 2 6.5 DSC1 1823 0.9885 2 2 2.6 RP6-213H19.1 51765 0.9995 2 2 6.5 ARL6IP4 51329 0.9999 2 2 8.3 ERH 2079 0.9999 2 2 17.3 CRNN 49860 1 2 2 11.5 DLAT 1737 0.9954 2 2 4.5 SF3B5 83443 1 2 2 27.9 DSG1 1828 0.9997 2 2 3.7 RPS26P25 728937 0.9972 2 2 20.9 CALML5 51806 1 2 2 13 QARS 5859 0.9994 2 2 2.5 216

EXOSC6 118460 0.9994 2 2 8.5 RPS24 6229 0.9995 2 2 11.5 LGALS7B 653499 0.9995 2 2 18.4 PPIA 5478 0.9998 2 2 10.9 ALDH3A2 224 0.9993 2 2 5.2 GTPBP4 23560 0.9999 2 2 2.7 SF3B4 10262 0.9999 2 2 5 PFKFB3 5209 0.9999 2 2 5.6 RRP1B 23076 0.9998 2 2 3.3 LOC100287887 100287887 0.9994 2 2 9.4 RCN1 5954 0.9996 2 2 6.9 DDX46 9879 0.9988 2 2 2.2

217

Table S2-2. Proteins identified in FLAG-COPS5 purifications after background removal. (COPS4 and DDB1 spectra were detected in FLAG alone controls, but these proteins were not filtered out due to substantial increase in spectra in FLAG-COPS5 APs). Sample Separation Method and replicate identification are in the first column. Samples coming from the same batch of purifications are indicated in superscripts. Hit Gene and Hit ID are the HUGO Symbol and Entrez Gene ID, respectively; "Prophet" is the ProteinProphet probability; "n pep" is the number of unique peptides; "n spec" is the spectral count, "% cover" is the hit sequence coverage, in percentage; "BioGRID" indicates whether an interaction is reported in the BioGRID database [202] (v3.0.67) (PN = physical interaction, not high-throughput; PH = physical interaction, high-throughput). Sample Separation n n % Method and ID Hit Gene Hit ID Prophet pep spec cover BioGRID RP LTQ (Biological replicate A)1 COPS5 10987 1 296 71 65.6 RP LTQ (Biological replicate A)1 DDB1 1642 1 140 54 33.3 [PN] RP LTQ (Biological replicate A)1 COPS4 51138 1 260 70 70.9 [PH] RP LTQ (Biological replicate A)1 COPS8 10920 1 211 31 67.9 [PN] RP LTQ (Biological replicate A)1 COPS2 9318 1 179 53 53.0 [PH] RP LTQ (Biological replicate A)1 GPS1 2873 1 107 29 46.8 [PH] RP LTQ (Biological replicate A)1 COPS7A 50813 1 100 25 65.5 [PH] RP LTQ (Biological replicate A)1 COPS3 8533 1 86 31 57.4 [PH] RP LTQ (Biological replicate A)1 CUL3 8452 1 77 33 40.0 [PH] RP LTQ (Biological replicate A)1 CUL2 8453 1 65 24 34.4 [PH] RP LTQ (Biological replicate A)1 CUL4B 8450 1 58 22 27.0 [PH] RP LTQ (Biological replicate A)1 MYEOV2 150678 1 53 9 100.0 RP LTQ (Biological replicate A)1 CUL4A 8451 1 43 18 26.9 [PH] RP LTQ (Biological replicate A)1 COPS6 10980 1 38 17 54.4 [PH] RP LTQ (Biological replicate A)1 CUL1 8454 1 28 16 32.7 [PH] RP LTQ (Biological replicate A)1 DCAF11 80344 1 26 11 20.7 [PH] RP LTQ (Biological replicate A)1 DDB2 1643 1 22 9 21.1 [PH] RP LTQ (Biological replicate A)1 KLHDC5 57542 1 19 12 36.4 RP LTQ (Biological replicate A)1 COPS7B 64708 1 18 12 44.7 [PH] RP LTQ (Biological replicate A)1 LRRC14 9684 1 13 8 23.9 [PH] RP LTQ (Biological replicate A)1 PPIL5 122769 1 9 4 11.6 [PH] RP LTQ (Biological replicate A)1 FBXO17 115290 1 9 6 27.3 [PH] RP LTQ (Biological replicate A)1 SKP1 6500 1 8 5 33.1 RP LTQ (Biological replicate A)1 MEPCE 56257 1 8 7 16.0 RP LTQ (Biological replicate A)1 HIST1H2AE 3012 0.9981 7 2 21.9 RP LTQ (Biological replicate A)1 RPL27A 6157 0.9998 7 2 14.2 RP LTQ (Biological replicate A)1 PRPF4 9128 0.9998 6 2 4.0 RP LTQ (Biological replicate A)1 CRBN 51185 1 6 5 20.6 RP LTQ (Biological replicate A)1 MYOZ1 58529 0.9983 5 2 8.4 RP LTQ (Biological replicate A)1 ERCC8 1161 1 5 4 11.9 [PH] RP LTQ (Biological replicate A)1 VPRBP 9730 1 4 3 4.4 [PH] RP LTQ (Biological replicate A)1 C17orf79 55352 0.9982 3 2 15.8 RP LTQ (Biological replicate A)1 BTBD1 53339 0.9997 3 3 11.0 RP LTQ (Biological replicate A)1 LRRC28 123355 0.9967 3 3 14.2 218

RP LTQ (Biological replicate A)1 BTBD2 55643 0.9954 3 3 10.5 [PH] RP LTQ (Biological replicate A)1 TCEB1 6921 1 3 2 17.9 RP LTQ (Biological replicate A)1 PRPS2 5634 1 3 3 10.1 RP LTQ (Biological replicate A)1 DTL 51514 1 3 3 5.9 RP LTQ (Biological replicate A)1 APOB 338 0.9973 2 2 0.9 RP LTQ (Biological replicate A)1 HIST1H1T 3010 1 2 2 9.7 RP LTQ (Biological replicate A)1 SF3B14 51639 1 2 2 20.8 RP LTQ (Biological replicate A)1 FEM1B 10116 0.9993 2 2 4.9 RP LTQ (Biological replicate A)1 KLHL8 57563 1 2 2 8.1 RP LTQ (Biological replicate B)2 COPS5 10987 1 517 71 53.0 RP LTQ (Biological replicate B)2 COPS4 51138 1 231 55 57.1 [PH] RP LTQ (Biological replicate B)2 DDB1 1642 1 169 50 31.1 [PN] RP LTQ (Biological replicate B)2 COPS2 9318 1 188 49 50.3 [PH] RP LTQ (Biological replicate B)2 COPS3 8533 1 163 44 48.5 [PH] RP LTQ (Biological replicate B)2 GPS1 2873 1 143 31 33.0 [PH] RP LTQ (Biological replicate B)2 COPS8 10920 1 136 27 67.9 [PN;PH] RP LTQ (Biological replicate B)2 MYEOV2 150678 1 79 6 100.0 RP LTQ (Biological replicate B)2 COPS7A 50813 1 74 22 55.6 [PH;PN] RP LTQ (Biological replicate B)2 CUL2 8453 1 65 29 27.5 [PH] RP LTQ (Biological replicate B)2 CUL4A 8451 1 58 22 28.1 [PH;PN] RP LTQ (Biological replicate B)2 COPS6 10980 1 57 15 43.4 [PH] RP LTQ (Biological replicate B)2 CUL4B 8450 1 52 25 28.8 [PH] RP LTQ (Biological replicate B)2 CUL3 8452 1 49 25 38.0 [PH] RP LTQ (Biological replicate B)2 DDB2 1643 1 47 11 25.3 [PH] RP LTQ (Biological replicate B)2 COPS7B 64708 1 26 6 23.9 [PH] RP LTQ (Biological replicate B)2 CUL1 8454 1 26 16 27.3 [PH] RP LTQ (Biological replicate B)2 LRRC14 9684 1 20 7 15.6 [PH] RP LTQ (Biological replicate B)2 KLHDC5 57542 1 17 8 25.0 RP LTQ (Biological replicate B)2 DCAF11 80344 1 14 9 21.1 [PH] RP LTQ (Biological replicate B)2 FBXO17 115290 1 8 4 27.0 [PH] RP LTQ (Biological replicate B)2 SART3 9733 1 6 4 7.5 RP LTQ (Biological replicate B)2 SKP1 6500 1 6 4 33.1 RP LTQ (Biological replicate B)2 TCEB1 6921 1 6 4 17.9 RP LTQ (Biological replicate B)2 ZYG11B 79699 0.9926 4 3 5.0 RP LTQ (Biological replicate B)2 TPD52L1 7164 0.9509 3 2 10.7 RP LTQ (Biological replicate B)2 PTBP1 5725 0.9963 3 2 8.3 RP LTQ (Biological replicate B)2 BTBD2 55643 0.9694 3 3 11.2 [PH] RP LTQ (Biological replicate B)2 DIP2C 22982 0.9991 3 3 1.0 RP LTQ (Biological replicate B)2 LSM2 57819 0.9612 2 2 20.0 RP LTQ (Biological replicate C)3 COPS5 10987 1 503 74 69.8 RP LTQ (Biological replicate C)3 COPS4 51138 1 335 68 70.7 [PH] RP LTQ (Biological replicate C)3 DDB1 1642 1 198 59 37.4 [PN] RP LTQ (Biological replicate C)3 COPS7A 50813 1 244 24 63.3 [PH;PN] RP LTQ (Biological replicate C)3 COPS8 10920 1 187 25 67.9 [PN;PH] RP LTQ (Biological replicate C)3 COPS2 9318 1 179 52 50.6 [PH] RP LTQ (Biological replicate C)3 COPS3 8533 1 173 37 55.8 [PH] RP LTQ (Biological replicate C)3 COPS6 10980 1 97 27 57.5 [PH] RP LTQ (Biological replicate C)3 GPS1 2873 1 77 27 33.0 [PH] RP LTQ (Biological replicate C)3 CUL4B 8450 1 75 22 30.8 [PH] RP LTQ (Biological replicate C)3 CUL1 8454 1 75 27 36.5 [PH] RP LTQ (Biological replicate C)3 CUL3 8452 1 68 29 33.7 [PH] RP LTQ (Biological replicate C)3 CUL2 8453 1 63 35 41.1 [PH] RP LTQ (Biological replicate C)3 COPS7B 64708 1 50 15 42.4 [PH] RP LTQ (Biological replicate C)3 MYEOV2 150678 1 49 8 100.0 219

RP LTQ (Biological replicate C)3 CUL4A 8451 1 48 22 38.3 [PH;PN] RP LTQ (Biological replicate C)3 DDB2 1643 1 46 19 39.1 [PH] RP LTQ (Biological replicate C)3 LRRC14 9684 1 41 9 16.0 [PH] RP LTQ (Biological replicate C)3 KLHDC5 57542 1 33 14 38.0 RP LTQ (Biological replicate C)3 SKP1 6500 1 31 6 43.6 RP LTQ (Biological replicate C)3 FBXO17 115290 1 22 9 27.0 [PH] RP LTQ (Biological replicate C)3 VPRBP 9730 1 21 10 9.0 [PH] RP LTQ (Biological replicate C)3 ZYG11B 79699 1 19 8 13.0 RP LTQ (Biological replicate C)3 DCAF11 80344 1 17 11 23.3 [PH] RP LTQ (Biological replicate C)3 KPNB1 3837 1 14 4 5.8 RP LTQ (Biological replicate C)3 BTBD2 55643 1 12 8 12.0 [PH] RP LTQ (Biological replicate C)3 BTBD1 53339 1 12 7 17.1 RP LTQ (Biological replicate C)3 MYOZ1 58529 0.9646 10 2 4.3 RP LTQ (Biological replicate C)3 CCT8 10694 1 9 5 13.3 RP LTQ (Biological replicate C)3 CRBN 51185 1 9 5 20.6 RP LTQ (Biological replicate C)3 DTL 51514 0.9554 7 2 5.2 RP LTQ (Biological replicate C)3 TCEB2 6923 0.9993 7 3 13.6 RP LTQ (Biological replicate C)3 ERCC8 1161 1 7 4 23.5 [PH] RP LTQ (Biological replicate C)3 ASB6 140459 1 6 4 10.5 RP LTQ (Biological replicate C)3 UBB 7314 0.9999 6 2 32.8 RP LTQ (Biological replicate C)3 CUL5 8065 0.9999 6 5 10.3 RP LTQ (Biological replicate C)3 MEPCE 56257 0.9998 6 4 14.4 RP LTQ (Biological replicate C)3 RPSA 3921 1 6 3 13.6 RP LTQ (Biological replicate C)3 EIF3B 8662 1 6 4 7.7 RP LTQ (Biological replicate C)3 PRKDC 5591 0.9986 5 5 3.0 RP LTQ (Biological replicate C)3 KLHL15 80311 1 5 5 11.6 [PH] RP LTQ (Biological replicate C)3 DDX3X 1654 0.9998 4 4 12.2 RP LTQ (Biological replicate C)3 SMC1A 8243 0.9738 4 2 1.8 RP LTQ (Biological replicate C)3 LRRC28 123355 0.9909 4 4 12.3 RP LTQ (Biological replicate C)3 SLC30A9 10463 1 4 4 9.0 RP LTQ (Biological replicate C)3 EFTUD2 9343 1 4 4 7.0 RP LTQ (Biological replicate C)3 TCEB1 6921 1 4 4 28.6 RP LTQ (Biological replicate C)3 CCT7 10574 0.9953 3 2 4.1 RP LTQ (Biological replicate C)3 PRMT1 3276 0.9878 3 2 6.9 RP LTQ (Biological replicate C)3 CKAP5 9793 0.9606 3 3 3.2 RP LTQ (Biological replicate C)3 LARP7 51574 0.9944 3 3 5.8 RP LTQ (Biological replicate C)3 DCAF16 54876 0.9799 3 3 19.0 RP LTQ (Biological replicate C)3 FUS 2521 0.9712 3 2 8.2 RP LTQ (Biological replicate C)3 EIF3A 8661 1 3 3 5.7 RP LTQ (Biological replicate C)3 KLHL12 59349 0.9994 3 3 8.3 RP LTQ (Biological replicate C)3 ZSWIM6 57688 0.9987 2 2 2.3 RP LTQ (Biological replicate C)3 CCT2 10576 0.9754 2 2 6.4 RP LTQ (Biological replicate C)3 KLHL24 54800 0.9796 2 2 4.0 [PH] RP LTQ (Biological replicate C)3 FBXL14 144699 0.9874 2 2 7.4 RP LTQ (Biological replicate C)3 SART3 9733 0.9816 2 2 3.7 RP LTQ (Biological replicate C)3 DENR 8562 1 2 2 8.6 RP LTQ (Biological replicate C)3 SSB 6741 1 2 2 6.4 RP LTQ (Biological replicate C)3 C17orf79 55352 1 2 2 7.1 RP LTQ (Biological replicate C)3 ZYX 7791 0.9621 2 2 6.3 RP LTQ (Biological replicate C)3 KCTD9 54793 0.9657 2 2 6.7 RP LTQ (Biological replicate C)3 DDX20 11218 0.9521 2 2 3.0 RP LTQ (Biological replicate C)3 TIMM50 92609 0.989 2 2 7.2 RP LTQ (Biological replicate C)3 CPSF6 11052 0.9971 2 2 6.7 RP LTQ (Biological replicate C)3 FEM1B 10116 0.998 2 2 5.9 220

RP LTQ (Biological replicate C)3 CCT4 10575 0.9986 2 2 5.4 RP LTQ (Biological replicate C)3 LOC652147 652147 0.9901 2 2 1.4 RP LTQ (Biological replicate C)3 WARS 7453 0.9919 2 2 8.4 RP LTQ (Biological replicate C)3 EXOSC2 23404 0.992 2 2 9.2 RP LTQ (Biological replicate C)3 HNRNPUL1 11100 0.9923 2 2 6.1 RP LTQ (Biological replicate D)4 COPS5 10987 1 127 27 43.1 RP LTQ (Biological replicate D)4 COPS4 51138 1 131 28 46.1 [PH] RP LTQ (Biological replicate D)4 DDB1 1642 1 101 30 18.8 [PN] RP LTQ (Biological replicate D)4 COPS3 8533 1 173 26 48.2 [PH] RP LTQ (Biological replicate D)4 GPS1 2873 1 117 19 35.4 [PH] RP LTQ (Biological replicate D)4 CUL3 8452 1 84 9 9.4 [PH] RP LTQ (Biological replicate D)4 COPS2 9318 1 61 18 28.0 [PH] RP LTQ (Biological replicate D)4 COPS7A 50813 1 54 17 39.6 [PH;PN] RP LTQ (Biological replicate D)4 CUL4B 8450 1 49 21 26.6 [PH] RP LTQ (Biological replicate D)4 CUL4A 8451 1 43 14 16.5 [PH;PN] RP LTQ (Biological replicate D)4 COPS7B 64708 1 40 16 26.1 [PH] RP LTQ (Biological replicate D)4 CUL2 8453 1 38 17 19.2 [PH] RP LTQ (Biological replicate D)4 COPS6 10980 1 17 6 22.3 [PH] RP LTQ (Biological replicate D)4 COPS8 10920 1 16 3 33.0 [PN;PH] RP LTQ (Biological replicate D)4 TUBB1 81027 0.9853 15 2 2.0 RP LTQ (Biological replicate D)4 VPRBP 9730 1 14 9 7.1 [PH] RP LTQ (Biological replicate D)4 DCAF11 80344 1 12 4 6.5 [PH] RP LTQ (Biological replicate D)4 DDA1 79016 1 10 4 34.3 RP LTQ (Biological replicate D)4 CUL1 8454 1 8 6 11.7 [PH] RP LTQ (Biological replicate D)4 SKP1 6500 0.9995 7 2 24.5 RP LTQ (Biological replicate D)4 LRRC14 9684 0.9991 7 3 9.5 [PH] RP LTQ (Biological replicate D)4 TCEB1 6921 0.9842 7 2 21.4 RP LTQ (Biological replicate D)4 ZYG11B 79699 0.9999 6 4 7.3 RP LTQ (Biological replicate D)4 DTL 51514 1 6 4 9.5 RP LTQ (Biological replicate D)4 MYEOV2 150678 1 6 3 100.0 RP LTQ (Biological replicate D)4 RPSA 3921 1 6 3 12.2 RP LTQ (Biological replicate D)4 DDB2 1643 1 6 4 14.5 [PH] RP LTQ (Biological replicate D)4 KLHDC5 57542 1 6 2 9.5 RP LTQ (Biological replicate D)4 PRPS2 5634 1 5 3 4.7 RP LTQ (Biological replicate D)4 CCT2 10576 0.9786 4 2 4.5 RP LTQ (Biological replicate D)4 BTBD2 55643 1 4 3 8.6 [PH] RP LTQ (Biological replicate D)4 RPL22 6146 0.9924 3 2 21.9 RP LTQ (Biological replicate D)4 BTBD1 53339 0.9996 3 3 10.1 RP LTQ (Biological replicate D)4 SEC31B 25956 0.9841 2 2 1.2 RP LTQ (Biological replicate D)4 VARS 7407 1 2 2 3.2 RP LTQ (Biological replicate D)4 EIF3I 8668 0.9926 2 2 6.8 RP ORB7 COPS4 51138 1 161 60 83.5 [PH] RP ORB7 COPS5 10987 1 176 55 71.0 RP ORB7 DDB1 1642 1 114 65 37.8 [PN] RP ORB7 COPS3 8533 1 109 35 51.8 [PH;PN] RP ORB7 COPS2 9318 1 98 46 60.3 [PH] RP ORB7 GPS1 2873 1 71 32 35.6 [PH] RP ORB7 CUL2 8453 1 60 42 44.8 [PH] RP ORB7 COPS6 10980 1 58 26 40.1 [PH] RP ORB7 CUL3 8452 1 55 37 50.7 [PH] RP ORB7 COPS8 10920 1 54 20 54.1 [PN;PH] RP ORB7 CUL4A 8451 1 44 32 50.2 [PH;PN] RP ORB7 CUL1 8454 1 38 34 42.3 [PH;PN] RP ORB7 COPS7A 50813 1 34 19 50.9 [PH;PN] 221

RP ORB7 MEPCE 56257 1 33 24 38.8 RP ORB7 CUL4B 8450 1 32 28 40.8 [PH] RP ORB7 DDB2 1643 1 26 18 42.4 [PH] RP ORB7 COPS7B 64708 1 25 14 37.5 [PH] RP ORB7 DCAF11 80344 1 23 19 35.9 [PH] RP ORB7 LRRC14 9684 1 21 15 34.3 [PH] RP ORB7 KLHDC5 57542 1 18 15 33.3 RP ORB7 VPRBP 9730 1 15 14 10.6 [PH] RP ORB7 TCEB1 6921 1 10 6 46.4 RP ORB7 MYEOV2 150678 0.9998 9 4 100.0 RP ORB7 FBXO17 115290 1 9 7 31.3 [PH] RP ORB7 DTL 51514 1 8 7 11.8 RP ORB7 ZYG11B 79699 1 7 7 13.7 RP ORB7 CRBN 51185 1 7 7 26.7 RP ORB7 BTBD2 55643 1 7 6 18.1 [PH] RP ORB7 LARP7 51574 1 6 6 12.0 RP ORB7 CUL5 8065 1 6 6 10.0 RP ORB7 TCEB2 6923 1 5 4 30.5 RP ORB7 PPIL5 122769 1 4 4 9.2 [PH] RP ORB7 BTBD1 53339 1 4 4 11.8 RP ORB7 KLHL24 54800 0.9948 3 3 5.7 [PH] RP ORB7 KLHL15 80311 0.9999 3 3 5.3 [PH] RP ORB7 SART3 9733 0.9995 3 3 3.5 RP ORB7 SKP1 6500 1 3 3 16.0 RP ORB7 ASB3 51130 1 2 2 5.4 RP ORB7 UBE2M 9040 0.9949 2 2 9.8 RP ORB7 KLHL18 23276 0.9994 2 2 3.5 RP/RP LTQ1 COPS5 10987 1 307 62 70.1 RP/RP LTQ1 COPS4 51138 1 416 79 65.5 [PH] RP/RP LTQ1 COPS2 9318 1 299 52 51.0 [PH] RP/RP LTQ1 COPS8 10920 1 270 26 73.2 [PN;PH] RP/RP LTQ1 CUL3 8452 1 180 37 37.6 [PH] RP/RP LTQ1 COPS7B 64708 1 180 24 35.2 [PH] RP/RP LTQ1 COPS7A 50813 1 179 31 57.8 [PH;PN] RP/RP LTQ1 GPS1 2873 1 168 33 44.6 [PH] RP/RP LTQ1 COPS3 8533 1 164 36 34.3 [PH] RP/RP LTQ1 COPS6 10980 1 97 13 32.4 [PH] RP/RP LTQ1 CUL2 8453 1 78 24 20.5 [PH] RP/RP LTQ1 CUL4B 8450 1 74 25 32.3 [PH] RP/RP LTQ1 CUL4A 8451 1 72 19 32.8 [PH;PN] RP/RP LTQ1 CUL1 8454 1 50 23 29.0 [PH] RP/RP LTQ1 KLHDC5 57542 1 45 11 19.6 RP/RP LTQ1 MEPCE 56257 1 41 11 19.0 RP/RP LTQ1 DDB2 1643 1 39 13 23.2 [PH] RP/RP LTQ1 DCAF11 80344 1 33 13 21.9 [PH] RP/RP LTQ1 UBB 7314 1 31 4 44.7 RP/RP LTQ1 MYEOV2 150678 1 17 8 100.0 RP/RP LTQ1 H2AFV 94239 0.9995 15 3 22.8 RP/RP LTQ1 FBXO17 115290 1 15 6 16.5 [PH] RP/RP LTQ1 CRBN 51185 1 13 5 18.6 RP/RP LTQ1 VPRBP 9730 1 12 8 7.0 [PH] RP/RP LTQ1 SLC30A9 10463 1 12 6 13.4 RP/RP LTQ1 LRRC14 9684 1 10 6 12.6 [PH] RP/RP LTQ1 LARP7 51574 1 10 8 16.8 222

RP/RP LTQ1 DTL 51514 1 10 7 10.0 RP/RP LTQ1 BTBD2 55643 1 9 4 11.2 [PH] RP/RP LTQ1 MYOZ1 58529 0.9996 8 3 8.0 RP/RP LTQ1 SART3 9733 1 7 5 5.3 RP/RP LTQ1 PMS1 5378 0.9736 7 4 6.6 RP/RP LTQ1 SKP1 6500 1 7 5 33.1 RP/RP LTQ1 RHOBTB1 9886 1 7 6 7.0 RP/RP LTQ1 TCEB1 6921 1 6 4 34.8 RP/RP LTQ1 CCNT2 905 0.9984 6 2 1.5 RP/RP LTQ1 ZYG11B 79699 1 5 4 8.7 RP/RP LTQ1 ZBTB33 10009 0.9908 4 2 2.2 RP/RP LTQ1 CUL5 8065 1 4 3 5.3 RP/RP LTQ1 GPR64 10149 0.955 4 2 0.7 RP/RP LTQ1 THOC4 10189 0.9974 3 2 8.3 RP/RP LTQ1 KCTD3 51133 0.9999 3 2 6.1 RP/RP LTQ1 KLHL15 80311 0.9999 3 3 5.3 [PH] RP/RP LTQ1 KLHL8 57563 1 3 3 5.6 RP/RP LTQ1 EXOSC8 11340 1 2 2 10.1 RP/RP LTQ1 HNRNPUL1 11100 0.9768 2 2 3.0 RP/RP LTQ1 ERCC8 1161 0.9999 2 2 7.3 [PH] RP/RP LTQ1 RABL2B 11158 0.9864 2 2 3.9 RP/RP LTQ1 SSB 6741 0.9989 2 2 5.4 RP/RP LTQ1 TCEB2 6923 0.9998 2 2 14.4 RP/RP LTQ1 DDB1 1642 1 299 64 30.8 [PN] GeLC LTQ6 COPS5 10987 1 269 39 59.3 GeLC LTQ6 COPS4 51138 1 426 52 75.9 [PH] GeLC LTQ6 GPS1 2873 1 279 46 44.8 [PH] GeLC LTQ6 COPS2 9318 1 246 38 52.6 [PH] GeLC LTQ6 COPS3 8533 1 143 29 42.1 [PH] GeLC LTQ6 COPS6 10980 1 130 20 41.6 [PH] GeLC LTQ6 COPS7A 50813 1 110 14 44.7 [PH;PN] GeLC LTQ6 COPS8 10920 1 94 12 60.8 [PN;PH] GeLC LTQ6 CUL3 8452 1 87 38 49.7 [PH] GeLC LTQ6 COPS7B 64708 1 79 21 37.9 [PH] GeLC LTQ6 CUL2 8453 1 77 32 37.9 [PH] GeLC LTQ6 CUL4B 8450 1 67 28 38.7 [PH] GeLC LTQ6 CUL4A 8451 1 63 25 47.2 [PH;PN] GeLC LTQ6 DTL 51514 1 55 11 20.3 GeLC LTQ6 LRRC14 9684 1 42 26 44.4 [PH] GeLC LTQ6 CUL1 8454 1 37 28 38.8 [PH] GeLC LTQ6 MEPCE 56257 1 37 17 25.5 GeLC LTQ6 DDB2 1643 1 32 13 33.5 [PH] GeLC LTQ6 KLHDC5 57542 1 23 10 23.0 GeLC LTQ6 VPRBP 9730 1 20 13 12.3 [PH] GeLC LTQ6 UBB 7314 0.9996 18 3 44.7 GeLC LTQ6 BTBD2 55643 1 17 5 17.3 [PH] GeLC LTQ6 DCAF11 80344 1 15 9 25.6 [PH] GeLC LTQ6 CRBN 51185 1 14 5 19.0 GeLC LTQ6 PPIL5 122769 1 10 8 20.5 [PH] GeLC LTQ6 LARP7 51574 1 6 5 12.9 GeLC LTQ6 BTBD1 53339 0.9985 6 3 15.6 GeLC LTQ6 CUL5 8065 0.9998 6 6 11.0 GeLC LTQ6 DCAF10 79269 1 6 3 8.9 GeLC LTQ6 FBXO17 115290 0.9993 6 3 30.2 [PH] 223

GeLC LTQ6 ATP1A1 476 0.9947 4 3 5.0 GeLC LTQ6 KLHL15 80311 0.997 4 4 10.6 [PH] GeLC LTQ6 KLHL24 54800 1 4 4 13.5 [PH] GeLC LTQ6 TCEB1 6921 0.996 4 2 33.0 GeLC LTQ6 SLC30A9 10463 1 4 4 10.4 GeLC LTQ6 KLHL9 55958 0.9817 4 3 6.2 [PH] GeLC LTQ6 NAT10 55226 0.9689 4 2 2.5 GeLC LTQ6 FEM1B 10116 0.9957 3 3 4.6 GeLC LTQ6 KLHL8 57563 0.9981 3 3 9.0 GeLC LTQ6 CCT8 10694 0.9991 3 3 9.5 GeLC LTQ6 KLHL12 59349 1 3 3 6.2 GeLC LTQ6 KIAA1543 57662 1 3 2 3.5 GeLC LTQ6 APPBP2 10513 0.9607 2 2 3.1 GeLC LTQ6 WDR83 84292 0.9888 2 2 7.3 GeLC LTQ6 ZSWIM6 57688 0.9834 2 2 1.7 GeLC LTQ6 HIST1H4J 8363 0.996 2 2 26.2 GeLC LTQ6 DDB1 1642 1 241 69 40.6 [PN] MudPIT ORB7 COPS4 51138 1 290 44 45.3 [PH] MudPIT ORB7 COPS5 10987 1 54 13 18.6 MudPIT ORB7 DDB1 1642 1 126 29 17.5 [PN] MudPIT ORB7 COPS2 9318 1 191 12 25.3 [PH] MudPIT ORB7 COPS3 8533 1 187 15 16.8 [PH;PN] MudPIT ORB7 COPS6 10980 1 135 4 8.6 [PH] MudPIT ORB7 COPS7A 50813 1 122 13 26.2 [PH;PN] MudPIT ORB7 COPS7B 64708 1 88 21 23.1 [PH] MudPIT ORB7 CUL3 8452 1 77 22 21.0 [PH] MudPIT ORB7 CUL4B 8450 1 76 24 30.5 [PH] MudPIT ORB7 GPS1 2873 1 59 10 13.6 [PH] MudPIT ORB7 CUL2 8453 1 58 19 22.0 [PH] MudPIT ORB7 CUL4A 8451 1 56 14 25.0 [PH;PN] MudPIT ORB7 CUL1 8454 1 41 18 18.8 [PH;PN] MudPIT ORB7 DDB2 1643 1 35 9 16.6 [PH] MudPIT ORB7 BTBD2 55643 1 28 9 12.8 [PH] MudPIT ORB7 VPRBP 9730 1 25 13 7.2 [PH] MudPIT ORB7 LRRC14 9684 1 21 8 14.0 [PH] MudPIT ORB7 KLHDC5 57542 1 19 8 12.1 MudPIT ORB7 DCAF11 80344 1 19 9 14.8 [PH] MudPIT ORB7 MEPCE 56257 1 17 5 9.7 MudPIT ORB7 COPS8 10920 1 14 3 24.4 [PN;PH] MudPIT ORB7 TCEB1 6921 0.9758 12 3 27.7 MudPIT ORB7 BTBD1 53339 0.9889 12 6 11.9 MudPIT ORB7 UBB 7314 0.9677 10 2 23.6 MudPIT ORB7 CUL5 8065 1 10 8 14.7 MudPIT ORB7 FEM1B 10116 1 10 5 7.8 MudPIT ORB7 LARP7 51574 1 9 7 13.2 MudPIT ORB7 TCEB2 6923 0.9999 9 4 24.6 MudPIT ORB7 DTL 51514 1 9 6 10.8 MudPIT ORB7 ZYG11B 79699 1 9 7 11.6 MudPIT ORB7 SLC30A9 10463 0.9999 8 4 9.2 MudPIT ORB7 KLHL13 90293 1 7 5 7.5 MudPIT ORB7 DDX23 9416 0.9996 7 4 6.1 MudPIT ORB7 LRRC28 123355 1 7 4 13.4 MudPIT ORB7 SKP1 6500 0.9999 6 2 7.4 MudPIT ORB7 KLHL15 80311 0.9892 6 4 5.5 [PH] 224

MudPIT ORB7 SNRNP200 23020 1 6 5 3.0 MudPIT ORB7 EFTUD2 9343 1 5 5 6.5 MudPIT ORB7 TIMM50 92609 0.9988 5 2 11.0 MudPIT ORB7 CCT8 10694 1 5 4 8.8 MudPIT ORB7 CDK9 1025 1 5 3 11.8 MudPIT ORB7 CAD 790 1 5 5 2.6 MudPIT ORB7 SART3 9733 0.9988 5 3 3.5 MudPIT ORB7 RANGAP1 5905 0.9999 5 4 8.0 MudPIT ORB7 ATP1A1 476 1 5 4 6.5 MudPIT ORB7 NEDD8 4738 0.9761 4 2 24.7 [PH] MudPIT ORB7 FBXO17 115290 0.9998 4 2 13.3 [PH] MudPIT ORB7 PSMC2 5701 0.9965 4 3 6.9 MudPIT ORB7 KLHL23 151230 0.9946 4 3 5.9 MudPIT ORB7 RHOBTB1 9886 0.9996 4 4 6.8 MudPIT ORB7 PRPF3 9129 1 4 4 6.1 MudPIT ORB7 ERCC8 1161 0.9776 4 2 7.6 [PH] MudPIT ORB7 EIF2S2 8894 0.9999 4 4 11.7 MudPIT ORB7 APPBP2 10513 0.9798 3 2 2.9 MudPIT ORB7 AIMP1 9255 0.9943 3 3 9.3 MudPIT ORB7 SRRM2 23524 0.9987 3 3 1.4 MudPIT ORB7 CCT5 22948 1 3 3 5.5 MudPIT ORB7 KLHL8 57563 0.9978 3 2 3.2 MudPIT ORB7 FBL 2091 0.9977 3 2 5.9 MudPIT ORB7 KIAA1543 57662 0.9996 3 3 4.1 MudPIT ORB7 GCN1L1 10985 0.9989 3 3 1.2 MudPIT ORB7 EXOSC8 11340 0.973 2 2 8.0 MudPIT ORB7 EMD 2010 0.9863 2 2 12.6 MudPIT ORB7 SMC2 10592 0.9561 2 2 3.4 MudPIT ORB7 KBTBD4 55709 0.9638 2 2 3.9 MudPIT ORB7 BAG2 9532 0.9669 2 2 12.3 MudPIT ORB7 EIF4A1 1973 0.9712 2 2 8.9 MudPIT ORB7 SHKBP1 92799 0.9716 2 2 2.7 MudPIT ORB7 PDHA1 5160 0.9998 2 2 5.6 MudPIT ORB7 CAPRIN1 4076 0.9792 2 2 4.3 MudPIT ORB7 MRPS22 56945 0.9994 2 2 6.1 MudPIT ORB7 KLHL24 54800 0.9888 2 2 4.5 [PH] MudPIT ORB7 CKAP5 9793 0.9977 2 2 2.8 MudPIT ORB7 PSMD3 5709 0.9919 2 2 5.4 MudPIT ORB7 MDN1 23195 0.9979 2 2 0.4 MudPIT ORB7 KLHL17 339451 0.9981 2 2 2.6 MudPIT ORB7 RAD50 10111 0.9856 2 2 1.7 MudPIT ORB7 DDX20 11218 0.9989 2 2 3.4 MudPIT ORB7 RBM14 10432 0.999 2 2 5.8 MudPIT ORB7 CCDC8 83987 0.9818 2 2 6.5 MudPIT ORB7 SNRPA1 6627 0.9992 2 2 10.2 MudPIT ORB7 RRP12 23223 0.9811 2 2 1.9 RP TripleTOF 56005 COPS4 51138 1 623 66 67.5 [PH] RP TripleTOF 56005 COPS5 10987 1 412 57 62.3 RP TripleTOF 56005 DDB1 1642 1 188 52 34.2 [PN] RP TripleTOF 56005 COPS3 8533 1 316 34 42.8 [PH;PN] RP TripleTOF 56005 COPS2 9318 1 206 54 47.9 [PH] RP TripleTOF 56005 COPS8 10920 1 185 23 67.9 [PN;PH] RP TripleTOF 56005 CUL3 8452 1 125 46 48.3 [PH] RP TripleTOF 56005 COPS7A 50813 1 117 22 57.8 [PH;PN] 225

RP TripleTOF 56005 COPS6 10980 1 101 23 56.0 [PH] RP TripleTOF 56005 GPS1 2873 1 93 26 32.0 [PH] RP TripleTOF 56005 CUL1 8454 1 59 25 39.2 [PH;PN] RP TripleTOF 56005 COPS7B 64708 1 57 13 45.1 [PH] RP TripleTOF 56005 CUL4B 8450 1 54 23 34.9 [PH] RP TripleTOF 56005 CUL4A 8451 1 47 24 41.6 [PH;PN] RP TripleTOF 56005 CUL2 8453 1 40 22 35.0 [PH] RP TripleTOF 56005 DCAF11 80344 1 36 14 34.4 [PH] RP TripleTOF 56005 DDB2 1643 1 27 12 35.4 [PH] RP TripleTOF 56005 MEPCE 56257 1 27 13 30.6 RP TripleTOF 56005 KLHDC5 57542 1 26 13 26.9 RP TripleTOF 56005 TCEB1 6921 1 17 5 45.5 RP TripleTOF 56005 LRRC14 9684 1 17 11 22.5 [PH] RP TripleTOF 56005 VPRBP 9730 1 9 6 4.7 [PH] RP TripleTOF 56005 BTBD2 55643 1 8 5 21.1 [PH] RP TripleTOF 56005 FBXO17 115290 1 7 4 32.7 [PH] RP TripleTOF 56005 CUL5 8065 1 7 6 8.8 RP TripleTOF 56005 PPIL5 122769 1 7 4 10.6 [PH] RP TripleTOF 56005 CRBN 51185 1 6 3 8.8 RP TripleTOF 56005 BTBD1 53339 1 6 4 19.5 RP TripleTOF 56005 SP1 6667 0.9978 6 2 2.2 RP TripleTOF 56005 MYEOV2 150678 1 5 3 64.9 RP TripleTOF 56005 EIF4E2 9470 1 5 3 16.3 RP TripleTOF 56005 RPS7 6201 1 4 3 37.1 RP TripleTOF 56005 LOC728622 728622 0.9955 4 2 20.0 RP TripleTOF 56005 DCAF16 54876 0.9995 4 2 16.7 RP TripleTOF 56005 LOC100292021 100292021 0.9945 4 2 8.2 RP TripleTOF 56005 RIF1 55183 0.9926 4 2 1.4 RP TripleTOF 56005 DTL 51514 0.9999 4 3 4.2 RP TripleTOF 56005 NME4 4833 0.9734 3 2 15.5 RP TripleTOF 56005 TCEB2 6923 1 3 2 14.4 RP TripleTOF 56005 RBMXL1 494115 0.9982 3 2 13.8 RP TripleTOF 56005 KLHL15 80311 0.9999 3 3 6.0 [PH] RP TripleTOF 56005 LRRC28 123355 0.9997 3 2 10.6 RP TripleTOF 56005 LARP7 51574 0.9996 2 2 5.0 RP TripleTOF 56005 EFTUD2 9343 0.9985 2 2 2.3 RP TripleTOF 56005 KLHL18 23276 0.9998 2 2 3.5 RP TripleTOF 56005 SLC30A9 10463 0.9999 2 2 3.7 RP TripleTOF 56005 KLHL8 57563 0.9886 2 2 3.7 RP TripleTOF 56005 PRPF4 9128 0.9986 2 2 5.4 RP TripleTOF 56005 LOC731751 731751 0.9702 2 2 1.0 RP ORB (Technical replicate 1)8 COPS5 10987 1 92 37 67.4 RP ORB (Technical replicate 1)8 COPS4 51138 1 110 49 72.7 [PH] RP ORB (Technical replicate 1)8 DDB1 1642 1 54 38 31.3 [PN] RP ORB (Technical replicate 1)8 COPS2 9318 1 75 29 49.4 [PH] RP ORB (Technical replicate 1)8 COPS3 8533 1 64 22 31.2 [PH] RP ORB (Technical replicate 1)8 GPS1 2873 1 52 19 31.0 [PH] RP ORB (Technical replicate 1)8 COPS6 10980 1 32 18 42.8 [PH] RP ORB (Technical replicate 1)8 CUL3 8452 1 30 24 30.6 [PH] RP ORB (Technical replicate 1)8 COPS8 10920 1 29 11 41.1 [PN;PH] RP ORB (Technical replicate 1)8 COPS7A 50813 1 28 16 50.2 [PH;PN] RP ORB (Technical replicate 1)8 CUL2 8453 1 27 19 23.1 [PH] RP ORB (Technical replicate 1)8 CUL4A 8451 1 27 20 39.5 [PH;PN] RP ORB (Technical replicate 1)8 CUL4B 8450 1 21 17 31.2 [PH] 226

RP ORB (Technical replicate 1)8 DDB2 1643 1 16 12 32.8 [PH] RP ORB (Technical replicate 1)8 MEPCE 56257 1 16 14 25.3 RP ORB (Technical replicate 1)8 COPS7B 64708 1 14 8 23.1 [PH] RP ORB (Technical replicate 1)8 DCAF11 80344 1 12 12 30.4 [PH] RP ORB (Technical replicate 1)8 CUL1 8454 1 12 12 16.0 [PH] RP ORB (Technical replicate 1)8 KLHDC5 57542 1 11 8 18.0 RP ORB (Technical replicate 1)8 LRRC14 9684 1 10 9 15.8 [PH] RP ORB (Technical replicate 1)8 BTBD2 55643 1 8 7 13.7 [PH] RP ORB (Technical replicate 1)8 FBXO17 115290 0.9999 3 3 10.1 [PH] RP ORB (Technical replicate 1)8 TCEB1 6921 1 3 3 28.6 RP ORB (Technical replicate 1)8 PPIL5 122769 0.9983 2 2 4.3 [PH] RP ORB (Technical replicate 1)8 SKP1 6500 1 2 2 15.3 RP ORB (Technical replicate 1)8 LARP7 51574 0.9999 2 2 7.4 RP ORB (Technical replicate 2)8 COPS5 10987 1 141 38 67.7 RP ORB (Technical replicate 2)8 COPS4 51138 1 225 40 70.2 [PH] RP ORB (Technical replicate 2)8 DDB1 1642 1 71 29 28.2 [PN] RP ORB (Technical replicate 2)8 COPS6 10980 1 87 17 47.7 [PH] RP ORB (Technical replicate 2)8 COPS2 9318 1 87 32 47.2 [PH] RP ORB (Technical replicate 2)8 COPS7A 50813 1 76 12 43.3 [PH;PN] RP ORB (Technical replicate 2)8 COPS3 8533 1 68 17 46.8 [PH] RP ORB (Technical replicate 2)8 DDB2 1643 1 55 7 22.2 [PH] RP ORB (Technical replicate 2)8 GPS1 2873 1 50 23 40.3 [PH] RP ORB (Technical replicate 2)8 CUL3 8452 1 47 14 23.4 [PH] RP ORB (Technical replicate 2)8 CUL4B 8450 1 43 6 18.2 [PH] RP ORB (Technical replicate 2)8 COPS8 10920 1 38 10 63.2 [PN;PH] RP ORB (Technical replicate 2)8 COPS7B 64708 1 37 6 22.0 [PH] RP ORB (Technical replicate 2)8 CUL2 8453 1 24 11 18.5 [PH] RP ORB (Technical replicate 2)8 CUL4A 8451 1 18 13 24.8 [PH;PN] RP ORB (Technical replicate 2)8 MYEOV2 150678 1 12 4 100.0 RP ORB (Technical replicate 2)8 DCAF11 80344 1 12 7 10.6 [PH] RP ORB (Technical replicate 2)8 CUL1 8454 1 9 7 11.7 [PH] RP ORB (Technical replicate 2)8 KLHDC5 57542 1 7 4 15.2 RP ORB (Technical replicate 2)8 MEPCE 56257 0.9936 6 3 4.1 RP ORB (Technical replicate 2)8 LRRC14 9684 0.977 4 2 7.5 [PH] RP ORB (Technical replicate 2)8 FBXO17 115290 1 4 3 17.3 [PH] RP ORB (Technical replicate 2)8 OR6K6 128371 0.9753 3 2 3.5 RP ORB (Technical replicate 2)8 PPIL5 122769 0.9997 3 3 7.2 [PH] RP ORB (Technical replicate 2)8 TCEB1 6921 1 3 2 17.9 RP ORB (Technical replicate 2)8 EIF4E2 9470 0.9999 2 2 9.8 RP LTQ (Technical replicate 3)8 COPS5 10987 1 200 60 65.9 RP LTQ (Technical replicate 3)8 COPS4 51138 1 157 46 64.0 [PH] RP LTQ (Technical replicate 3)8 DDB1 1642 1 72 31 23.9 [PN] RP LTQ (Technical replicate 3)8 COPS2 9318 1 110 25 42.2 [PH] RP LTQ (Technical replicate 3)8 COPS3 8533 1 58 19 36.9 [PH] RP LTQ (Technical replicate 3)8 COPS7A 50813 1 53 22 56.0 [PH;PN] RP LTQ (Technical replicate 3)8 MYEOV2 150678 1 53 7 100.0 RP LTQ (Technical replicate 3)8 COPS8 10920 1 51 22 82.3 [PN;PH] RP LTQ (Technical replicate 3)8 CUL3 8452 1 28 18 33.1 [PH] RP LTQ (Technical replicate 3)8 GPS1 2873 1 24 18 33.6 [PH] RP LTQ (Technical replicate 3)8 CUL2 8453 1 19 16 29.5 [PH] RP LTQ (Technical replicate 3)8 COPS7B 64708 1 16 10 37.5 [PH] RP LTQ (Technical replicate 3)8 CUL4B 8450 1 15 14 18.7 [PH] RP LTQ (Technical replicate 3)8 COPS6 10980 1 15 11 33.9 [PH] RP LTQ (Technical replicate 3)8 CUL4A 8451 1 14 12 27.5 [PH;PN] 227

RP LTQ (Technical replicate 3)8 CUL1 8454 1 10 8 14.6 [PH] RP LTQ (Technical replicate 3)8 LRRC14 9684 0.9999 7 3 8.1 [PH] RP LTQ (Technical replicate 3)8 KLHDC5 57542 1 6 6 27.3 RP LTQ (Technical replicate 3)8 DDB2 1643 1 6 6 16.9 [PH] RP LTQ (Technical replicate 3)8 DCAF11 80344 1 3 3 11.0 [PH] RP LTQ (Technical replicate 3)8 MRS2 57380 0.9849 2 2 9.0 RP LTQ (Technical replicate 3)8 VPRBP 9730 0.9716 2 2 3.1 [PH]

228

Table S2-3. Proteins identified in FLAG-EIF4A2 purifications after background removal. Sample separation method and replicate identification are in the first column. "Hit Gene" and "Hit ID" are the HUGO Symbol and Entrez Gene ID, respectively; "Prophet" is the ProteinProphet probability; "n pep" is the number of unique peptides; "n spec" is the spectral count, "% cover" is the hit sequence coverage, in percentage; "BioGRID" indicates whether an interaction is reported in the BioGRID database [202] (v3.0.67) (PN = physical interaction, not high-throughput; PH = physical interaction, high-throughput). Sample Separation n n % Method and ID Hit Gene Hit ID Prophet pep spec cover BioGRID EIF4A2_RP (1) EIF4G1 1981 1 69 34 27 [PH] EIF4A2_RP (1) EIF4A2 1974 1 68 24 42.5 EIF4A2_RP (1) EIF3A 8661 1 57 41 25.8 [PH] EIF4A2_RP (1) EIF3C 8663 1 29 21 26.4 EIF4A2_RP (1) EIF3B 8662 1 26 18 20.1 EIF4A2_RP (1) EIF3L 51386 1 24 16 28.7 [PH] EIF4A2_RP (1) EIF4G3 8672 1 23 20 18.3 [PH] EIF4A2_RP (1) PDCD4 27250 1 19 12 27.9 [PH] EIF4A2_RP (1) EIF3E 3646 1 17 11 28.1 EIF4A2_RP (1) ATXN2L 11273 1 13 11 14.6 EIF4A2_RP (1) EIF4G2 1982 1 12 12 19.4 [PH] EIF4A2_RP (1) NUFIP2 57532 1 12 11 22.9 EIF4A2_RP (1) BAT2L2 23215 1 11 10 4.9 EIF4A2_RP (1) EIF3G 8666 1 10 9 21.6 EIF4A2_RP (1) EIF3I 8668 1 9 7 28.3 EIF4A2_RP (1) LOC100131693 100131693 1 9 5 25.3 EIF4A2_RP (1) EIF3H 8667 1 9 7 22.7 [PH] EIF4A2_RP (1) EIF3F 8665 1 8 6 19.9 [PH] EIF4A2_RP (1) BAT2 7916 1 8 6 4.9 EIF4A2_RP (1) EIF3D 8664 1 7 6 9.9 [PH] EIF4A2_RP (1) LARP1 23367 1 7 7 9.3 EIF4A2_RP (1) ATXN2 6311 1 7 7 7.8 EIF4A2_RP (1) BAT2L1 84726 1 6 6 5.5 EIF4A2_RP (1) SRRM2 23524 1 6 6 4.5 EIF4A2_RP (1) HNRPDL 9987 0.9995 5 4 10.5 EIF4A2_RP (1) ELAVL1 1994 0.9996 5 5 17.5 EIF4A2_RP (1) EIF3M 10480 0.9983 5 4 6.4 [PH] EIF4A2_RP (1) CSDA 8531 0.9971 4 3 18.3 EIF4A2_RP (1) G3BP1 10146 0.9998 4 3 9.9 EIF4A2_RP (1) FXR2 9513 1 4 3 8.9 EIF4A2_RP (1) C10orf137 26098 1 4 4 4.7 EIF4A2_RP (1) LARP4 113251 0.9978 3 2 8.3 EIF4A2_RP (1) CAPRIN1 4076 0.9913 3 2 2.6 EIF4A2_RP (1) MOV10 4343 0.9999 3 3 3.2 EIF4A2_RP (1) IGF2BP3 10643 0.9743 3 3 7.1 229

EIF4A2_RP (1) FIP1L1 81608 0.9593 2 2 4.6 EIF4A2_RP (1) DDX50 79009 0.9924 2 2 11.9 EIF4A2_RP (1) USP10 9100 0.9657 2 2 2.8 EIF4A2_RP (1) SUGP2 10147 0.9828 2 2 2.4 EIF4A2_RP (1) THOC4 10189 0.9999 2 2 8 EIF4A2_RP (1) UPF1 5976 0.9991 2 2 3.8 EIF4A2_RP (1) HNRNPF 3185 0.9975 2 2 4.3 EIF4A2_RP (2) EIF3A 8661 1 80 43 30.2 [PH] EIF4A2_RP (2) EIF4G1 1981 1 75 45 34.6 [PH] EIF4A2_RP (2) EIF4A2 1974 1 74 23 39.6 EIF4A2_RP (2) EIF3C 8663 1 42 25 26.6 EIF4A2_RP (2) EIF3B 8662 1 34 21 29.7 EIF4A2_RP (2) EIF3L 51386 1 30 19 38.5 [PH] EIF4A2_RP (2) EIF4G3 8672 1 29 21 17.5 [PH] EIF4A2_RP (2) EIF3E 3646 1 28 18 37.8 EIF4A2_RP (2) EIF4G2 1982 1 27 16 26.5 [PH] EIF4A2_RP (2) BAT2 7916 1 25 19 16.8 EIF4A2_RP (2) PDCD4 27250 1 24 13 24.5 [PH] EIF4A2_RP (2) EIF3I 8668 1 16 10 33.8 EIF4A2_RP (2) BAT2L2 23215 1 16 15 8 EIF4A2_RP (2) EIF3F 8665 1 14 7 24.1 [PH] EIF4A2_RP (2) EIF3D 8664 1 13 8 18.6 [PH] EIF4A2_RP (2) EIF3G 8666 1 13 6 24.4 EIF4A2_RP (2) EIF3H 8667 1 11 8 31.8 [PH] EIF4A2_RP (2) EIF3J 8669 1 9 7 19.8 EIF4A2_RP (2) EIF3M 10480 0.9997 8 4 10.4 [PH] EIF4A2_RP (2) LARP1 23367 1 5 5 9.2 EIF4A2_RP (2) ABCE1 6059 0.9786 4 3 7.7 EIF4A2_RP (2) G3BP1 10146 0.9933 4 4 10.3 EIF4A2_RP (2) DDX3X 1654 0.9859 4 3 6.8 [PH] EIF4A2_RP (2) RPL27A 6157 0.998 3 2 14.2 EIF4A2_RP (2) EIF3K 27335 0.9945 3 2 13.3 [PH] EIF4A2_RP (2) SERBP1 26135 0.9845 3 2 8.3 EIF4A2_RP (2) EIF2S2 8894 0.999 3 3 13.5 EIF4A2_RP (2) EIF4A1 1973 1 3 3 24.1 EIF4A2_RP (2) RPSA 3921 1 3 3 16.3 EIF4A2_RP (2) RPS10 6204 1 3 3 14.5 EIF4A2_RP (2) SRSF2 6427 0.9977 2 2 11.3 EIF4A2_RP (2) CSDA 8531 0.983 2 2 24.4 EIF4A2_RP (2) MTDH 92140 0.9809 2 2 4.6 EIF4A2_RP (2) ATXN2 6311 1 2 2 4.6 EIF4A2_RP (2) UPF1 5976 0.9993 2 2 4 EIF4A2_RP (2) GNB2L1 10399 1 2 2 7.6 EIF4A2_RP (2) NUFIP2 57532 0.9871 2 2 6.9 EIF4A2_RP (2) C22orf28 51493 0.9887 2 2 5.9 EIF4A2_RP (2) LOC100131693 100131693 0.9995 2 2 21.7 EIF4A2_RP (2) C10orf137 26098 0.9997 2 2 4.6 EIF4A2_RP/RP (1) EIF4G1 1981 1 200 40 26.7 [PH] EIF4A2_RP/RP (1) EIF3A 8661 1 148 41 26.3 [PH] EIF4A2_RP/RP (1) EIF4A2 1974 1 109 22 40.5 230

EIF4A2_RP/RP (1) EIF4G3 8672 1 99 30 20.1 [PH] EIF4A2_RP/RP (1) EIF3B 8662 1 84 25 20.8 EIF4A2_RP/RP (1) EIF3L 51386 1 80 20 32.1 [PH] EIF4A2_RP/RP (1) EIF3C 8663 1 73 28 25.5 EIF4A2_RP/RP (1) PDCD4 27250 1 73 15 28.2 [PH] EIF4A2_RP/RP (1) EIF3E 3646 1 53 15 33.3 EIF4A2_RP/RP (1) BAT2L2 23215 1 43 13 9.5 EIF4A2_RP/RP (1) EIF3G 8666 1 39 7 23.4 EIF4A2_RP/RP (1) EIF3I 8668 1 33 10 26.2 EIF4A2_RP/RP (1) EIF3H 8667 1 32 10 25 [PH] EIF4A2_RP/RP (1) NUFIP2 57532 1 32 13 23.9 EIF4A2_RP/RP (1) BAT2L1 84726 1 28 13 12.5 EIF4A2_RP/RP (1) EIF4G2 1982 1 27 15 19.6 [PH] EIF4A2_RP/RP (1) LARP1 23367 1 23 10 10.1 EIF4A2_RP/RP (1) EIF4A1 1973 0.9999 21 5 23.6 EIF4A2_RP/RP (1) LOC100131693 100131693 1 19 4 15.2 EIF4A2_RP/RP (1) BAT2 7916 1 18 11 9.3 EIF4A2_RP/RP (1) EIF3D 8664 1 15 7 11.7 [PH] EIF4A2_RP/RP (1) SRRM2 23524 1 15 12 7 EIF4A2_RP/RP (1) C10orf137 26098 1 13 7 8.6 EIF4A2_RP/RP (1) EIF3F 8665 1 11 7 21.6 [PH] EIF4A2_RP/RP (1) LARP4 113251 0.9998 11 6 13.5 EIF4A2_RP/RP (1) ELAVL1 1994 1 11 5 12.6 EIF4A2_RP/RP (1) CSDA 8531 1 11 4 28.7 EIF4A2_RP/RP (1) G3BP1 10146 0.9996 11 3 7.1 EIF4A2_RP/RP (1) ATXN2 6311 1 10 8 9 EIF4A2_RP/RP (1) DDX50 79009 1 10 10 18.5 EIF4A2_RP/RP (1) UPF1 5976 1 10 8 10.6 EIF4A2_RP/RP (1) MOV10 4343 1 10 7 7.9 EIF4A2_RP/RP (1) IGF2BP2 10644 0.9999 8 5 13.5 EIF4A2_RP/RP (1) SRSF9 8683 0.9971 8 6 25.3 EIF4A2_RP/RP (1) CAPRIN1 4076 0.9726 7 2 4.3 EIF4A2_RP/RP (1) FXR2 9513 1 7 5 11.9 EIF4A2_RP/RP (1) IGF2BP3 10643 0.9924 6 4 10.7 EIF4A2_RP/RP (1) FMR1 2332 0.999 5 2 7.9 EIF4A2_RP/RP (1) EIF3J 8669 0.9979 5 4 23.3 EIF4A2_RP/RP (1) LSM12 124801 0.9999 5 2 18.5 EIF4A2_RP/RP (1) ZFR 51663 1 5 4 6.3 EIF4A2_RP/RP (1) LARP4B 23185 0.9851 4 2 3 EIF4A2_RP/RP (1) FAM120A 23196 0.9859 4 3 5.6 EIF4A2_RP/RP (1) ATXN2L 11273 1 4 4 11.6 EIF4A2_RP/RP (1) USP10 9100 0.9705 4 3 4.5 EIF4A2_RP/RP (1) DDX3X 1654 0.9998 4 2 7.6 [PH] EIF4A2_RP/RP (1) HNRNPF 3185 0.9991 4 2 4.3 EIF4A2_RP/RP (1) DHX36 170506 0.953 3 2 2.7 EIF4A2_RP/RP (1) HNRPDL 9987 0.9686 3 2 5.7 EIF4A2_RP/RP (1) CUX1 1523 0.9834 3 2 1 EIF4A2_RP/RP (1) SRSF5 6430 0.9994 3 3 10.3 EIF4A2_RP/RP (1) RBM14 10432 0.9998 3 3 8.4 EIF4A2_RP/RP (1) FIP1L1 81608 0.9994 3 2 5.8 231

EIF4A2_RP/RP (1) PURB 5814 0.9998 3 2 10.3 EIF4A2_RP/RP (1) C22orf28 51493 0.9964 3 2 7.9 EIF4A2_RP/RP (1) LARP1B 55132 0.9992 3 2 5.9 EIF4A2_RP/RP (1) ZC3H18 124245 0.9997 2 2 3.6 EIF4A2_RP/RP (1) SRRT 51593 0.9568 2 2 4.6 EIF4A2_RP/RP (1) DDX1 1653 0.9909 2 2 2.7 EIF4A2_RP/RP (1) RPS7 6201 0.9822 2 2 8.8 EIF4A2_RP/RP (1) RBM39 9584 1 2 2 5.2 EIF4A2_RP/RP (1) BIRC6 57448 0.952 2 2 0.4 EIF4A2_RP/RP (2) EIF4G1 1981 1 152 40 26.8 [PH] EIF4A2_RP/RP (2) EIF4A2 1974 1 130 29 44.5 EIF4A2_RP/RP (2) EIF3A 8661 1 110 41 26.6 [PH] EIF4A2_RP/RP (2) EIF3C 8663 1 83 31 21.4 EIF4A2_RP/RP (2) EIF3L 51386 1 73 21 29.4 [PH] EIF4A2_RP/RP (2) EIF3B 8662 1 70 28 23.5 EIF4A2_RP/RP (2) EIF4G3 8672 1 57 28 17.9 [PH] EIF4A2_RP/RP (2) PDCD4 27250 1 51 9 18.8 [PH] EIF4A2_RP/RP (2) EIF3E 3646 1 43 16 31.5 EIF4A2_RP/RP (2) BAT2 7916 1 38 20 15.2 EIF4A2_RP/RP (2) EIF3I 8668 1 30 12 35.4 EIF4A2_RP/RP (2) EIF3H 8667 1 27 8 19.6 [PH] EIF4A2_RP/RP (2) EIF3J 8669 1 26 9 23.6 EIF4A2_RP/RP (2) EIF3D 8664 1 26 9 10.9 [PH] EIF4A2_RP/RP (2) EIF4G2 1982 1 24 12 19.6 [PH] EIF4A2_RP/RP (2) EIF3G 8666 1 15 8 22.2 EIF4A2_RP/RP (2) LOC100131693 100131693 1 12 4 18.4 EIF4A2_RP/RP (2) BAT2L2 23215 1 12 8 5.7 EIF4A2_RP/RP (2) BAT2L1 84726 1 10 8 5.9 EIF4A2_RP/RP (2) EIF2S2 8894 1 8 6 22.2 EIF4A2_RP/RP (2) EIF3F 8665 1 8 4 16.8 [PH] EIF4A2_RP/RP (2) EIF3K 27335 0.9992 8 4 17.9 [PH] EIF4A2_RP/RP (2) RPS7 6201 0.9945 8 4 14.4 EIF4A2_RP/RP (2) DDX3X 1654 1 7 4 8.6 [PH] EIF4A2_RP/RP (2) LARP1 23367 1 6 5 6.9 EIF4A2_RP/RP (2) RPS10 6204 1 5 3 9.1 EIF4A2_RP/RP (2) EIF4A1 1973 1 5 3 24.1 EIF4A2_RP/RP (2) RPSA 3921 1 5 2 7.1 EIF4A2_RP/RP (2) EIF5B 9669 1 5 5 6.8 EIF4A2_RP/RP (2) RPS15 6209 0.9987 4 2 8.3 EIF4A2_RP/RP (2) C22orf28 51493 0.9996 4 3 8.7 EIF4A2_RP/RP (2) C10orf137 26098 0.9996 4 3 6.1 EIF4A2_RP/RP (2) LARP4 113251 0.9986 4 3 5.7 EIF4A2_RP/RP (2) NUFIP2 57532 0.9966 4 4 11.2 EIF4A2_RP/RP (2) MOV10 4343 0.9852 4 4 5.5 EIF4A2_RP/RP (2) ELAVL1 1994 0.9996 4 2 7.4 EIF4A2_RP/RP (2) RPS21 6227 1 4 3 13.3 EIF4A2_RP/RP (2) DHX29 54505 1 4 4 3.8 EIF4A2_RP/RP (2) ASCC3 10973 1 3 3 2.1 EIF4A2_RP/RP (2) IGF2BP2 10644 0.9986 3 2 4.5 EIF4A2_RP/RP (2) PA2G4 5036 0.9834 3 3 8.6 232

EIF4A2_RP/RP (2) DDX50 79009 0.9969 3 3 13.4 EIF4A2_RP/RP (2) SRRM2 23524 1 3 2 1.1 EIF4A2_RP/RP (2) GNB2L1 10399 0.9876 3 2 7.9 EIF4A2_RP/RP (2) FMR1 2332 0.9916 3 2 6 EIF4A2_RP/RP (2) OBSL1 23363 1 3 3 2 EIF4A2_RP/RP (2) ABCE1 6059 0.9999 3 3 5.2 EIF4A2_RP/RP (2) UPF1 5976 0.9995 3 3 4.8 EIF4A2_RP/RP (2) HADHA 3030 0.9999 3 3 5 EIF4A2_RP/RP (2) SERBP1 26135 0.9989 3 2 5.4 EIF4A2_RP/RP (2) PURB 5814 0.9985 3 2 10.3 EIF4A2_RP/RP (2) ABCF1 23 0.9557 2 2 3.1 EIF4A2_RP/RP (2) G3BP1 10146 0.9562 2 2 3.6 EIF4A2_RP/RP (2) PRMT1 3276 0.9884 2 2 6.1 EIF4A2_RP/RP (2) HNRNPF 3185 0.9939 2 2 6.3 EIF4A2_RP/RP (2) ATXN2 6311 0.9999 2 2 2 EIF4A2_RP/RP (2) MTDH 92140 0.999 2 2 6.9 EIF4A2_RP/RP (2) FXR2 9513 0.999 2 2 7.1 EIF4A2_RP/RP (2) LARP4B 23185 0.9983 2 2 5.6 EIF4A2_RP/RP (2) CSDA 8531 0.9982 2 2 24.4

233

Table S2-4. Proteins identified in FLAG-RAF1 purifications after background removal. Sample separation method and replicate identification are in the first column. "Hit Gene" and "Hit ID" are the HUGO Symbol and Entrez Gene ID, respectively; "Prophet" is the ProteinProphet probability; "n pep" is the number of unique peptides; "n spec" is the spectral count, "% cover" is the hit sequence coverage, in percentage; "BioGRID" indicates whether an interaction is reported in the BioGRID database [202] (v3.0.67) (PN = physical interaction, not high-throughput; PH = physical interaction, high-throughput). Sample Separation n n % Method and ID Hit Gene Hit ID Prophet pep spec cover BioGRID RAF1_RP (1) RAF1 5894 1 84 27 43.2 RAF1_RP (1) YWHAE 7531 1 33 14 42.7 [PN] RAF1_RP (1) YWHAG 7532 1 14 9 39.7 [PH;PN] RAF1_RP (1) GCN1L1 10985 1 13 12 5.4 RAF1_RP (1) TIMM50 92609 0.9999 12 6 16.7 RAF1_RP (1) CDC37 11140 1 12 8 21.4 RAF1_RP (1) YWHAZ 7534 1 9 4 22.9 [PN] RAF1_RP (1) YWHAB 7529 1 8 5 42.7 [PN] RAF1_RP (1) KRAS 3845 0.9989 7 5 31.4 [PN] RAF1_RP (1) YWHAH 7533 1 7 6 28.9 [PN] RAF1_RP (1) ATAD3A 55210 0.9999 6 5 9.6 RAF1_RP (1) ATP1A1 476 1 6 5 5.2 RAF1_RP (1) LOC731751 731751 0.9999 5 5 1.7 RAF1_RP (1) CAD 790 1 5 5 2.9 RAF1_RP (1) SSR4 6748 0.9987 4 2 12.1 RAF1_RP (1) HAX1 10456 0.9997 4 3 18.6 RAF1_RP (1) STIP1 10963 0.9999 4 4 13.8 RAF1_RP (1) RPS27L 51065 0.9911 3 2 28.6 RAF1_RP (1) DNAJA1 3301 0.9849 3 3 11.6 RAF1_RP (1) EMD 2010 0.9857 3 3 12.2 RAF1_RP (1) GRAMD1A 57655 0.9881 2 2 3.8 RAF1_RP (1) YWHAQ 10971 0.9916 2 2 20.4 [PH;PN] RAF1_RP (1) SEC61B 10952 0.9984 2 2 27.1 RAF1_RP (2) RAF1 5894 1 116 38 47.2 RAF1_RP (2) YWHAE 7531 1 60 26 71.4 [PN] RAF1_RP (2) GCN1L1 10985 1 28 25 15.5 RAF1_RP (2) PRKDC 5591 1 26 26 10 RAF1_RP (2) TIMM50 92609 1 24 12 22.4 RAF1_RP (2) CDC37 11140 1 21 13 31.2 RAF1_RP (2) YWHAZ 7534 1 16 11 51.8 [PN] RAF1_RP (2) YWHAB 7529 1 15 10 59.8 [PN] RAF1_RP (2) YWHAG 7532 1 15 10 46.2 [PH;PN] RAF1_RP (2) YWHAH 7533 1 13 9 47.2 [PN] RAF1_RP (2) STIP1 10963 1 11 9 22.1 RAF1_RP (2) FKBP5 2289 1 8 8 28.2 RAF1_RP (2) CALU 813 1 8 8 32.1 RAF1_RP (2) EMD 2010 1 6 4 17.7 RAF1_RP (2) HRAS 3265 0.9983 6 4 31.2 [PN] 234

RAF1_RP (2) DNAJA2 10294 0.9999 6 6 14.3 RAF1_RP (2) YWHAQ 10971 1 6 4 40.4 [PH;PN] RAF1_RP (2) ATP1A1 476 1 6 6 15.2 RAF1_RP (2) KRAS 3845 0.984 5 4 30.3 [PN] RAF1_RP (2) XPOT 11260 1 5 5 6.8 RAF1_RP (2) CSE1L 1434 1 5 4 7.4 RAF1_RP (2) DNAJA1 3301 1 5 4 19.1 RAF1_RP (2) SLC25A11 8402 0.9994 4 3 12.2 RAF1_RP (2) FDFT1 2222 1 4 3 15.1 RAF1_RP (2) NDUFS3 4722 0.9999 4 3 17 RAF1_RP (2) FANCD2 2177 0.9999 4 4 2.1 RAF1_RP (2) SDF4 51150 0.9999 4 4 16.4 RAF1_RP (2) CAD 790 1 4 4 3.7 RAF1_RP (2) PNKD 25953 0.9836 3 2 18.3 RAF1_RP (2) FKBP8 23770 0.9994 3 3 9.7 RAF1_RP (2) TELO2 9894 0.959 3 3 5.9 RAF1_RP (2) FASN 2194 0.9979 3 3 1.6 RAF1_RP (2) RPN1 6184 0.9953 3 3 4.8 RAF1_RP (2) HAX1 10456 1 3 3 23.8 RAF1_RP (2) XPO1 7514 0.9998 3 3 5.6 RAF1_RP (2) ARL1 400 0.9998 2 2 7.7 RAF1_RP (2) C12orf23 90488 0.9865 2 2 30.2 RAF1_RP (2) KPNB1 3837 0.9774 2 2 3.2 RAF1_RP (2) DNAJA3 9093 0.9734 2 2 7.1 RAF1_RP (2) RANGAP1 5905 0.9654 2 2 4.3 RAF1_RP (2) COPB1 1315 0.9624 2 2 3 RAF1_RP (2) GLUD1 2746 0.9919 2 2 6.3 RAF1_RP (2) CCT8 10694 0.9923 2 2 4.4 RAF1_RP (2) DCAF7 10238 0.9994 2 2 8.2 RAF1_RP (2) SLC1A5 6510 0.9989 2 2 7.6 RAF1_RP (2) SSR4 6748 0.9996 2 2 13.9 RAF1_RP (2) NAP1L1 4673 0.9975 2 2 9.5 RAF1_RP (2) RUVBL1 8607 0.9961 2 2 7.7 RAF1_RP (2) SRSF2 6427 0.9929 2 2 11.3 RAF1_RP (2) NDUFA5 4698 0.9929 2 2 16.4 RAF1_RP (2) ATAD3C 219293 0.9916 2 2 6.8 RAF1_RP/RP (1) RAF1 5894 1 119 17 29.5 RAF1_RP/RP (1) YWHAE 7531 1 94 20 55.3 [PN] RAF1_RP/RP (1) YWHAG 7532 1 52 14 36 [PH;PN] RAF1_RP/RP (1) CDC37 11140 1 50 13 26.7 RAF1_RP/RP (1) TIMM50 92609 1 46 8 9 RAF1_RP/RP (1) YWHAZ 7534 1 38 8 30.6 [PN] RAF1_RP/RP (1) ATP1A1 476 1 35 14 15.4 RAF1_RP/RP (1) YWHAB 7529 1 34 10 48.8 [PN] RAF1_RP/RP (1) YWHAH 7533 1 30 8 29.3 [PN] RAF1_RP/RP (1) LOC731751 731751 1 17 10 3.6 RAF1_RP/RP (1) EMD 2010 1 16 5 16.1 RAF1_RP/RP (1) YWHAQ 10971 1 13 5 31.4 [PH;PN] RAF1_RP/RP (1) GCN1L1 10985 1 13 11 7.5 RAF1_RP/RP (1) DNAJA2 10294 1 13 5 10.7 RAF1_RP/RP (1) NRAS 4893 1 9 5 31.2 [PN] RAF1_RP/RP (1) CAD 790 1 8 4 3.1 RAF1_RP/RP (1) KRAS 3845 0.9997 8 4 30.2 [PN] RAF1_RP/RP (1) ATAD3A 55210 0.9999 6 4 10.4 235

RAF1_RP/RP (1) FANCD2 2177 1 6 4 4.4 RAF1_RP/RP (1) SV2A 9900 1 6 2 3.2 RAF1_RP/RP (1) HNRNPF 3185 0.9791 6 3 10.1 RAF1_RP/RP (1) CCT8 10694 1 5 5 12.2 RAF1_RP/RP (1) HAX1 10456 1 5 3 17.2 RAF1_RP/RP (1) PNKD 25953 0.9939 5 2 19.7 RAF1_RP/RP (1) SEC61A1 29927 0.9961 4 2 5.9 RAF1_RP/RP (1) SCO2 9997 0.9567 4 2 10.5 RAF1_RP/RP (1) CCT5 22948 0.9951 4 2 3.3 RAF1_RP/RP (1) TARS2 80222 0.9942 4 3 5.2 RAF1_RP/RP (1) RPS27L 51065 0.986 4 2 28.6 RAF1_RP/RP (1) DNAJA1 3301 0.9994 4 3 6.5 RAF1_RP/RP (1) CAND2 23066 1 4 3 5.8 RAF1_RP/RP (1) GLUD2 2747 1 4 3 7.5 RAF1_RP/RP (1) GRAMD1A 57655 0.9996 4 2 6.9 RAF1_RP/RP (1) RBBP7 5931 0.9995 4 3 7.1 RAF1_RP/RP (1) CTNNA2 1496 0.995 3 2 2 RAF1_RP/RP (1) PSMC2 5701 0.9961 3 2 5.5 RAF1_RP/RP (1) CHST14 113189 0.9932 3 3 5.1 RAF1_RP/RP (1) APOL2 23780 0.9543 3 2 6.5 RAF1_RP/RP (1) RBM39 9584 1 3 2 5.2 RAF1_RP/RP (1) MTDH 92140 1 3 3 8.8 RAF1_RP/RP (1) APP 351 0.9547 3 2 3.1 RAF1_RP/RP (1) HAUS5 23354 0.9951 3 2 3.3 RAF1_RP/RP (1) PSMD3 5709 0.9985 3 2 6.2 RAF1_RP/RP (1) TM9SF1 10548 0.9994 3 3 6.3 RAF1_RP/RP (1) CSE1L 1434 0.998 3 2 2 RAF1_RP/RP (1) DNAJA3 9093 0.9995 3 2 6.5 RAF1_RP/RP (1) RPN1 6184 0.9975 3 3 6.8 RAF1_RP/RP (1) ARAF 369 0.9972 3 2 11.7 RAF1_RP/RP (1) AKAP8L 26993 0.9998 3 3 8.4 RAF1_RP/RP (1) ALG1 56052 0.9808 2 2 4.1 RAF1_RP/RP (1) SDF4 51150 1 2 2 11.5 RAF1_RP/RP (1) ARF1 375 0.9701 2 2 12.7 RAF1_RP/RP (1) TRIP13 9319 0.9625 2 2 4.2 RAF1_RP/RP (1) P4HA1 5033 0.9997 2 2 6.2 RAF1_RP/RP (1) FANCI 55215 0.9986 2 2 1.9 RAF1_RP/RP (1) MTOR 2475 0.9986 2 2 1.7 RAF1_RP/RP (1) ATR 545 0.998 2 2 0.8 RAF1_RP/RP (1) WNK1 65125 0.998 2 2 1 RAF1_RP/RP (1) FASTKD5 60493 0.993 2 2 3.3 RAF1_RP/RP (1) QPCTL 54814 0.9918 2 2 6.9 RAF1_RP/RP (1) SLC25A3 5250 0.9867 2 2 9.7 RAF1_RP/RP (1) ABCC1 4363 0.9956 2 2 1.3 RAF1_RP/RP (1) UBB 7314 0.9897 2 2 23.6 RAF1_RP/RP (2) YWHAE 7531 1 174 28 69.8 [PN] RAF1_RP/RP (2) RAF1 5894 1 174 25 36.9 RAF1_RP/RP (2) CDC37 11140 1 45 16 28.6 RAF1_RP/RP (2) PRKDC 5591 1 44 31 9.3 RAF1_RP/RP (2) YWHAB 7529 1 29 8 45.9 [PN] RAF1_RP/RP (2) YWHAZ 7534 1 29 9 33.5 [PN] RAF1_RP/RP (2) TIMM50 92609 1 25 6 15.8 RAF1_RP/RP (2) YWHAG 7532 1 24 9 47.4 [PH;PN] RAF1_RP/RP (2) GCN1L1 10985 1 23 19 11.8 236

RAF1_RP/RP (2) YWHAH 7533 1 21 8 29.3 [PN] RAF1_RP/RP (2) FKBP5 2289 1 20 9 24.1 RAF1_RP/RP (2) EMD 2010 1 16 7 29.5 RAF1_RP/RP (2) ATP1A1 476 1 12 8 12.6 RAF1_RP/RP (2) STIP1 10963 1 11 9 15.3 RAF1_RP/RP (2) YWHAQ 10971 0.9999 11 4 25.7 [PH;PN] RAF1_RP/RP (2) DNAJA1 3301 0.9997 8 3 11.8 RAF1_RP/RP (2) NRAS 4893 0.9992 7 5 31.2 [PN] RAF1_RP/RP (2) RANBP2 5903 1 7 6 2.7 RAF1_RP/RP (2) SEC61A1 29927 1 7 4 11.3 RAF1_RP/RP (2) GLUD2 2747 1 7 6 11.6 RAF1_RP/RP (2) PSMC2 5701 1 6 4 14.8 RAF1_RP/RP (2) DNAJA2 10294 1 6 4 10.4 RAF1_RP/RP (2) CALU 813 0.9594 6 5 17.1 RAF1_RP/RP (2) RPN1 6184 0.9623 6 4 8.7 RAF1_RP/RP (2) MDN1 23195 1 6 6 1.5 RAF1_RP/RP (2) SDF4 51150 1 6 3 12.9 RAF1_RP/RP (2) CAD 790 1 6 5 3.8 RAF1_RP/RP (2) AKAP8L 26993 0.9998 5 4 7.4 RAF1_RP/RP (2) GEMIN4 50628 0.9762 5 4 4.5 RAF1_RP/RP (2) SV2A 9900 0.9999 5 2 3.4 RAF1_RP/RP (2) CCT8 10694 0.9996 5 4 8.6 RAF1_RP/RP (2) ARF1 375 0.9994 5 3 24.3 RAF1_RP/RP (2) HAX1 10456 1 5 5 21.5 RAF1_RP/RP (2) LETM1 3954 0.9999 4 2 3.4 RAF1_RP/RP (2) KRAS 3845 0.9954 4 3 30.2 [PN] RAF1_RP/RP (2) CSE1L 1434 1 4 4 4.5 RAF1_RP/RP (2) PHGDH 26227 1 4 3 7.1 RAF1_RP/RP (2) ATAD3A 55210 0.9997 4 4 11 RAF1_RP/RP (2) CHST14 113189 1 4 2 4.8 RAF1_RP/RP (2) CCT2 10576 1 4 4 9.5 RAF1_RP/RP (2) ATP2A1 487 1 4 3 2.4 RAF1_RP/RP (2) PSMD3 5709 0.9999 4 4 12.9 RAF1_RP/RP (2) SLC25A11 8402 0.9979 4 3 11.8 RAF1_RP/RP (2) SLC3A2 6520 1 4 3 8.5 RAF1_RP/RP (2) PPP6R3 55291 0.9994 3 3 5.7 RAF1_RP/RP (2) QPCTL 54814 0.993 3 3 13.9 RAF1_RP/RP (2) SALL2 6297 0.9829 3 2 2.8 RAF1_RP/RP (2) FARSA 2193 0.9608 3 3 6.9 RAF1_RP/RP (2) LMNB1 4001 0.9995 3 3 3.8 RAF1_RP/RP (2) POLR2B 5431 0.9979 3 3 3.2 RAF1_RP/RP (2) MTOR 2475 0.998 3 3 1.5 RAF1_RP/RP (2) SNRNP200 23020 0.998 3 3 2.6 RAF1_RP/RP (2) GANAB 23193 0.9855 3 3 3.3 RAF1_RP/RP (2) HNRNPF 3185 0.9985 3 3 12 RAF1_RP/RP (2) XPOT 11260 0.9988 3 3 2.2 RAF1_RP/RP (2) TELO2 9894 0.9999 3 3 6.3 RAF1_RP/RP (2) RANGAP1 5905 1 3 3 8.7 RAF1_RP/RP (2) SLC6A15 55117 0.9998 3 2 8.7 RAF1_RP/RP (2) MARCKS 4082 0.9998 3 2 9.6 RAF1_RP/RP (2) WDR6 11180 1 3 3 2.1 RAF1_RP/RP (2) NUP107 57122 0.9999 3 3 3.5 RAF1_RP/RP (2) CAND2 23066 0.9998 3 3 5.1 RAF1_RP/RP (2) PSMD6 9861 0.9999 3 3 5.7 237

RAF1_RP/RP (2) SEC13 6396 0.9866 2 2 8.4 RAF1_RP/RP (2) RAP1A 5906 0.987 2 2 11.4 [PN] RAF1_RP/RP (2) RPAP1 26015 0.9999 2 2 1.7 RAF1_RP/RP (2) GNAS 2778 0.9926 2 2 8.7 RAF1_RP/RP (2) EXOC4 60412 0.9872 2 2 2.4 RAF1_RP/RP (2) IGBP1 3476 0.9879 2 2 6.5 RAF1_RP/RP (2) HAUS6 54801 0.9511 2 2 2.4 RAF1_RP/RP (2) BAG2 9532 0.9515 2 2 13.3 RAF1_RP/RP (2) C5orf15 56951 0.9573 2 2 9.8 RAF1_RP/RP (2) NUDC 10726 1 2 2 9.1 RAF1_RP/RP (2) TMCO7 79613 0.9999 2 2 2.7 RAF1_RP/RP (2) FASN 2194 0.9691 2 2 0.9 RAF1_RP/RP (2) AK2 204 0.9737 2 2 10.3 RAF1_RP/RP (2) SSR4 6748 1 2 2 13.9 RAF1_RP/RP (2) LUZP1 7798 0.9775 2 2 3.1 RAF1_RP/RP (2) RPS10 6204 0.9793 2 2 9.1 RAF1_RP/RP (2) UBE3C 9690 0.9999 2 2 2 RAF1_RP/RP (2) DNAJA3 9093 0.9999 2 2 5.2 RAF1_RP/RP (2) SMC4 10051 0.9942 2 2 1.9 RAF1_RP/RP (2) ALG1 56052 0.9984 2 2 3.9 RAF1_RP/RP (2) EFHD2 79180 1 2 2 8.8 RAF1_RP/RP (2) CKAP5 9793 0.9987 2 2 1.2 RAF1_RP/RP (2) DCAF8 50717 0.9989 2 2 4.9 RAF1_RP/RP (2) COX4I1 1327 0.999 2 2 13.6 RAF1_RP/RP (2) PSMG1 8624 0.999 2 2 5.2 RAF1_RP/RP (2) FANCI 55215 0.9991 2 2 1.9 RAF1_RP/RP (2) EIF4G1 1981 0.9992 2 2 2.6 RAF1_RP/RP (2) MTDH 92140 0.9998 2 2 8.9 RAF1_RP/RP (2) TECR 9524 1 2 2 12.7 RAF1_RP/RP (2) WNK1 65125 0.9998 2 2 1 RAF1_RP/RP (2) TUBGCP2 10844 0.9966 2 2 2.8 RAF1_RP/RP (2) FAR1 84188 0.9967 2 2 4.9 RAF1_RP/RP (2) CDK1 983 0.9967 2 2 15.4 RAF1_RP/RP (2) TDRKH 11022 0.9968 2 2 3.9 RAF1_RP/RP (2) SCO2 9997 0.9971 2 2 13.9 RAF1_RP/RP (2) EIF2S2 8894 0.9987 2 2 6.6 RAF1_RP/RP (2) MAGED1 9500 0.9932 2 2 3 RAF1_RP/RP (2) SEC16A 9919 0.9975 2 2 1.9 RAF1_RP/RP (2) SNX1 6642 0.9977 2 2 4.4 RAF1_RP/RP (2) FANCD2 2177 0.9978 2 2 3.9 RAF1_RP/RP (2) ESYT2 57488 0.9995 2 2 3.5

238

Table S2-5. Proteins identified in FLAG-MEPCE purifications after background removal. Sample Separation Method is in the first column. "Hit Gene" and "Hit ID" are the HUGO Symbol and Entrez Gene ID, respectively; "Prophet" is the ProteinProphet probability; "n pep" is the number of unique peptides; "n spec" is the spectral count, "% cover" is the hit sequence coverage, in percentage; "BioGRID" indicates whether an interaction is reported in the BioGRID database [202] (v3.0.67) (PN = physical interaction, not high-throughput; PH = physical interaction, high-throughput). Sample Separation n n % Method and ID Hit Gene Hit ID Prophet pep spec cover BioGRID MEPCE_RP MEPCE 56257 1 151 31 36 MEPCE_RP LARP7 51574 1 40 25 38.8 [PH] MEPCE_RP SART3 9733 1 36 24 26.7 [PH] MEPCE_RP PRPF4 9128 1 18 14 25.1 [PH] MEPCE_RP CDK9 1025 1 14 7 17.5 [PH] MEPCE_RP PRPF3 9129 1 12 11 18.9 [PH] MEPCE_RP SNRNP200 23020 1 12 11 8.3 [PH] MEPCE_RP SART1 9092 1 11 10 21.5 [PH] MEPCE_RP CCNT1 904 1 10 9 15.6 [PH] MEPCE_RP HEXIM1 10614 1 8 6 16.7 [PH] MEPCE_RP EFTUD2 9343 1 6 5 6.6 [PH] MEPCE_RP HNRNPUL1 11100 0.9962 5 3 10.1 [PH] MEPCE_RP METT10D 79066 0.9714 5 3 6.4 [PH] MEPCE_RP LSM4 25804 0.9567 5 2 20.9 [PH] MEPCE_RP KPNA2 3838 1 5 4 11.9 [PH] MEPCE_RP RSL1D1 26156 0.9918 4 4 12.4 MEPCE_RP RBMXL1 494115 0.9995 4 3 7.4 MEPCE_RP PPIH 10465 0.9974 4 3 15.3 [PH] MEPCE_RP SRSF2 6427 0.9981 4 2 11.3 MEPCE_RP DDX23 9416 0.9999 4 4 9.5 [PH] MEPCE_RP LSM6 11157 0.9922 3 2 45 MEPCE_RP RPS15 6209 0.9973 3 2 9 MEPCE_RP USP39 10713 0.9998 3 2 4.1 [PH] MEPCE_RP MKI67 4288 0.9812 2 2 1.9 MEPCE_RP CSDA 8531 0.9648 2 2 17.5 [PH] MEPCE_RP SNRNP40 9410 0.9988 2 2 5.6 MEPCE_RP RPS10 6204 0.9999 2 2 9.1 MEPCE_RP MTDH 92140 0.9998 2 2 3.8 MEPCE_RP RPS12 6206 0.9889 2 2 13.6 MEPCE_RP/RP MEPCE 56257 1 213 31 27.9 MEPCE_RP/RP LARP7 51574 1 68 30 37.8 [PH] MEPCE_RP/RP SART3 9733 1 59 32 26.8 [PH] MEPCE_RP/RP SNRNP200 23020 1 31 21 9.6 [PH] MEPCE_RP/RP PRPF3 9129 1 25 13 21.4 [PH] MEPCE_RP/RP DDX23 9416 1 18 14 18.7 [PH] MEPCE_RP/RP PRPF4 9128 1 16 10 24.3 [PH] MEPCE_RP/RP CDK9 1025 1 14 6 17.5 [PH] 239

MEPCE_RP/RP EFTUD2 9343 1 14 10 11.1 [PH] MEPCE_RP/RP SART1 9092 1 12 10 17.1 [PH] MEPCE_RP/RP SSB 6741 1 10 5 12 MEPCE_RP/RP HEXIM1 10614 1 10 5 12.3 [PH] MEPCE_RP/RP CCNT1 904 1 10 8 11.6 [PH] MEPCE_RP/RP RPS7 6201 0.9844 8 4 16.5 MEPCE_RP/RP MTDH 92140 1 8 6 12.7 MEPCE_RP/RP SRSF5 6430 0.9763 7 3 7.7 MEPCE_RP/RP LSM6 11157 0.9929 6 4 33.8 MEPCE_RP/RP PRPF4B 8899 1 6 5 4 MEPCE_RP/RP CSDA 8531 1 6 5 30.4 [PH] MEPCE_RP/RP KPNA2 3838 1 6 5 13.4 [PH] MEPCE_RP/RP METT10D 79066 1 6 3 4.3 [PH] MEPCE_RP/RP MKI67 4288 1 5 5 3 MEPCE_RP/RP LOC731751 731751 1 5 4 1.6 MEPCE_RP/RP RPL22 6146 1 5 4 31.2 MEPCE_RP/RP HP1BP3 50809 0.9994 4 4 7.4 MEPCE_RP/RP LUC7L 55692 0.9964 4 3 12.6 MEPCE_RP/RP MOV10 4343 0.9548 3 2 2.9 MEPCE_RP/RP G3BP1 10146 0.9974 3 2 4.5 MEPCE_RP/RP RPL36AL 6166 0.9979 3 2 17 MEPCE_RP/RP RBM39 9584 1 3 2 6.7 MEPCE_RP/RP ZC3HAV1 56829 1 3 3 6.7 MEPCE_RP/RP EBNA1BP2 10969 0.9965 3 2 6.9 MEPCE_RP/RP NOLC1 9221 1 3 3 6.6 MEPCE_RP/RP RPS12 6206 0.9811 3 2 13.6 MEPCE_RP/RP CCNT2 905 1 3 3 9.8 [PH] MEPCE_RP/RP PA2G4 5036 0.9829 3 3 5.1 MEPCE_RP/RP SNRPD2 6633 0.9847 3 3 25 [PH] MEPCE_RP/RP SRPK2 6733 0.9908 3 3 4.2 MEPCE_RP/RP KPNB1 3837 0.998 3 3 3.2 [PH] MEPCE_RP/RP PRPF19 27339 0.9999 3 3 5.4 MEPCE_RP/RP SERBP1 26135 0.9999 3 3 8.3 MEPCE_RP/RP RPS10 6204 0.9999 2 2 14.5 MEPCE_RP/RP EIF2S2 8894 0.9997 2 2 6.3 MEPCE_RP/RP SRSF2 6427 0.9999 2 2 18.1 MEPCE_RP/RP PURB 5814 1 2 2 10.3 MEPCE_RP/RP MAP4 4134 0.9983 2 2 3.3 MEPCE_RP/RP HNRNPUL1 11100 0.9991 2 2 5 [PH] MEPCE_RP/RP ADAR 103 1 2 2 2.1 MEPCE_RP/RP LUC7L3 51747 0.9999 2 2 7.4 [PH] MEPCE_RP/RP RBM34 23029 0.9972 2 2 8.4 MEPCE_RP/RP WIBG 84305 0.999 2 2 20.2 MEPCE_RP/RP STAU1 6780 0.9996 2 2 4.2 MEPCE_RP/RP RFC1 5981 0.9996 2 2 2.6 MEPCE_RP/RP SNRNP27 11017 1 2 2 22.6 [PH]

240

Table S2-6. SAINT analysis of proteins identified in FLAG-EIF4A2 or FLAG-RAF1 purifications. SAINT was used to score the probability for each of the biological replicates (iProb; "|" serves as a delimiter); these probabilities were averaged (AvgP). "Sample ID" is our internal sample labeling; "s spec" are the spectral counts in the individual relicates; "n spec in controls" refers to the spectral counts for each bait across the 7 controls used for the semi-supervised modeling. Data is sorted by Sample description, then by AvgP. SAINT scores ≥0.9 across the biological replicates for each bait / separation method are highlighted: EIF4A2 separated by 1D LC-MS/MS (EIF4A2_RP; blue); EIF4A2 separated by 2D (EIF4A2_RPRP; green); RAF1 separated by 1D (RAF1_RP; orange) or by 2D (RAF_RPRP; yellow). Sample description Hit HUGO Sample ID n spec iProb n spec in controls AvgP EIF4A2_RP EIF3B 10018|10192 26|34 1.00|1.00 0|0|0|0|0|0|0 1 EIF4A2_RP EIF3A 10018|10192 57|80 1.00|1.00 0|0|0|0|0|0|0 1 EIF4A2_RP EIF3L 10018|10192 24|30 1.00|1.00 0|0|0|0|0|0|0 1 EIF4A2_RP EIF3E 10018|10192 17|28 1.00|1.00 0|0|0|0|0|0|0 1 EIF4A2_RP EIF3G 10018|10192 10|13 1.00|1.00 0|0|0|0|0|0|0 1 EIF4A2_RP EIF4G3 10018|10192 23|29 1.00|1.00 0|0|0|0|0|0|0 1 EIF4A2_RP EIF4G2 10018|10192 12|27 1.00|1.00 0|0|0|0|0|0|0 1 EIF4A2_RP BAT2L2 10018|10192 11|16 1.00|1.00 0|0|0|0|0|0|0 1 EIF4A2_RP PDCD4 10018|10192 19|24 1.00|1.00 0|0|0|0|0|0|0 1 EIF4A2_RP EIF4G1 10018|10192 69|75 1.00|1.00 0|0|0|0|0|0|0 1 EIF4A2_RP EIF3H 10018|10192 9|11 1.00|1.00 0|0|0|0|0|0|0 1 EIF4A2_RP EIF3C 10018|10192 29|42 1.00|1.00 0|0|0|0|0|0|0 1 EIF4A2_RP PABPC4 10018|10192 18|29 1.00|1.00 0|0|2|0|0|0|0 0.999 EIF4A2_RP EIF3I 10018|10192 9|16 1.00|1.00 0|0|0|0|0|0|0 0.999 EIF4A2_RP EIF3D 10018|10192 7|13 1.00|1.00 0|0|0|0|0|0|0 0.998 EIF4A2_RP EIF3F 10018|10192 8|14 1.00|1.00 0|0|0|0|0|0|0 0.998 EIF4A2_RP PABPC1 10018|10192 23|27 1.00|1.00 0|0|5|0|0|5|0 0.998 EIF4A2_RP BAT2 10018|10192 8|25 1.00|1.00 0|0|0|0|0|0|0 0.998 EIF4A2_RP LARP1 10018|10192 7|5 1.00|1.00 0|0|0|0|0|0|0 0.997 EIF4A2_RP RPS15 10018|10192 2|2 1.00|1.00 0|0|0|0|0|0|0 0.996 EIF4A2_RP HSPA1L 10018|10192 2|3 0.99|1.00 0|0|0|0|0|0|0 0.996 EIF4A2_RP RPL22 10018|10192 1|1 1.00|0.99 0|0|0|0|0|0|0 0.995 EIF4A2_RP EIF3M 10018|10192 5|8 0.99|0.99 0|0|0|0|0|0|0 0.993 EIF4A2_RP SFPQ 10018|10192 7|3 0.99|0.99 0|0|0|0|0|0|0 0.992 EIF4A2_RP NUFIP2 10018|10192 12|2 1.00|0.98 0|0|0|0|0|0|0 0.992 EIF4A2_RP SRSF2 10018|10192 1|2 0.99|1.00 0|0|0|0|0|0|0 0.992 EIF4A2_RP C10orf137 10018|10192 4|2 1.00|0.98 0|0|0|0|0|0|0 0.99 EIF4A2_RP BAT2L1 10018|10192 6|3 1.00|0.98 0|0|0|0|0|0|0 0.988 EIF4A2_RP EIF3J 10018|10192 1|9 0.98|1.00 0|0|0|0|0|0|0 0.988 EIF4A2_RP ATXN2 10018|10192 7|2 1.00|0.98 0|0|0|0|0|0|0 0.988 EIF4A2_RP XRCC5 10018|10192 2|2 0.99|0.99 0|0|0|0|0|0|0 0.987 EIF4A2_RP G3BP1 10018|10192 4|4 0.99|0.99 0|0|1|0|0|0|0 0.987 EIF4A2_RP EIF4A1 10018|10192 1|3 0.98|1.00 0|0|0|0|0|0|0 0.986 EIF4A2_RP UPF1 10018|10192 2|2 0.99|0.98 0|0|0|0|0|0|0 0.986 241

EIF4A2_RP EIF4A3 10018|10192 1|1 0.98|0.99 0|0|0|0|0|0|0 0.986 EIF4A2_RP RGS1 10018|10192 1|1 0.98|0.99 0|0|0|0|0|0|0 0.985 EIF4A2_RP LOC100131693 10018|10192 9|2 1.00|0.96 0|0|0|0|0|0|0 0.982 EIF4A2_RP MSI2 10018|10192 1|1 0.99|0.98 0|0|0|0|0|0|0 0.981 EIF4A2_RP LOC100132983 10018|10192 2|1 0.99|0.97 0|0|0|0|0|0|0 0.981 EIF4A2_RP HNRPDL 10018|10192 5|1 0.97|0.99 0|0|0|0|0|0|0 0.98 EIF4A2_RP CSDA 10018|10192 4|2 0.99|0.96 0|0|1|0|0|0|0 0.975 EIF4A2_RP RBM39 10018|10192 1|1 0.96|0.97 0|0|0|0|0|0|0 0.964 EIF4A2_RP DDX50 10018|10192 2|2 0.96|0.95 1|0|0|0|0|0|0 0.954 EIF4A2_RP LRPPRC 10018|10192 3|3 0.95|0.94 0|0|4|0|0|0|0 0.944 EIF4A2_RP OBSL1 10018|10192 1|1 0.96|0.93 0|0|0|0|0|0|0 0.943 EIF4A2_RP HNRNPL 10018|10192 2|6 0.91|0.97 1|0|1|0|0|0|0 0.94 EIF4A2_RP ATXN2L 10018|10192 13|1 0.97|0.90 0|0|0|0|0|0|0 0.936 EIF4A2_RP EIF2S1 10018|10192 1|2 0.89|0.95 0|0|1|0|0|0|0 0.921 EIF4A2_RP DDX6 10018|10192 1|1 0.91|0.88 0|0|1|0|0|0|0 0.895 EIF4A2_RP RPS7 10018|10192 2|1 0.94|0.84 0|0|0|0|0|2|0 0.888 EIF4A2_RP EWSR1 10018|10192 1|1 0.90|0.88 0|0|1|0|0|0|0 0.886 EIF4A2_RP DHX9 10018|10192 7|4 0.93|0.72 0|0|6|0|0|2|0 0.828 EIF4A2_RP IGF2BP1 10018|10192 2|4 0.74|0.89 0|0|1|0|0|0|2 0.816 EIF4A2_RP HNRNPD 10018|10192 5|4 0.90|0.72 1|1|2|0|0|1|1 0.811 EIF4A2_RP YBX1 10018|10192 7|11 0.76|0.85 0|0|5|1|2|0|2 0.805 EIF4A2_RP RPS27 10018|10192 1|4 0.56|0.96 0|0|2|0|0|1|0 0.761 EIF4A2_RP DDX17 10018|10192 4|4 0.73|0.64 0|1|3|0|1|0|2 0.685 EIF4A2_RP RPS14 10018|10192 4|11 0.37|0.96 0|0|4|2|0|3|2 0.668 EIF4A2_RP HNRNPA2B1 10018|10192 10|9 0.73|0.53 2|0|5|0|1|6|1 0.63 EIF4A2_RP HNRNPA3 10018|10192 5|1 0.76|0.31 0|0|4|0|0|0|1 0.533 EIF4A2_RP HNRNPK 10018|10192 3|2 0.57|0.25 0|1|4|0|0|2|0 0.411 EIF4A2_RP RPS4X 10018|10192 2|10 0.01|0.76 1|2|5|0|0|4|0 0.382 EIF4A2_RP HNRNPA1 10018|10192 5|5 0.40|0.32 1|3|3|0|0|3|2 0.36 EIF4A2_RP RPS18 10018|10192 7|20 0.00|0.71 2|0|9|0|1|7|6 0.355 EIF4A2_RP RPS11 10018|10192 2|3 0.24|0.45 0|0|3|0|0|3|1 0.341 EIF4A2_RP RPS16 10018|10192 2|7 0.00|0.66 4|1|2|0|0|6|0 0.33 EIF4A2_RP RPS3A 10018|10192 3|7 0.03|0.58 0|0|6|0|1|4|2 0.306 EIF4A2_RP RPS9 10018|10192 3|9 0.00|0.60 2|0|4|0|2|6|3 0.301 EIF4A2_RP SRSF1 10018|10192 9|1 0.51|0.05 0|0|3|0|0|2|2 0.28 EIF4A2_RP HNRNPM 10018|10192 16|7 0.52|0.00 2|5|7|0|0|5|6 0.26 EIF4A2_RP HNRNPC 10018|10192 2|2 0.22|0.25 6|0|2|0|0|0|0 0.231 EIF4A2_RP RPS8 10018|10192 3|11 0.00|0.41 3|0|6|0|4|5|3 0.206 EIF4A2_RP HNRNPR 10018|10192 3|3 0.17|0.13 2|0|3|0|0|3|1 0.15 EIF4A2_RP RPL19 10018|10192 2|2 0.07|0.07 1|0|1|3|0|1|2 0.074 EIF4A2_RP HNRNPAB 10018|10192 2|1 0.11|0.02 1|0|2|0|0|1|3 0.067 EIF4A2_RP RPL24 10018|10192 1|1 0.04|0.04 2|0|0|0|1|4|0 0.04 EIF4A2_RP RPL10A 10018|10192 1|1 0.02|0.02 1|0|2|0|0|2|1 0.016 EIF4A2_RP RPS2 10018|10192 2|4 0.00|0.03 4|0|1|1|2|3|3 0.013 EIF4A2_RP NPM1 10018|10192 2|5 0.00|0.00 14|8|15|6|1|3|6 0 EIF4A2_RP WDR77 10018|10192 6|2 0.00|0.00 20|11|14|0|11|14|6 0 EIF4A2_RP KIF11 10018|10192 4|0 0.00|0.00 100|19|78|36|19|43|38 0 EIF4A2_RP TRIM21 10018|10192 0|1 0.00|0.00 0|0|6|0|1|0|2 0 EIF4A2_RP RPS3 10018|10192 2|15 0.00|0.00 3|0|6|2|1|9|3 0 EIF4A2_RP RPLP0 10018|10192 0|1 0.00|0.00 4|2|3|0|1|4|0 0 EIF4A2_RP PRMT5 10018|10192 9|5 0.00|0.00 59|15|55|14|17|43|30 0 EIF4A2_RP TUBB 10018|10192 1|5 0.00|0.00 25|1|1|0|3|8|3 0 EIF4A2_RP CLNS1A 10018|10192 1|1 0.00|0.00 8|6|19|6|5|15|8 0 EIF4A2_RP RPLP2 10018|10192 0|6 0.00|0.00 0|0|1|0|0|4|2 0 242

EIF4A2_RP NCL 10018|10192 3|6 0.00|0.00 4|1|14|16|6|12|6 0 EIF4A2_RP STK38 10018|10192 7|10 0.00|0.00 32|10|32|11|3|25|12 0 EIF4A2_RP DHX15 10018|10192 1|0 0.00|0.00 0|2|13|1|1|5|5 0 EIF4A2_RP TUBA1C 10018|10192 4|3 0.00|0.00 42|0|10|8|5|16|7 0 EIF4A2_RP PPM1B 10018|10192 6|0 0.00|0.00 21|33|2|31|2|40|28 0 EIF4A2_RP KRT1 10018|10192 0|1 0.00|0.00 192|250|68|43|29|31|51 0 EIF4A2_RP MAP1B 10018|10192 1|2 0.00|0.00 0|0|10|1|0|7|6 0 EIF4A2_RP RPL18 10018|10192 3|2 0.00|0.00 4|1|5|1|2|3|3 0 EIF4A2_RP RPS6 10018|10192 1|8 0.00|0.00 2|1|4|0|0|0|3 0 EIF4A2_RP QPCT 10018|10192 3|3 0.00|0.00 9|5|15|10|0|11|4 0 EIF4A2_RP PTBP1 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP LARS 10018|10192 2|1 0.00|0.00 0|3|7|0|0|3|1 0 EIF4A2_RP RPL10 10018|10192 0|2 0.00|0.00 0|0|0|0|1|2|1 0 EIF4A2_RP RPS26 10018|10192 0|2 0.00|0.00 2|0|2|0|0|1|0 0 EIF4A2_RP HNRNPH1 10018|10192 1|0 0.00|0.00 0|0|3|0|0|1|0 0 EIF4A2_RP RPS15A 10018|10192 0|3 0.00|0.00 0|0|1|0|0|2|0 0 EIF4A2_RP RPLP1 10018|10192 1|0 0.00|0.00 0|1|0|0|0|1|0 0 EIF4A2_RP RPL9 10018|10192 0|1 0.00|0.00 0|0|0|0|1|0|0 0 EIF4A2_RP RPL23 10018|10192 1|0 0.00|0.00 1|0|1|1|0|0|0 0 EIF4A2_RP RPS19 10018|10192 0|6 0.00|0.00 0|0|2|0|0|4|0 0 EIF4A2_RP SUB1 10018|10192 1|3 0.00|0.00 5|0|15|6|1|8|3 0 EIF4A2_RP RPS17 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP RPL7A 10018|10192 1|1 0.00|0.00 4|0|5|0|1|9|4 0 EIF4A2_RP XRCC6 10018|10192 0|1 0.00|0.00 0|0|0|0|0|2|1 0 EIF4A2_RP ACTB 10018|10192 3|5 0.00|0.00 65|33|48|59|23|15|47 0 EIF4A2_RP THOC4 10018|10192 2|0 0.00|0.00 0|0|0|0|0|0|1 0 EIF4A2_RP RPL12 10018|10192 1|0 0.00|0.00 1|0|3|0|0|1|2 0 EIF4A2_RP RPL11 10018|10192 1|0 0.00|0.00 1|0|1|0|1|0|1 0 EIF4A2_RP SRSF3 10018|10192 3|0 0.00|0.00 2|0|3|0|0|2|2 0 EIF4A2_RP DDX26B 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP RPS24 10018|10192 0|6 0.00|0.00 1|0|0|0|1|2|0 0 EIF4A2_RP HSPA5 10018|10192 4|1 0.00|0.00 3|5|11|0|2|10|3 0 EIF4A2_RP RPL4 10018|10192 2|1 0.00|0.00 3|0|4|5|0|7|2 0 EIF4A2_RP CMBL 10018|10192 1|0 0.00|0.00 15|5|9|5|2|8|5 0 EIF4A2_RP ILF3 10018|10192 6|1 0.00|0.00 0|0|7|0|0|2|2 0 EIF4A2_RP SYNCRIP 10018|10192 0|3 0.00|0.00 0|0|0|0|0|1|0 0 EIF4A2_RP ILF2 10018|10192 1|0 0.00|0.00 0|0|2|0|0|1|0 0 EIF4A2_RP PTS 10018|10192 1|0 0.00|0.00 2|0|4|1|0|3|1 0 EIF4A2_RP RPS28 10018|10192 0|2 0.00|0.00 0|0|1|0|0|1|1 0 EIF4A2_RP HIST1H1C 10018|10192 4|5 0.00|0.00 8|0|8|0|4|7|0 0 EIF4A2_RP RPSA 10018|10192 0|3 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP IARS 10018|10192 2|0 0.00|0.00 0|1|3|0|0|2|4 0 EIF4A2_RP SRSF7 10018|10192 3|1 0.00|0.00 0|0|3|0|0|4|3 0 EIF4A2_RP DDX3X 10018|10192 0|4 0.00|0.00 0|0|0|1|0|0|0 0 EIF4A2_RP SF3B2 10018|10192 1|0 0.00|0.00 2|0|7|3|0|7|5 0 EIF4A2_RP PARP1 10018|10192 0|1 0.00|0.00 0|0|1|0|0|0|0 0 EIF4A2_RP RPL13 10018|10192 2|3 0.00|0.00 4|0|7|3|2|6|2 0 EIF4A2_RP LOC100291837 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP THRAP3 10018|10192 4|0 0.00|0.00 16|0|9|7|2|7|4 0 EIF4A2_RP RPS10 10018|10192 0|3 0.00|0.00 0|0|0|0|0|1|0 0 EIF4A2_RP RPL6 10018|10192 5|3 0.00|0.00 8|0|11|2|0|8|5 0 EIF4A2_RP RPS20 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP RPL3 10018|10192 2|1 0.00|0.00 0|0|7|0|0|8|4 0 EIF4A2_RP RPL31 10018|10192 2|0 0.00|0.00 1|0|1|0|1|1|2 0 243

EIF4A2_RP EIF2S2 10018|10192 0|3 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP HNRNPU 10018|10192 7|5 0.00|0.00 7|3|7|0|1|9|4 0 EIF4A2_RP RPS13 10018|10192 0|1 0.00|0.00 2|0|1|0|1|2|0 0 EIF4A2_RP PABPN1 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP HNRNPA0 10018|10192 1|0 0.00|0.00 0|0|2|0|0|0|0 0 EIF4A2_RP HEATR1 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP RPL23A 10018|10192 2|4 0.00|0.00 2|0|8|0|0|5|3 0 EIF4A2_RP LUC7L2 10018|10192 0|2 0.00|0.00 0|0|6|0|0|1|2 0 EIF4A2_RP HNRNPF 10018|10192 2|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP RPL15 10018|10192 2|2 0.00|0.00 3|0|5|3|1|5|0 0 EIF4A2_RP SRRM2 10018|10192 6|0 0.00|0.00 0|0|1|4|0|0|2 0 EIF4A2_RP RPL36 10018|10192 0|1 0.00|0.00 0|0|2|0|1|2|0 0 EIF4A2_RP DYRK1B 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|17 0 EIF4A2_RP RBM14 10018|10192 2|0 0.00|0.00 0|0|1|0|0|0|1 0 EIF4A2_RP ELAVL1 10018|10192 5|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP EIF3K 10018|10192 0|3 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP TTN 10018|10192 0|1 0.00|0.00 1|0|1|0|0|2|0 0 EIF4A2_RP MLL2 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP RPL8 10018|10192 1|1 0.00|0.00 1|0|7|5|1|5|3 0 EIF4A2_RP BCLAF1 10018|10192 3|0 0.00|0.00 7|0|5|2|0|3|3 0 EIF4A2_RP NFKB1 10018|10192 2|0 0.00|0.00 0|0|0|0|4|0|19 0 EIF4A2_RP RPL27A 10018|10192 0|3 0.00|0.00 1|0|1|0|0|2|1 0 EIF4A2_RP PRSS1 10018|10192 3|0 0.00|0.00 3|0|7|0|2|5|6 0 EIF4A2_RP CIT 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP RPS12 10018|10192 0|2 0.00|0.00 0|0|1|0|0|0|1 0 EIF4A2_RP TUBB4 10018|10192 4|0 0.00|0.00 2|2|0|0|0|0|0 0 EIF4A2_RP RPL26 10018|10192 1|0 0.00|0.00 0|0|4|0|0|2|1 0 EIF4A2_RP RPL29 10018|10192 0|2 0.00|0.00 0|0|4|0|0|2|0 0 EIF4A2_RP RPS5 10018|10192 0|1 0.00|0.00 2|0|0|0|0|2|0 0 EIF4A2_RP RPL27 10018|10192 0|1 0.00|0.00 0|0|1|0|1|2|3 0 EIF4A2_RP RPS25 10018|10192 0|2 0.00|0.00 1|0|0|0|0|1|1 0 EIF4A2_RP LOC100292021 10018|10192 1|0 0.00|0.00 0|0|0|0|1|0|0 0 EIF4A2_RP RPS23 10018|10192 0|7 0.00|0.00 0|0|5|0|0|2|0 0 EIF4A2_RP RPL34 10018|10192 1|0 0.00|0.00 0|0|3|0|0|4|0 0 EIF4A2_RP DDX21 10018|10192 6|3 0.00|0.00 0|0|15|0|0|9|2 0 EIF4A2_RP NCOR1 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP DHX29 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP RBMXL2 10018|10192 0|1 0.00|0.00 1|0|0|0|1|0|0 0 EIF4A2_RP FAM83C 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP LARP4 10018|10192 3|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP FAM120A 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP KIF16B 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP WBP11 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP TTBK2 10018|10192 0|2 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP FXR2 10018|10192 4|0 0.00|0.00 0|0|0|0|0|1|0 0 EIF4A2_RP MKI67 10018|10192 1|0 0.00|0.00 0|0|2|0|0|0|0 0 EIF4A2_RP RBMX 10018|10192 7|0 0.00|0.00 0|0|0|0|0|2|0 0 EIF4A2_RP CAPRIN1 10018|10192 3|0 0.00|0.00 0|0|1|0|0|0|0 0 EIF4A2_RP SMC5 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP SERBP1 10018|10192 0|3 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP DDX3Y 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP MATR3 10018|10192 3|0 0.00|0.00 0|0|3|0|0|0|2 0 EIF4A2_RP DHX30 10018|10192 3|0 0.00|0.00 0|0|2|0|0|0|0 0 EIF4A2_RP RSL1D1 10018|10192 2|0 0.00|0.00 0|0|2|0|0|0|0 0 244

EIF4A2_RP GNL3 10018|10192 2|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP SMCHD1 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP MTDH 10018|10192 0|2 0.00|0.00 0|0|1|0|0|0|0 0 EIF4A2_RP SACS 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP PTBP2 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP CDC5L 10018|10192 2|0 0.00|0.00 0|0|1|0|0|0|0 0 EIF4A2_RP ABCE1 10018|10192 0|4 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP C22orf28 10018|10192 0|2 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP FMR1 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP NACA 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP GNB2L1 10018|10192 0|2 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP MOV10 10018|10192 3|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP USP10 10018|10192 2|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP HNRNPUL2 10018|10192 1|0 0.00|0.00 0|0|2|0|0|0|0 0 EIF4A2_RP C14orf166 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP LSM12 10018|10192 3|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP NUDCD1 10018|10192 0|3 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP FIP1L1 10018|10192 2|0 0.00|0.00 0|0|1|0|0|0|0 0 EIF4A2_RP IGF2BP3 10018|10192 3|0 0.00|0.00 0|0|1|0|0|0|0 0 EIF4A2_RP CUX1 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP TAF15 10018|10192 1|0 0.00|0.00 0|0|1|0|0|1|0 0 EIF4A2_RP FAM178B 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP U2AF1 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP NCBP1 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP PCBP1 10018|10192 2|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP MYCL1 10018|10192 0|2 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP RBMS1 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP TRA2A 10018|10192 2|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP C12orf41 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP MAST1 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP SUGP2 10018|10192 2|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP G3BP2 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP MATK 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP ZCCHC3 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP CCNA1 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP MRPL47 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP SRRM1 10018|10192 1|0 0.00|0.00 0|0|2|0|0|0|0 0 EIF4A2_RP LOC643446 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP CHD8 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP MSI1 10018|10192 2|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP ERI1 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP C2orf53 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP ELP3 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP LOC100293516 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP FER1L6 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP CRMP1 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP SOX30 10018|10192 2|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP BVES 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP EBF2 10018|10192 2|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP COL28A1 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP PLEKHM2 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP FGF5 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP MKRN1 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP DTX4 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 245

EIF4A2_RP HSPA2 10018|10192 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP SUPT7L 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP TJP2 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP PHLDB2 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP PSMB6 10018|10192 0|1 0.00|0.00 0|0|1|0|0|0|0 0 EIF4A2_RP NISCH 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP ARPC1A 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP MFSD1 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP XPO7 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP LOC729570 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP SLC4A4 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP ALPK3 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP C20orf12 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP GRM5 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP LOC644717 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP ATP10B 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP PTPDC1 10018|10192 0|2 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP CAPS 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RP CD22 10018|10192 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP EIF3B 10199|10025 70|84 1.00|1.00 0|0|0|0|0|0|0 1 EIF4A2_RPRP EIF3A 10199|10025 110|148 1.00|1.00 0|0|0|0|0|0|0 1 EIF4A2_RPRP EIF3L 10199|10025 73|80 1.00|1.00 0|0|0|0|0|0|0 1 EIF4A2_RPRP EIF3I 10199|10025 30|33 1.00|1.00 0|0|0|0|0|0|0 1 EIF4A2_RPRP EIF3E 10199|10025 43|53 1.00|1.00 0|0|0|0|0|0|0 1 EIF4A2_RPRP EIF4G3 10199|10025 57|99 1.00|1.00 0|0|0|0|0|0|0 1 EIF4A2_RPRP EIF4G2 10199|10025 24|27 1.00|1.00 0|0|0|0|0|0|0 1 EIF4A2_RPRP PDCD4 10199|10025 51|73 1.00|1.00 0|0|0|0|0|0|0 1 EIF4A2_RPRP EIF4G1 10199|10025 152|200 1.00|1.00 0|0|0|0|0|0|0 1 EIF4A2_RPRP EIF3H 10199|10025 27|32 1.00|1.00 0|0|0|0|0|0|0 1 EIF4A2_RPRP EIF3C 10199|10025 83|73 1.00|1.00 0|0|0|0|0|0|0 1 EIF4A2_RPRP SRSF5 10199|10025 3|3 1.00|1.00 0|0|0|0|0|0|0 0.999 EIF4A2_RPRP EIF3D 10199|10025 26|15 1.00|1.00 0|0|0|0|0|0|0 0.999 EIF4A2_RPRP BAT2L1 10199|10025 10|28 1.00|1.00 0|0|0|0|0|0|0 0.999 EIF4A2_RPRP EIF3G 10199|10025 15|39 1.00|1.00 0|0|0|0|0|0|0 0.999 EIF4A2_RPRP NUDCD1 10199|10025 22|16 1.00|1.00 0|0|0|0|0|0|0 0.999 EIF4A2_RPRP EIF3F 10199|10025 8|11 1.00|1.00 0|0|0|0|0|0|0 0.998 EIF4A2_RPRP LOC100131693 10199|10025 12|19 1.00|1.00 0|0|0|0|0|0|0 0.998 EIF4A2_RPRP PURB 10199|10025 3|3 0.99|1.00 0|0|0|0|0|0|0 0.996 EIF4A2_RPRP MOV10 10199|10025 4|10 1.00|1.00 0|0|0|0|0|0|0 0.996 EIF4A2_RPRP LARP1 10199|10025 6|23 0.99|1.00 0|0|0|0|0|0|0 0.995 EIF4A2_RPRP NONO 10199|10025 5|6 0.99|0.99 0|0|0|0|0|0|0 0.994 EIF4A2_RPRP EIF3K 10199|10025 8|4 0.99|0.99 0|0|0|0|0|0|0 0.993 EIF4A2_RPRP SFPQ 10199|10025 8|19 0.99|0.99 0|0|0|0|0|0|0 0.992 EIF4A2_RPRP C10orf137 10199|10025 4|13 0.99|1.00 0|0|0|0|0|0|0 0.992 EIF4A2_RPRP ELAVL1 10199|10025 4|11 0.99|0.99 0|0|0|0|0|0|0 0.991 EIF4A2_RPRP BAT2L2 10199|10025 12|43 0.98|1.00 0|0|0|0|0|0|0 0.991 EIF4A2_RPRP IGF2BP2 10199|10025 3|8 0.99|0.99 0|0|0|0|0|0|0 0.991 EIF4A2_RPRP FMR1 10199|10025 3|5 0.99|0.99 0|0|0|0|0|0|0 0.991 EIF4A2_RPRP PABPC4 10199|10025 24|82 0.98|1.00 0|0|2|0|0|0|0 0.989 EIF4A2_RPRP EIF4A1 10199|10025 5|21 1.00|0.98 0|0|0|0|0|0|0 0.989 EIF4A2_RPRP HNRNPF 10199|10025 2|4 0.99|0.99 0|0|0|0|0|0|0 0.988 EIF4A2_RPRP EIF3J 10199|10025 26|5 0.99|0.98 0|0|0|0|0|0|0 0.988 EIF4A2_RPRP UPF1 10199|10025 3|10 0.98|0.99 0|0|0|0|0|0|0 0.988 EIF4A2_RPRP C22orf28 10199|10025 4|3 0.99|0.98 0|0|0|0|0|0|0 0.985 246

EIF4A2_RPRP LSM12 10199|10025 1|5 0.98|0.99 0|0|0|0|0|0|0 0.985 EIF4A2_RPRP LARP4 10199|10025 4|11 0.99|0.98 0|0|0|0|0|0|0 0.984 EIF4A2_RPRP LARP4B 10199|10025 2|4 0.97|0.99 0|0|0|0|0|0|0 0.983 EIF4A2_RPRP FAM134B 10199|10025 3|4 0.99|0.98 0|0|0|0|0|0|0 0.982 EIF4A2_RPRP BAT2 10199|10025 38|18 1.00|0.96 0|0|0|0|0|0|0 0.979 EIF4A2_RPRP SMCHD1 10199|10025 3|5 0.98|0.97 0|0|0|0|0|0|0 0.977 EIF4A2_RPRP SRP9 10199|10025 1|1 0.98|0.97 0|0|0|0|0|0|0 0.975 EIF4A2_RPRP PCBP2 10199|10025 1|3 0.96|0.99 0|0|0|0|0|0|0 0.975 EIF4A2_RPRP NUFIP2 10199|10025 4|32 0.97|0.97 0|0|0|0|0|0|0 0.974 EIF4A2_RPRP HNRPDL 10199|10025 1|3 0.96|0.99 0|0|0|0|0|0|0 0.973 EIF4A2_RPRP ATXN2 10199|10025 2|10 0.94|1.00 0|0|0|0|0|0|0 0.97 EIF4A2_RPRP SRSF9 10199|10025 1|8 0.95|0.99 0|0|0|0|0|0|0 0.97 EIF4A2_RPRP XRCC5 10199|10025 2|2 0.97|0.97 0|0|0|0|0|0|0 0.969 EIF4A2_RPRP RPL22 10199|10025 1|1 0.98|0.95 0|0|0|0|0|0|0 0.965 EIF4A2_RPRP PTBP1 10199|10025 2|1 0.98|0.95 0|0|0|0|0|0|0 0.963 EIF4A2_RPRP USP10 10199|10025 1|4 0.93|0.99 0|0|0|0|0|0|0 0.962 EIF4A2_RPRP DDX3X 10199|10025 7|4 0.99|0.93 0|0|0|1|0|0|0 0.961 EIF4A2_RPRP PABPC1 10199|10025 26|85 0.91|1.00 0|0|5|0|0|5|0 0.956 EIF4A2_RPRP LRPPRC 10199|10025 8|14 0.95|0.95 0|0|4|0|0|0|0 0.954 EIF4A2_RPRP LARP1B 10199|10025 1|3 0.95|0.96 0|0|0|0|0|0|0 0.953 EIF4A2_RPRP DDX50 10199|10025 3|10 0.94|0.96 1|0|0|0|0|0|0 0.95 EIF4A2_RPRP VAC14 10199|10025 1|2 0.94|0.96 0|0|0|0|0|0|0 0.949 EIF4A2_RPRP EIF2S1 10199|10025 7|5 0.97|0.92 0|0|1|0|0|0|0 0.947 EIF4A2_RPRP C14orf166 10199|10025 1|1 0.95|0.94 0|0|0|0|0|0|0 0.947 EIF4A2_RPRP FXR2 10199|10025 2|7 0.92|0.97 0|0|0|0|0|1|0 0.943 EIF4A2_RPRP DPP7 10199|10025 1|1 0.95|0.93 0|0|0|0|0|0|0 0.939 EIF4A2_RPRP CAPRIN1 10199|10025 2|7 0.90|0.97 0|0|1|0|0|0|0 0.934 EIF4A2_RPRP G3BP1 10199|10025 2|11 0.87|0.99 0|0|1|0|0|0|0 0.931 EIF4A2_RPRP DHX29 10199|10025 4|1 0.98|0.88 0|0|0|0|0|0|0 0.929 EIF4A2_RPRP CSDA 10199|10025 2|11 0.85|0.98 0|0|1|0|0|0|0 0.918 EIF4A2_RPRP DHX30 10199|10025 2|3 0.88|0.85 0|0|2|0|0|0|0 0.865 EIF4A2_RPRP EIF3M 10199|10025 2|1 0.93|0.76 0|0|0|0|0|0|0 0.848 EIF4A2_RPRP HNRNPL 10199|10025 3|6 0.82|0.87 1|0|1|0|0|0|0 0.843 EIF4A2_RPRP ATR 10199|10025 1|1 0.89|0.79 0|0|0|0|0|0|0 0.842 EIF4A2_RPRP RPS7 10199|10025 8|2 0.95|0.71 0|0|0|0|0|2|0 0.833 EIF4A2_RPRP EWSR1 10199|10025 1|3 0.79|0.87 0|0|1|0|0|0|0 0.83 EIF4A2_RPRP RPS10 10199|10025 5|1 0.92|0.70 0|0|0|0|0|1|0 0.811 EIF4A2_RPRP ZC3H18 10199|10025 1|2 0.78|0.78 0|0|0|2|0|0|0 0.778 EIF4A2_RPRP MTDH 10199|10025 2|1 0.87|0.63 0|0|1|0|0|0|0 0.751 EIF4A2_RPRP RPS15A 10199|10025 5|5 0.82|0.67 0|0|1|0|0|2|0 0.743 EIF4A2_RPRP RPL9 10199|10025 1|1 0.77|0.70 0|0|0|0|1|0|0 0.736 EIF4A2_RPRP RPS27L 10199|10025 2|1 0.85|0.61 0|0|1|0|0|0|0 0.729 EIF4A2_RPRP IGF2BP1 10199|10025 3|7 0.65|0.73 0|0|1|0|0|0|2 0.691 EIF4A2_RPRP PARP1 10199|10025 1|1 0.75|0.63 0|0|1|0|0|0|0 0.689 EIF4A2_RPRP PGAM5 10199|10025 1|3 0.62|0.75 0|0|3|0|0|0|0 0.686 EIF4A2_RPRP RPS14 10199|10025 28|9 0.99|0.38 0|0|4|2|0|3|2 0.684 EIF4A2_RPRP DHX9 10199|10025 7|9 0.71|0.60 0|0|6|0|0|2|0 0.656 EIF4A2_RPRP RPLP2 10199|10025 13|5 0.96|0.35 0|0|1|0|0|4|2 0.654 EIF4A2_RPRP HNRNPUL2 10199|10025 1|1 0.67|0.61 0|0|2|0|0|0|0 0.642 EIF4A2_RPRP MATR3 10199|10025 2|21 0.38|0.81 0|0|3|0|0|0|2 0.597 EIF4A2_RPRP HNRNPA3 10199|10025 2|12 0.36|0.79 0|0|4|0|0|0|1 0.573 EIF4A2_RPRP SRRM2 10199|10025 3|15 0.37|0.70 0|0|1|4|0|0|2 0.536 EIF4A2_RPRP DDX17 10199|10025 6|9 0.51|0.52 0|1|3|0|1|0|2 0.515 EIF4A2_RPRP SRSF1 10199|10025 3|21 0.21|0.81 0|0|3|0|0|2|2 0.508 247

EIF4A2_RPRP RPS18 10199|10025 27|22 0.61|0.36 2|0|9|0|1|7|6 0.481 EIF4A2_RPRP YBX1 10199|10025 8|17 0.36|0.52 0|0|5|1|2|0|2 0.441 EIF4A2_RPRP RPS19 10199|10025 5|3 0.63|0.25 0|0|2|0|0|4|0 0.439 EIF4A2_RPRP HNRNPK 10199|10025 5|7 0.45|0.41 0|1|4|0|0|2|0 0.43 EIF4A2_RPRP RPS9 10199|10025 12|13 0.54|0.27 2|0|4|0|2|6|3 0.406 EIF4A2_RPRP RPS11 10199|10025 9|3 0.66|0.08 0|0|3|0|0|3|1 0.369 EIF4A2_RPRP HNRNPA1 10199|10025 5|18 0.10|0.61 1|3|3|0|0|3|2 0.358 EIF4A2_RPRP ILF3 10199|10025 5|12 0.22|0.41 0|0|7|0|0|2|2 0.319 EIF4A2_RPRP HNRNPR 10199|10025 6|8 0.32|0.23 2|0|3|0|0|3|1 0.272 EIF4A2_RPRP RPS3A 10199|10025 12|5 0.52|0.00 0|0|6|0|1|4|2 0.259 EIF4A2_RPRP HNRNPAB 10199|10025 3|8 0.11|0.39 1|0|2|0|0|1|3 0.248 EIF4A2_RPRP HNRNPM 10199|10025 10|41 0.01|0.47 2|5|7|0|0|5|6 0.239 EIF4A2_RPRP HNRNPD 10199|10025 4|3 0.36|0.08 1|1|2|0|0|1|1 0.221 EIF4A2_RPRP HNRNPA2B1 10199|10025 7|17 0.09|0.32 2|0|5|0|1|6|1 0.207 EIF4A2_RPRP RPS3 10199|10025 15|10 0.39|0.01 3|0|6|2|1|9|3 0.2 EIF4A2_RPRP RPS8 10199|10025 13|10 0.37|0.02 3|0|6|0|4|5|3 0.196 EIF4A2_RPRP H1FX 10199|10025 1|4 0.05|0.29 0|0|2|0|0|1|1 0.171 EIF4A2_RPRP RPL26 10199|10025 1|8 0.01|0.22 0|0|4|0|0|2|1 0.112 EIF4A2_RPRP HIST1H1C 10199|10025 8|20 0.00|0.21 8|0|8|0|4|7|0 0.106 EIF4A2_RPRP ARL6IP4 10199|10025 1|1 0.06|0.06 0|0|3|0|0|2|0 0.058 EIF4A2_RPRP RPS4X 10199|10025 10|2 0.10|0.00 1|2|5|0|0|4|0 0.05 EIF4A2_RPRP SNRPF 10199|10025 1|1 0.07|0.03 0|1|2|0|0|0|1 0.048 EIF4A2_RPRP RPL36 10199|10025 1|1 0.03|0.02 0|0|2|0|1|2|0 0.025 EIF4A2_RPRP RPL23A 10199|10025 6|7 0.03|0.01 2|0|8|0|0|5|3 0.02 EIF4A2_RPRP LUC7L2 10199|10025 2|2 0.02|0.01 0|0|6|0|0|1|2 0.015 EIF4A2_RPRP RPL35A 10199|10025 3|2 0.03|0.00 0|1|4|0|0|3|2 0.014 EIF4A2_RPRP RPL27 10199|10025 1|2 0.00|0.02 0|0|1|0|1|2|3 0.011 EIF4A2_RPRP RPS2 10199|10025 9|2 0.01|0.00 4|0|1|1|2|3|3 0.006 EIF4A2_RPRP RPL3 10199|10025 3|6 0.00|0.00 0|0|7|0|0|8|4 0.002 EIF4A2_RPRP DDX21 10199|10025 6|5 0.00|0.00 0|0|15|0|0|9|2 0.002 EIF4A2_RPRP SRSF8 10199|10025 1|1 0.00|0.00 0|0|2|1|0|2|1 0.001 EIF4A2_RPRP NPM1 10199|10025 6|14 0.00|0.00 14|8|15|6|1|3|6 0 EIF4A2_RPRP WDR77 10199|10025 4|7 0.00|0.00 20|11|14|0|11|14|6 0 EIF4A2_RPRP KIF11 10199|10025 1|11 0.00|0.00 100|19|78|36|19|43|38 0 EIF4A2_RPRP TRIM21 10199|10025 2|0 0.00|0.00 0|0|6|0|1|0|2 0 EIF4A2_RPRP RPLP0 10199|10025 0|4 0.00|0.00 4|2|3|0|1|4|0 0 EIF4A2_RPRP PRMT5 10199|10025 8|18 0.00|0.00 59|15|55|14|17|43|30 0 EIF4A2_RPRP HSPA8 10199|10025 2|4 0.00|0.00 4|2|9|7|0|8|4 0 EIF4A2_RPRP IVNS1ABP 10199|10025 0|4 0.00|0.00 5|8|13|4|0|9|7 0 EIF4A2_RPRP SKP1 10199|10025 1|0 0.00|0.00 0|0|1|0|0|0|0 0 EIF4A2_RPRP TUBB 10199|10025 8|7 0.00|0.00 25|1|1|0|3|8|3 0 EIF4A2_RPRP CLNS1A 10199|10025 1|6 0.00|0.00 8|6|19|6|5|15|8 0 EIF4A2_RPRP SF3B3 10199|10025 0|1 0.00|0.00 3|3|9|0|2|6|4 0 EIF4A2_RPRP NCL 10199|10025 12|21 0.00|0.00 4|1|14|16|6|12|6 0 EIF4A2_RPRP STK38 10199|10025 3|17 0.00|0.00 32|10|32|11|3|25|12 0 EIF4A2_RPRP DHX15 10199|10025 2|8 0.00|0.00 0|2|13|1|1|5|5 0 EIF4A2_RPRP TUBA1C 10199|10025 3|5 0.00|0.00 42|0|10|8|5|16|7 0 EIF4A2_RPRP PPM1B 10199|10025 2|15 0.00|0.00 21|33|2|31|2|40|28 0 EIF4A2_RPRP KRT1 10199|10025 0|3 0.00|0.00 192|250|68|43|29|31|51 0 EIF4A2_RPRP MAP1B 10199|10025 1|11 0.00|0.00 0|0|10|1|0|7|6 0 EIF4A2_RPRP HSPA1B 10199|10025 5|7 0.00|0.00 17|6|9|0|4|16|6 0 EIF4A2_RPRP RPL18 10199|10025 2|1 0.00|0.00 4|1|5|1|2|3|3 0 EIF4A2_RPRP EIF4B 10199|10025 1|0 0.00|0.00 0|0|2|0|0|2|0 0 EIF4A2_RPRP RPS6 10199|10025 10|1 0.00|0.00 2|1|4|0|0|0|3 0 248

EIF4A2_RPRP QPCT 10199|10025 0|2 0.00|0.00 9|5|15|10|0|11|4 0 EIF4A2_RPRP RBM10 10199|10025 1|3 0.00|0.00 7|3|22|6|2|12|8 0 EIF4A2_RPRP ACTA2 10199|10025 16|0 0.00|0.00 21|0|1|0|0|0|0 0 EIF4A2_RPRP SPTBN1 10199|10025 0|1 0.00|0.00 0|3|25|3|0|25|10 0 EIF4A2_RPRP LARS 10199|10025 3|1 0.00|0.00 0|3|7|0|0|3|1 0 EIF4A2_RPRP RPL10A 10199|10025 1|0 0.00|0.00 1|0|2|0|0|2|1 0 EIF4A2_RPRP RPL10 10199|10025 1|0 0.00|0.00 0|0|0|0|1|2|1 0 EIF4A2_RPRP RPS26 10199|10025 3|0 0.00|0.00 2|0|2|0|0|1|0 0 EIF4A2_RPRP IRS4 10199|10025 2|0 0.00|0.00 0|0|4|0|0|2|0 0 EIF4A2_RPRP RPS16 10199|10025 5|0 0.00|0.00 4|1|2|0|0|6|0 0 EIF4A2_RPRP RPL23 10199|10025 0|2 0.00|0.00 1|0|1|1|0|0|0 0 EIF4A2_RPRP TXN 10199|10025 1|0 0.00|0.00 0|0|4|0|0|2|0 0 EIF4A2_RPRP SUB1 10199|10025 6|4 0.00|0.00 5|0|15|6|1|8|3 0 EIF4A2_RPRP SNRPD3 10199|10025 0|1 0.00|0.00 6|0|2|0|4|4|4 0 EIF4A2_RPRP HSPA9 10199|10025 0|1 0.00|0.00 0|4|2|1|0|4|2 0 EIF4A2_RPRP RPS15 10199|10025 4|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP EEF1A1 10199|10025 1|1 0.00|0.00 3|3|5|0|1|7|4 0 EIF4A2_RPRP RPL7A 10199|10025 5|0 0.00|0.00 4|0|5|0|1|9|4 0 EIF4A2_RPRP XRCC6 10199|10025 0|1 0.00|0.00 0|0|0|0|0|2|1 0 EIF4A2_RPRP ACTB 10199|10025 0|6 0.00|0.00 65|33|48|59|23|15|47 0 EIF4A2_RPRP RBM15 10199|10025 0|2 0.00|0.00 0|0|0|0|0|0|1 0 EIF4A2_RPRP THOC4 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|1 0 EIF4A2_RPRP RPL12 10199|10025 0|1 0.00|0.00 1|0|3|0|0|1|2 0 EIF4A2_RPRP PA2G4 10199|10025 3|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP RPL11 10199|10025 1|0 0.00|0.00 1|0|1|0|1|0|1 0 EIF4A2_RPRP SRSF3 10199|10025 0|8 0.00|0.00 2|0|3|0|0|2|2 0 EIF4A2_RPRP ZFR 10199|10025 0|5 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP SPIN1 10199|10025 0|2 0.00|0.00 3|2|7|0|2|4|6 0 EIF4A2_RPRP FLNA 10199|10025 0|2 0.00|0.00 0|6|23|0|0|12|11 0 EIF4A2_RPRP RPS24 10199|10025 1|0 0.00|0.00 1|0|0|0|1|2|0 0 EIF4A2_RPRP SPTAN1 10199|10025 1|1 0.00|0.00 0|3|31|1|2|20|11 0 EIF4A2_RPRP HSPA5 10199|10025 2|9 0.00|0.00 3|5|11|0|2|10|3 0 EIF4A2_RPRP RPL4 10199|10025 4|5 0.00|0.00 3|0|4|5|0|7|2 0 EIF4A2_RPRP KRT10 10199|10025 2|1 0.00|0.00 143|154|31|55|28|22|31 0 EIF4A2_RPRP CMBL 10199|10025 1|5 0.00|0.00 15|5|9|5|2|8|5 0 EIF4A2_RPRP PTS 10199|10025 0|3 0.00|0.00 2|0|4|1|0|3|1 0 EIF4A2_RPRP RPS28 10199|10025 6|0 0.00|0.00 0|0|1|0|0|1|1 0 EIF4A2_RPRP KRT9 10199|10025 4|15 0.00|0.00 89|168|64|12|11|20|31 0 EIF4A2_RPRP RPSA 10199|10025 5|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP IARS 10199|10025 0|2 0.00|0.00 0|1|3|0|0|2|4 0 EIF4A2_RPRP SF3B1 10199|10025 0|5 0.00|0.00 3|1|14|0|1|9|5 0 EIF4A2_RPRP SRSF7 10199|10025 0|5 0.00|0.00 0|0|3|0|0|4|3 0 EIF4A2_RPRP RBBP4 10199|10025 1|0 0.00|0.00 2|0|2|0|0|0|0 0 EIF4A2_RPRP LTBP1 10199|10025 1|1 0.00|0.00 3|1|19|1|1|10|5 0 EIF4A2_RPRP SF3B2 10199|10025 0|3 0.00|0.00 2|0|7|3|0|7|5 0 EIF4A2_RPRP CPSF6 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP RPL13 10199|10025 7|4 0.00|0.00 4|0|7|3|2|6|2 0 EIF4A2_RPRP PRDX1 10199|10025 0|2 0.00|0.00 0|0|6|0|0|2|0 0 EIF4A2_RPRP LOC100291837 10199|10025 4|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP THRAP3 10199|10025 0|17 0.00|0.00 16|0|9|7|2|7|4 0 EIF4A2_RPRP PRMT1 10199|10025 2|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP RPL7 10199|10025 1|0 0.00|0.00 4|2|10|2|0|5|3 0 EIF4A2_RPRP RPL6 10199|10025 5|8 0.00|0.00 8|0|11|2|0|8|5 0 EIF4A2_RPRP EPRS 10199|10025 0|11 0.00|0.00 0|3|17|3|1|9|5 0 249

EIF4A2_RPRP RPS20 10199|10025 6|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP DYNC1H1 10199|10025 1|0 0.00|0.00 0|0|4|0|0|0|0 0 EIF4A2_RPRP RPL19 10199|10025 1|4 0.00|0.00 1|0|1|3|0|1|2 0 EIF4A2_RPRP RARS 10199|10025 1|0 0.00|0.00 0|3|6|0|0|7|3 0 EIF4A2_RPRP RIOK1 10199|10025 2|0 0.00|0.00 0|1|8|0|0|5|2 0 EIF4A2_RPRP EIF2S2 10199|10025 8|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP HNRNPU 10199|10025 0|8 0.00|0.00 7|3|7|0|1|9|4 0 EIF4A2_RPRP RPS13 10199|10025 1|0 0.00|0.00 2|0|1|0|1|2|0 0 EIF4A2_RPRP HSP90AA1 10199|10025 2|0 0.00|0.00 0|0|3|0|0|1|0 0 EIF4A2_RPRP DARS 10199|10025 3|0 0.00|0.00 0|2|9|0|0|9|5 0 EIF4A2_RPRP HNRNPA0 10199|10025 0|6 0.00|0.00 0|0|2|0|0|0|0 0 EIF4A2_RPRP RPL15 10199|10025 0|1 0.00|0.00 3|0|5|3|1|5|0 0 EIF4A2_RPRP SRPK2 10199|10025 0|1 0.00|0.00 0|0|1|0|0|0|1 0 EIF4A2_RPRP NAP1L1 10199|10025 1|0 0.00|0.00 0|0|0|0|0|1|0 0 EIF4A2_RPRP PRPF31 10199|10025 0|1 0.00|0.00 0|3|3|0|0|1|3 0 EIF4A2_RPRP DYRK1B 10199|10025 0|9 0.00|0.00 0|0|0|0|0|0|17 0 EIF4A2_RPRP RBM14 10199|10025 0|3 0.00|0.00 0|0|1|0|0|0|1 0 EIF4A2_RPRP PRDX6 10199|10025 0|1 0.00|0.00 0|0|0|0|0|1|0 0 EIF4A2_RPRP RPL5 10199|10025 2|0 0.00|0.00 0|0|1|0|0|0|0 0 EIF4A2_RPRP KIAA1967 10199|10025 1|4 0.00|0.00 0|0|7|0|0|2|0 0 EIF4A2_RPRP PRPF6 10199|10025 2|0 0.00|0.00 0|0|5|0|0|0|0 0 EIF4A2_RPRP MYH9 10199|10025 0|1 0.00|0.00 6|3|14|0|0|11|6 0 EIF4A2_RPRP BIRC6 10199|10025 0|2 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP TTN 10199|10025 1|0 0.00|0.00 1|0|1|0|0|2|0 0 EIF4A2_RPRP HADHA 10199|10025 3|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP RPL8 10199|10025 3|2 0.00|0.00 1|0|7|5|1|5|3 0 EIF4A2_RPRP BCLAF1 10199|10025 0|11 0.00|0.00 7|0|5|2|0|3|3 0 EIF4A2_RPRP RPL24 10199|10025 0|2 0.00|0.00 2|0|0|0|1|4|0 0 EIF4A2_RPRP RPL27A 10199|10025 0|2 0.00|0.00 1|0|1|0|0|2|1 0 EIF4A2_RPRP PRSS1 10199|10025 3|5 0.00|0.00 3|0|7|0|2|5|6 0 EIF4A2_RPRP AIMP1 10199|10025 0|1 0.00|0.00 0|0|1|0|1|1|0 0 EIF4A2_RPRP CPSF1 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP C3orf15 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP HNRNPC 10199|10025 0|5 0.00|0.00 6|0|2|0|0|0|0 0 EIF4A2_RPRP RPS12 10199|10025 3|0 0.00|0.00 0|0|1|0|0|0|1 0 EIF4A2_RPRP TUBB4 10199|10025 1|0 0.00|0.00 2|2|0|0|0|0|0 0 EIF4A2_RPRP SHROOM3 10199|10025 0|2 0.00|0.00 2|5|4|3|0|1|2 0 EIF4A2_RPRP RPL29 10199|10025 0|5 0.00|0.00 0|0|4|0|0|2|0 0 EIF4A2_RPRP RPS5 10199|10025 2|0 0.00|0.00 2|0|0|0|0|2|0 0 EIF4A2_RPRP KIAA1731 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP KRT5 10199|10025 0|2 0.00|0.00 24|57|9|3|3|2|9 0 EIF4A2_RPRP RPS25 10199|10025 4|0 0.00|0.00 1|0|0|0|0|1|1 0 EIF4A2_RPRP DDX5 10199|10025 0|7 0.00|0.00 0|0|5|0|0|2|0 0 EIF4A2_RPRP KRT6A 10199|10025 0|1 0.00|0.00 9|12|2|0|0|0|3 0 EIF4A2_RPRP RPS23 10199|10025 7|0 0.00|0.00 0|0|5|0|0|2|0 0 EIF4A2_RPRP ABCC5 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|4 0 EIF4A2_RPRP GATM 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP NOP2 10199|10025 2|0 0.00|0.00 0|0|4|0|0|2|0 0 EIF4A2_RPRP CHD4 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP VIM 10199|10025 1|0 0.00|0.00 2|0|5|0|0|1|2 0 EIF4A2_RPRP RPL28 10199|10025 0|1 0.00|0.00 1|0|3|0|0|5|1 0 EIF4A2_RPRP DCD 10199|10025 0|3 0.00|0.00 1|11|4|2|0|2|0 0 EIF4A2_RPRP SRSF6 10199|10025 0|2 0.00|0.00 1|0|1|0|0|4|2 0 EIF4A2_RPRP RPS27 10199|10025 4|0 0.00|0.00 0|0|2|0|0|1|0 0 250

EIF4A2_RPRP AIFM1 10199|10025 1|0 0.00|0.00 0|0|4|0|0|0|1 0 EIF4A2_RPRP KIAA0562 10199|10025 0|2 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP KARS 10199|10025 0|1 0.00|0.00 0|0|4|0|0|0|0 0 EIF4A2_RPRP PARG 10199|10025 2|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP CCDC124 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP ACTN4 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP DCHS1 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP GZMA 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP SRPR 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP RBM39 10199|10025 0|2 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP KLHL26 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP FAM120A 10199|10025 0|4 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP UBAP2L 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP KIAA1543 10199|10025 0|1 0.00|0.00 0|0|2|0|0|0|0 0 EIF4A2_RPRP FEZF1 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP RBMX 10199|10025 0|14 0.00|0.00 0|0|0|0|0|2|0 0 EIF4A2_RPRP EEF2 10199|10025 1|0 0.00|0.00 0|0|1|0|0|0|0 0 EIF4A2_RPRP RRP12 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP EIF2S3 10199|10025 2|0 0.00|0.00 0|0|1|0|0|0|0 0 EIF4A2_RPRP WIBG 10199|10025 2|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP SERBP1 10199|10025 3|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP RSL1D1 10199|10025 0|2 0.00|0.00 0|0|2|0|0|0|0 0 EIF4A2_RPRP C1orf25 10199|10025 0|1 0.00|0.00 0|0|1|0|0|0|0 0 EIF4A2_RPRP ZC3HAV1 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP USH1C 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP DNAH5 10199|10025 0|4 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP CDKL1 10199|10025 2|0 0.00|0.00 0|0|0|1|0|0|0 0 EIF4A2_RPRP SEC31A 10199|10025 0|1 0.00|0.00 0|0|2|0|0|1|0 0 EIF4A2_RPRP QRICH2 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP PNN 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP OBSL1 10199|10025 3|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP ZRANB2 10199|10025 5|0 0.00|0.00 0|0|2|0|0|0|1 0 EIF4A2_RPRP ASCC3 10199|10025 3|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP EIF5B 10199|10025 5|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP ABCE1 10199|10025 3|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP FXR1 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP NACA 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP GNB2L1 10199|10025 3|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP MSI2 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP AKAP1 10199|10025 3|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP PDAP1 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|1 0 EIF4A2_RPRP ABCF1 10199|10025 2|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP KHSRP 10199|10025 2|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP LRRFIP1 10199|10025 1|0 0.00|0.00 0|0|1|0|0|0|0 0 EIF4A2_RPRP RBM8A 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP KIAA1522 10199|10025 2|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP CHST8 10199|10025 2|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP KIF21B 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP HDGFRP2 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP WDR90 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP POMT2 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP FAM98A 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP LOC100132983 10199|10025 4|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP RPS4Y2 10199|10025 2|0 0.00|0.00 0|0|1|0|0|0|0 0 251

EIF4A2_RPRP FTSJ3 10199|10025 1|0 0.00|0.00 0|0|1|0|0|0|0 0 EIF4A2_RPRP LOC730144 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP CIRBP 10199|10025 1|0 0.00|0.00 0|0|1|0|0|0|0 0 EIF4A2_RPRP FUBP3 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP PGD 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP TNPO3 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP CRISPLD2 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP ABCC1 10199|10025 2|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP CCDC71 10199|10025 4|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP RDBP 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP PIK3R4 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP GPR179 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP PITPNM3 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP WWC1 10199|10025 2|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP SEMA6C 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP DOCK2 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP MAGOH 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP DCDC1 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP ETV3 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP OR2M3 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP CCBL2 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP LILRB1 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP RGS1 10199|10025 3|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP STMN3 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP RNASEL 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP ZBTB25 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP RIN2 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP TYW1 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP TTC6 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP LOC100294429 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP SMC3 10199|10025 1|0 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP ATXN2L 10199|10025 0|4 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP FIP1L1 10199|10025 0|3 0.00|0.00 0|0|1|0|0|0|0 0 EIF4A2_RPRP IGF2BP3 10199|10025 0|6 0.00|0.00 0|0|1|0|0|0|0 0 EIF4A2_RPRP DDX1 10199|10025 0|2 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP PUM2 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP CUX1 10199|10025 0|3 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP ZC3H4 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP GAR1 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP SRRT 10199|10025 0|2 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP DHX36 10199|10025 0|3 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP RBM25 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP YTHDF3 10199|10025 0|2 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP TAF15 10199|10025 0|3 0.00|0.00 0|0|1|0|0|1|0 0 EIF4A2_RPRP CALM1 10199|10025 0|2 0.00|0.00 0|0|0|0|0|0|1 0 EIF4A2_RPRP FAM178B 10199|10025 0|4 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP U2AF1 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP GRIPAP1 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP DDX6 10199|10025 0|2 0.00|0.00 0|0|1|0|0|0|0 0 EIF4A2_RPRP ONECUT1 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP UBE2CBP 10199|10025 0|2 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP LOC100288544 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP GLYR1 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP ASPRV1 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 252

EIF4A2_RPRP MCM5 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP PEG10 10199|10025 0|2 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP NCBP1 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP BRIX1 10199|10025 0|1 0.00|0.00 0|0|2|0|0|0|0 0 EIF4A2_RPRP PTPN20A 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP PCBP1 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP LOC100133899 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP C1orf113 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP MYCL1 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP RBMS1 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP SPATS2 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP WDR25 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP CCHCR1 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP TRA2A 10199|10025 0|2 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP MID1IP1 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP Mrps7 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP PDP1 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP CABLES1 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP DNAH14 10199|10025 0|2 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP SH3BP2 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP FOXP3 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP C12orf41 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP SLC5A2 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP COL17A1 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP MAST1 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP HNRNPH3 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP GNG12 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP CAB39 10199|10025 0|3 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP WTAP 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 EIF4A2_RPRP TRIM75 10199|10025 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP YWHAE 10185|9931 60|33 1.00|1.00 0|0|0|0|0|0|0 1 RAF1_RP TIMM50 10185|9931 24|12 1.00|1.00 0|0|0|0|0|0|0 1 RAF1_RP YWHAZ 10185|9931 16|9 1.00|1.00 0|0|1|0|0|0|0 1 RAF1_RP YWHAG 10185|9931 15|14 1.00|1.00 0|0|0|0|0|0|0 1 RAF1_RP YWHAH 10185|9931 13|7 1.00|1.00 0|0|0|0|0|0|0 1 RAF1_RP SSR4 10185|9931 2|4 1.00|1.00 0|0|0|0|0|0|0 1 RAF1_RP KRAS 10185|9931 5|7 1.00|1.00 0|0|0|0|0|0|0 1 RAF1_RP HSP90AA1 10185|9931 46|25 1.00|1.00 0|0|3|0|0|1|0 0.999 RAF1_RP HSP90AB1 10185|9931 46|20 1.00|1.00 0|0|4|1|0|0|0 0.999 RAF1_RP CDC37 10185|9931 21|12 1.00|1.00 0|0|0|0|0|0|0 0.999 RAF1_RP NDUFS3 10185|9931 4|2 1.00|1.00 0|0|0|0|0|0|0 0.999 RAF1_RP DNAJA1 10185|9931 5|3 1.00|1.00 0|0|0|0|0|0|0 0.998 RAF1_RP YWHAB 10185|9931 15|8 1.00|1.00 2|0|0|0|0|0|0 0.998 RAF1_RP YWHAQ 10185|9931 6|2 1.00|1.00 0|0|0|0|0|0|0 0.998 RAF1_RP STIP1 10185|9931 11|4 1.00|1.00 0|0|0|0|0|0|0 0.998 RAF1_RP HAX1 10185|9931 3|4 1.00|1.00 0|0|0|0|0|0|0 0.997 RAF1_RP FKBP5 10185|9931 8|2 1.00|1.00 0|0|0|0|0|0|0 0.997 RAF1_RP RUVBL2 10185|9931 2|3 1.00|1.00 0|0|0|0|0|0|0 0.996 RAF1_RP SEC61B 10185|9931 2|2 0.99|1.00 0|0|0|0|0|0|0 0.996 RAF1_RP GCN1L1 10185|9931 28|13 0.99|1.00 0|0|0|2|0|0|0 0.996 RAF1_RP DNAJA2 10185|9931 6|1 1.00|0.99 0|0|0|0|0|0|0 0.996 RAF1_RP SLC1A5 10185|9931 2|2 0.99|1.00 0|0|0|0|0|0|0 0.996 RAF1_RP SLC25A11 10185|9931 4|1 1.00|0.99 0|0|0|0|0|0|0 0.995 RAF1_RP ZNF764 10185|9931 4|1 1.00|0.99 0|0|0|0|0|0|0 0.995 253

RAF1_RP PNKD 10185|9931 3|1 1.00|0.99 0|0|0|0|0|0|0 0.995 RAF1_RP CCT8 10185|9931 2|1 0.99|0.99 0|0|0|0|0|0|0 0.994 RAF1_RP ATP1A1 10185|9931 6|6 0.99|1.00 0|0|0|0|0|0|1 0.994 RAF1_RP CAD 10185|9931 4|5 0.99|1.00 0|0|1|0|0|0|0 0.994 RAF1_RP TUBB6 10185|9931 7|6 0.99|1.00 0|0|0|0|0|0|0 0.994 RAF1_RP COX4I1 10185|9931 2|1 1.00|0.99 0|0|0|0|0|0|0 0.994 RAF1_RP RPN1 10185|9931 3|2 1.00|0.99 0|0|0|0|0|0|0 0.994 RAF1_RP IRS4 10185|9931 25|35 0.99|0.99 0|0|4|0|0|2|0 0.993 RAF1_RP XPO1 10185|9931 3|1 0.99|0.98 0|0|0|0|0|0|0 0.988 RAF1_RP QPCTL 10185|9931 1|2 0.98|0.99 0|0|0|0|0|0|0 0.987 RAF1_RP TRIM28 10185|9931 2|1 0.99|0.98 0|0|0|0|0|0|0 0.986 RAF1_RP HNRNPF 10185|9931 1|2 0.98|1.00 0|0|0|0|0|0|0 0.986 RAF1_RP FANCD2 10185|9931 4|1 1.00|0.98 0|0|0|0|0|0|0 0.986 RAF1_RP BAG2 10185|9931 2|1 0.99|0.97 0|0|0|0|0|0|0 0.984 RAF1_RP SV2A 10185|9931 1|2 0.97|1.00 0|0|0|0|0|0|0 0.984 RAF1_RP ATP5B 10185|9931 9|2 0.98|0.98 0|0|0|0|0|0|1 0.983 RAF1_RP IGBP1 10185|9931 1|1 0.99|0.98 0|0|0|0|0|0|0 0.983 RAF1_RP TBR1 10185|9931 1|1 0.97|0.99 0|0|0|0|0|0|0 0.983 RAF1_RP CSE1L 10185|9931 5|1 0.98|0.98 0|0|0|0|0|0|0 0.982 RAF1_RP EMD 10185|9931 6|3 0.98|0.98 0|0|2|0|0|0|0 0.982 RAF1_RP TRAP1 10185|9931 5|2 0.99|0.98 0|0|1|0|0|0|0 0.981 RAF1_RP CAND2 10185|9931 1|2 0.97|0.99 0|0|0|0|0|0|0 0.981 RAF1_RP AKAP8L 10185|9931 1|1 0.98|0.98 0|0|0|0|0|0|0 0.979 RAF1_RP FASN 10185|9931 3|1 0.98|0.97 0|0|0|0|0|0|0 0.978 RAF1_RP GRAMD1A 10185|9931 1|2 0.97|0.99 0|0|0|0|0|0|0 0.978 RAF1_RP XPOT 10185|9931 5|1 0.98|0.97 0|0|0|0|0|0|0 0.977 RAF1_RP TUBG1 10185|9931 1|1 0.98|0.98 0|0|0|0|0|0|0 0.977 RAF1_RP AIFM1 10185|9931 11|6 0.98|0.97 0|0|4|0|0|0|1 0.976 RAF1_RP ALDH3A2 10185|9931 4|3 0.98|0.98 0|0|2|0|0|0|0 0.976 RAF1_RP ATP2A2 10185|9931 11|2 0.99|0.96 0|0|2|0|0|0|0 0.973 RAF1_RP USP11 10185|9931 1|1 0.96|0.98 0|0|0|0|0|0|0 0.969 RAF1_RP CCT7 10185|9931 1|1 0.97|0.96 0|0|0|0|0|0|0 0.968 RAF1_RP AFG3L2 10185|9931 1|1 0.97|0.97 0|0|0|0|0|0|0 0.968 RAF1_RP CALU 10185|9931 8|1 0.99|0.92 0|0|0|0|0|0|1 0.958 RAF1_RP DYNC1H1 10185|9931 3|3 0.94|0.97 0|0|4|0|0|0|0 0.955 RAF1_RP HSPD1 10185|9931 9|4 0.95|0.91 0|0|2|0|0|3|0 0.931 RAF1_RP RPL23 10185|9931 3|4 0.86|0.98 1|0|1|1|0|0|0 0.921 RAF1_RP RPL21 10185|9931 3|3 0.86|0.97 0|0|1|0|0|2|0 0.912 RAF1_RP HSPA8 10185|9931 28|26 0.84|0.93 4|2|9|7|0|8|4 0.882 RAF1_RP HSPA9 10185|9931 8|13 0.64|0.98 0|4|2|1|0|4|2 0.808 RAF1_RP RPLP1 10185|9931 2|1 0.82|0.75 0|1|0|0|0|1|0 0.784 RAF1_RP RBBP4 10185|9931 2|4 0.60|0.93 2|0|2|0|0|0|0 0.765 RAF1_RP TUBB4 10185|9931 10|1 0.94|0.53 2|2|0|0|0|0|0 0.735 RAF1_RP SLC25A5 10185|9931 9|3 0.91|0.43 2|1|1|0|1|3|0 0.673 RAF1_RP RPS12 10185|9931 1|1 0.58|0.74 0|0|1|0|0|0|1 0.66 RAF1_RP KRT19 10185|9931 2|2 0.57|0.74 0|0|3|0|0|2|0 0.653 RAF1_RP TUBB2C 10185|9931 3|10 0.25|0.95 0|0|4|0|0|3|2 0.602 RAF1_RP KRT8 10185|9931 6|4 0.56|0.31 0|0|9|0|0|4|0 0.434 RAF1_RP ATP5A1 10185|9931 7|2 0.73|0.04 0|2|3|0|0|3|2 0.388 RAF1_RP TUBB2B 10185|9931 2|1 0.39|0.22 0|0|3|3|0|0|0 0.303 RAF1_RP RPL24 10185|9931 1|3 0.01|0.40 2|0|0|0|1|4|0 0.209 RAF1_RP HNRNPD 10185|9931 1|2 0.02|0.23 1|1|2|0|0|1|1 0.124 RAF1_RP TMPO 10185|9931 2|1 0.18|0.07 1|0|3|0|0|0|2 0.122 RAF1_RP RPL27A 10185|9931 1|1 0.10|0.12 1|0|1|0|0|2|1 0.108 254

RAF1_RP EEF1A1 10185|9931 5|8 0.00|0.15 3|3|5|0|1|7|4 0.077 RAF1_RP RPL10A 10185|9931 2|1 0.12|0.03 1|0|2|0|0|2|1 0.076 RAF1_RP RPL27 10185|9931 2|1 0.07|0.01 0|0|1|0|1|2|3 0.041 RAF1_RP HSPA1B 10185|9931 24|26 0.01|0.03 17|6|9|0|4|16|6 0.02 RAF1_RP TUBB 10185|9931 20|8 0.01|0.00 25|1|1|0|3|8|3 0.004 RAF1_RP NPM1 10185|9931 5|3 0.00|0.00 14|8|15|6|1|3|6 0 RAF1_RP XRCC5 10185|9931 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP WDR77 10185|9931 1|2 0.00|0.00 20|11|14|0|11|14|6 0 RAF1_RP HNRNPA1 10185|9931 2|1 0.00|0.00 1|3|3|0|0|3|2 0 RAF1_RP DDB1 10185|9931 2|0 0.00|0.00 0|1|2|0|0|2|0 0 RAF1_RP TRIM21 10185|9931 3|0 0.00|0.00 0|0|6|0|1|0|2 0 RAF1_RP RPS3 10185|9931 3|5 0.00|0.00 3|0|6|2|1|9|3 0 RAF1_RP RPLP0 10185|9931 5|1 0.00|0.00 4|2|3|0|1|4|0 0 RAF1_RP PRMT5 10185|9931 5|4 0.00|0.00 59|15|55|14|17|43|30 0 RAF1_RP CLNS1A 10185|9931 0|4 0.00|0.00 8|6|19|6|5|15|8 0 RAF1_RP RPLP2 10185|9931 7|0 0.00|0.00 0|0|1|0|0|4|2 0 RAF1_RP NCL 10185|9931 4|6 0.00|0.00 4|1|14|16|6|12|6 0 RAF1_RP STK38 10185|9931 5|5 0.00|0.00 32|10|32|11|3|25|12 0 RAF1_RP TUBA1C 10185|9931 2|20 0.00|0.00 42|0|10|8|5|16|7 0 RAF1_RP PPM1B 10185|9931 0|5 0.00|0.00 21|33|2|31|2|40|28 0 RAF1_RP KRT1 10185|9931 9|2 0.00|0.00 192|250|68|43|29|31|51 0 RAF1_RP HNRNPM 10185|9931 3|2 0.00|0.00 2|5|7|0|0|5|6 0 RAF1_RP MAP1B 10185|9931 1|4 0.00|0.00 0|0|10|1|0|7|6 0 RAF1_RP RPL18 10185|9931 1|1 0.00|0.00 4|1|5|1|2|3|3 0 RAF1_RP RPS6 10185|9931 1|1 0.00|0.00 2|1|4|0|0|0|3 0 RAF1_RP QPCT 10185|9931 2|3 0.00|0.00 9|5|15|10|0|11|4 0 RAF1_RP ACTA2 10185|9931 4|3 0.00|0.00 21|0|1|0|0|0|0 0 RAF1_RP LARS 10185|9931 6|1 0.00|0.00 0|3|7|0|0|3|1 0 RAF1_RP HNRNPR 10185|9931 0|1 0.00|0.00 2|0|3|0|0|3|1 0 RAF1_RP RPS4X 10185|9931 2|0 0.00|0.00 1|2|5|0|0|4|0 0 RAF1_RP HNRNPH1 10185|9931 0|1 0.00|0.00 0|0|3|0|0|1|0 0 RAF1_RP RPS15A 10185|9931 1|0 0.00|0.00 0|0|1|0|0|2|0 0 RAF1_RP RPS16 10185|9931 1|1 0.00|0.00 4|1|2|0|0|6|0 0 RAF1_RP RPL9 10185|9931 0|1 0.00|0.00 0|0|0|0|1|0|0 0 RAF1_RP UBB 10185|9931 1|0 0.00|0.00 2|0|2|2|0|2|1 0 RAF1_RP RPS19 10185|9931 1|0 0.00|0.00 0|0|2|0|0|4|0 0 RAF1_RP TXN 10185|9931 1|0 0.00|0.00 0|0|4|0|0|2|0 0 RAF1_RP LOC731751 10185|9931 0|5 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP RPL14 10185|9931 1|0 0.00|0.00 3|0|0|0|0|1|1 0 RAF1_RP C1QBP 10185|9931 2|0 0.00|0.00 1|0|0|0|0|2|0 0 RAF1_RP RPS15 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP RPL7A 10185|9931 4|0 0.00|0.00 4|0|5|0|1|9|4 0 RAF1_RP PABPC4 10185|9931 0|1 0.00|0.00 0|0|2|0|0|0|0 0 RAF1_RP XRCC6 10185|9931 2|0 0.00|0.00 0|0|0|0|0|2|1 0 RAF1_RP RCN2 10185|9931 10|0 0.00|0.00 0|0|2|0|0|3|0 0 RAF1_RP NME2 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP RPS24 10185|9931 1|0 0.00|0.00 1|0|0|0|1|2|0 0 RAF1_RP HSPA5 10185|9931 3|3 0.00|0.00 3|5|11|0|2|10|3 0 RAF1_RP KRT10 10185|9931 5|0 0.00|0.00 143|154|31|55|28|22|31 0 RAF1_RP KPNB1 10185|9931 2|0 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RP CMBL 10185|9931 0|2 0.00|0.00 15|5|9|5|2|8|5 0 RAF1_RP SYNCRIP 10185|9931 2|0 0.00|0.00 0|0|0|0|0|1|0 0 RAF1_RP RPS28 10185|9931 1|0 0.00|0.00 0|0|1|0|0|1|1 0 RAF1_RP HIST1H1C 10185|9931 5|0 0.00|0.00 8|0|8|0|4|7|0 0 255

RAF1_RP KRT9 10185|9931 0|2 0.00|0.00 89|168|64|12|11|20|31 0 RAF1_RP SLC30A9 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP IARS 10185|9931 1|1 0.00|0.00 0|1|3|0|0|2|4 0 RAF1_RP SF3B1 10185|9931 0|1 0.00|0.00 3|1|14|0|1|9|5 0 RAF1_RP TUBAL3 10185|9931 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP PRKDC 10185|9931 26|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP RPL13 10185|9931 4|1 0.00|0.00 4|0|7|3|2|6|2 0 RAF1_RP RPS9 10185|9931 2|3 0.00|0.00 2|0|4|0|2|6|3 0 RAF1_RP RPS18 10185|9931 1|3 0.00|0.00 2|0|9|0|1|7|6 0 RAF1_RP EIF4A1 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP RPS3A 10185|9931 0|1 0.00|0.00 0|0|6|0|1|4|2 0 RAF1_RP RPS10 10185|9931 1|0 0.00|0.00 0|0|0|0|0|1|0 0 RAF1_RP CCT2 10185|9931 0|1 0.00|0.00 0|0|0|3|0|0|0 0 RAF1_RP RPL7 10185|9931 3|0 0.00|0.00 4|2|10|2|0|5|3 0 RAF1_RP RPL6 10185|9931 2|2 0.00|0.00 8|0|11|2|0|8|5 0 RAF1_RP EPRS 10185|9931 1|0 0.00|0.00 0|3|17|3|1|9|5 0 RAF1_RP RPS20 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP RPL3 10185|9931 1|2 0.00|0.00 0|0|7|0|0|8|4 0 RAF1_RP RPL19 10185|9931 3|0 0.00|0.00 1|0|1|3|0|1|2 0 RAF1_RP RPS14 10185|9931 2|1 0.00|0.00 0|0|4|2|0|3|2 0 RAF1_RP RPL31 10185|9931 1|0 0.00|0.00 1|0|1|0|1|1|2 0 RAF1_RP CCT5 10185|9931 0|1 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RP KRT2 10185|9931 3|0 0.00|0.00 93|150|29|45|9|17|39 0 RAF1_RP HNRNPU 10185|9931 1|0 0.00|0.00 7|3|7|0|1|9|4 0 RAF1_RP RPS13 10185|9931 1|0 0.00|0.00 2|0|1|0|1|2|0 0 RAF1_RP RPS2 10185|9931 4|1 0.00|0.00 4|0|1|1|2|3|3 0 RAF1_RP DARS 10185|9931 0|1 0.00|0.00 0|2|9|0|0|9|5 0 RAF1_RP PRKAR1A 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP RPL23A 10185|9931 2|0 0.00|0.00 2|0|8|0|0|5|3 0 RAF1_RP NAP1L1 10185|9931 2|0 0.00|0.00 0|0|0|0|0|1|0 0 RAF1_RP RPL36 10185|9931 0|1 0.00|0.00 0|0|2|0|1|2|0 0 RAF1_RP DYRK1B 10185|9931 2|0 0.00|0.00 0|0|0|0|0|0|17 0 RAF1_RP IPO8 10185|9931 0|1 0.00|0.00 0|0|3|0|0|0|3 0 RAF1_RP YBX1 10185|9931 1|0 0.00|0.00 0|0|5|1|2|0|2 0 RAF1_RP KIAA1967 10185|9931 1|0 0.00|0.00 0|0|7|0|0|2|0 0 RAF1_RP EIF2S1 10185|9931 0|1 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RP SNRPF 10185|9931 1|0 0.00|0.00 0|1|2|0|0|0|1 0 RAF1_RP LOC100294459 10185|9931 2|0 0.00|0.00 6|0|9|9|2|6|2 0 RAF1_RP DRG1 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP RPL8 10185|9931 2|3 0.00|0.00 1|0|7|5|1|5|3 0 RAF1_RP NFKB1 10185|9931 2|0 0.00|0.00 0|0|0|0|4|0|19 0 RAF1_RP PNPLA6 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP PSMD2 10185|9931 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP LOC100292021 10185|9931 0|1 0.00|0.00 0|0|0|0|1|0|0 0 RAF1_RP LRPPRC 10185|9931 2|0 0.00|0.00 0|0|4|0|0|0|0 0 RAF1_RP RPS23 10185|9931 0|2 0.00|0.00 0|0|5|0|0|2|0 0 RAF1_RP DDX21 10185|9931 1|0 0.00|0.00 0|0|15|0|0|9|2 0 RAF1_RP UBR4 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|1 0 RAF1_RP RPS11 10185|9931 0|1 0.00|0.00 0|0|3|0|0|3|1 0 RAF1_RP SEC61A2 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP VIM 10185|9931 1|0 0.00|0.00 2|0|5|0|0|1|2 0 RAF1_RP ZBTB33 10185|9931 1|0 0.00|0.00 7|0|0|0|0|0|0 0 RAF1_RP RPS27 10185|9931 1|0 0.00|0.00 0|0|2|0|0|1|0 0 RAF1_RP GLUD2 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 256

RAF1_RP H1FX 10185|9931 0|1 0.00|0.00 0|0|2|0|0|1|1 0 RAF1_RP ATAD3A 10185|9931 0|6 0.00|0.00 0|0|3|0|0|0|1 0 RAF1_RP CLPB 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP WDR36 10185|9931 0|1 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RP ESYT1 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP SLC25A3 10185|9931 3|0 0.00|0.00 0|0|1|0|0|0|1 0 RAF1_RP SEC61A1 10185|9931 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP DENND4C 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP IRAK1 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP KIAA1543 10185|9931 1|0 0.00|0.00 0|0|2|0|0|0|0 0 RAF1_RP TUBA1B 10185|9931 15|0 0.00|0.00 0|9|0|0|0|0|0 0 RAF1_RP CCT3 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP ATP5C1 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP ASPH 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP PSMC2 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP PTPRD 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP RANGAP1 10185|9931 2|0 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RP MDN1 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP SLC25A6 10185|9931 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP RCN1 10185|9931 3|0 0.00|0.00 0|0|2|0|0|0|0 0 RAF1_RP EPB41L3 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|1 0 RAF1_RP SERBP1 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP PGAM5 10185|9931 2|0 0.00|0.00 0|0|3|0|0|0|0 0 RAF1_RP SDF4 10185|9931 4|0 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RP FANCI 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP USMG5 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP MON2 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP MASTL 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP RPS27L 10185|9931 0|3 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RP SRSF2 10185|9931 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP SOX30 10185|9931 0|4 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP NISCH 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP PTPDC1 10185|9931 11|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP NT5DC2 10185|9931 1|0 0.00|0.00 0|0|1|0|0|0|1 0 RAF1_RP RUVBL1 10185|9931 2|0 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RP LETM1 10185|9931 1|0 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RP TELO2 10185|9931 3|0 0.00|0.00 0|0|0|2|0|0|0 0 RAF1_RP WNK1 10185|9931 0|1 0.00|0.00 0|0|0|0|1|0|0 0 RAF1_RP C8orf30B 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|1 0 RAF1_RP LOC100289343 10185|9931 0|8 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP STON1 10185|9931 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP WDR6 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP TECR 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP SLC3A2 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP DNAJA3 10185|9931 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP NRAS 10185|9931 0|6 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP ALG1 10185|9931 0|2 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP MTOR 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP MAGED1 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP FLII 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP FKBP8 10185|9931 3|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP SALL2 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP SLC1A3 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP YME1L1 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 257

RAF1_RP CDC42EP1 10185|9931 3|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP DPM1 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP LOC730429 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP TUBA4A 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP MLLT11 10185|9931 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP COPB1 10185|9931 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP ARPC1B 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP TRIM14 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP TRIP13 10185|9931 0|2 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP RPN2 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP TIMM13 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP LGALS3BP 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP ABLIM2 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP ZNF507 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP PIK3R2 10185|9931 0|2 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP SCD5 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP NDUFA4 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP FDFT1 10185|9931 4|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP ARL1 10185|9931 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP DCAF7 10185|9931 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP HRAS 10185|9931 6|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP NDUFA5 10185|9931 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP GLUD1 10185|9931 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP ATAD3C 10185|9931 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP ATP5J2 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP C12orf23 10185|9931 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP STUB1 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP TIMM44 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP ACSL4 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP KIAA2022 10185|9931 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP LDHB 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP IPO9 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP KNTC1 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP NDUFA9 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP TCP11L1 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP NDUFS8 10185|9931 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP PTPMT1 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP NUP160 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP HEATR2 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP KIAA0368 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP CECR5 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP DOPEY2 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP DGKE 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP CYBA 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP NOC2L 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP CSNK2A1 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP SLC25A19 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP CEP110 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP LRRK2 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP ADCK4 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP C1orf106 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP SURF1 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP COL1A2 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP TRIM26 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 258

RAF1_RP XPO5 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP CAMK1 10185|9931 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP ADIPOR1 10185|9931 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP HLA-A 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP ZNF287 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP PFKM 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP PHF17 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP DOCK8 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP C14orf49 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP NEU1 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP FURIN 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP LOC100287476 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP PAPL 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP GUSB 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP MED1 10185|9931 0|2 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP AMDHD2 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RP KLHL7 10185|9931 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP YWHAE 10200|10007 174|94 1.00|1.00 0|0|0|0|0|0|0 1 RAF1_RPRP TIMM50 10200|10007 25|46 1.00|1.00 0|0|0|0|0|0|0 1 RAF1_RPRP HSP90AA1 10200|10007 91|88 1.00|1.00 0|0|3|0|0|1|0 1 RAF1_RPRP HSP90AB1 10200|10007 85|78 1.00|1.00 0|0|4|1|0|0|0 1 RAF1_RPRP YWHAZ 10200|10007 29|38 1.00|1.00 0|0|1|0|0|0|0 1 RAF1_RPRP YWHAB 10200|10007 29|34 1.00|1.00 2|0|0|0|0|0|0 1 RAF1_RPRP YWHAG 10200|10007 24|52 1.00|1.00 0|0|0|0|0|0|0 1 RAF1_RPRP YWHAH 10200|10007 21|30 1.00|1.00 0|0|0|0|0|0|0 1 RAF1_RPRP CDC37 10200|10007 45|50 1.00|1.00 0|0|0|0|0|0|0 1 RAF1_RPRP YWHAQ 10200|10007 11|13 1.00|1.00 0|0|0|0|0|0|0 0.999 RAF1_RPRP NRAS 10200|10007 7|9 1.00|1.00 0|0|0|0|0|0|0 0.999 RAF1_RPRP DNAJA1 10200|10007 8|4 1.00|1.00 0|0|0|0|0|0|0 0.998 RAF1_RPRP RUVBL2 10200|10007 3|6 1.00|1.00 0|0|0|0|0|0|0 0.998 RAF1_RPRP SOX30 10200|10007 6|9 1.00|1.00 0|0|0|0|0|0|0 0.997 RAF1_RPRP FKBP5 10200|10007 20|6 1.00|0.99 0|0|0|0|0|0|0 0.997 RAF1_RPRP MALT1 10200|10007 7|7 1.00|1.00 0|0|0|0|0|0|0 0.997 RAF1_RPRP CCT8 10200|10007 5|5 1.00|0.99 0|0|0|0|0|0|0 0.996 RAF1_RPRP SLC25A6 10200|10007 5|3 1.00|0.99 0|0|0|0|0|0|0 0.996 RAF1_RPRP NUDCD1 10200|10007 20|6 1.00|0.99 0|0|0|0|0|0|0 0.995 RAF1_RPRP ZNF764 10200|10007 4|8 0.99|1.00 0|0|0|0|0|0|0 0.995 RAF1_RPRP TUBB6 10200|10007 6|5 0.99|0.99 0|0|0|0|0|0|0 0.994 RAF1_RPRP ARF1 10200|10007 5|2 0.99|0.99 0|0|0|0|0|0|0 0.993 RAF1_RPRP SV2A 10200|10007 5|6 0.99|1.00 0|0|0|0|0|0|0 0.993 RAF1_RPRP KRAS 10200|10007 4|8 0.99|1.00 0|0|0|0|0|0|0 0.993 RAF1_RPRP AKAP8L 10200|10007 5|3 0.99|0.99 0|0|0|0|0|0|0 0.993 RAF1_RPRP HAX1 10200|10007 5|5 0.99|1.00 0|0|0|0|0|0|0 0.992 RAF1_RPRP HNRNPF 10200|10007 3|6 0.98|1.00 0|0|0|0|0|0|0 0.991 RAF1_RPRP PSMC2 10200|10007 6|3 0.99|0.99 0|0|0|0|0|0|0 0.991 RAF1_RPRP CHST14 10200|10007 4|3 0.99|0.99 0|0|0|0|0|0|0 0.991 RAF1_RPRP RPN1 10200|10007 6|3 0.99|0.99 0|0|0|0|0|0|0 0.991 RAF1_RPRP SCO2 10200|10007 2|4 0.99|0.99 0|0|0|0|0|0|0 0.99 RAF1_RPRP APOL2 10200|10007 4|3 0.99|0.99 0|0|0|0|0|0|0 0.99 RAF1_RPRP APP 10200|10007 4|3 0.99|0.99 0|0|0|0|0|0|0 0.99 RAF1_RPRP DNAJA2 10200|10007 6|13 0.99|0.99 0|0|0|0|0|0|0 0.989 RAF1_RPRP IRS4 10200|10007 35|66 0.97|1.00 0|0|4|0|0|2|0 0.987 RAF1_RPRP CSE1L 10200|10007 4|3 0.99|0.99 0|0|0|0|0|0|0 0.987 RAF1_RPRP WDR6 10200|10007 3|3 0.99|0.99 0|0|0|0|0|0|0 0.987 259

RAF1_RPRP SLC3A2 10200|10007 4|2 0.99|0.98 0|0|0|0|0|0|0 0.987 RAF1_RPRP ALG1 10200|10007 2|2 0.99|0.99 0|0|0|0|0|0|0 0.987 RAF1_RPRP FARSA 10200|10007 3|2 0.99|0.98 0|0|0|0|0|0|0 0.985 RAF1_RPRP PSMD3 10200|10007 4|3 0.98|0.99 0|0|0|0|0|0|0 0.984 RAF1_RPRP QPCTL 10200|10007 3|2 0.98|0.98 0|0|0|0|0|0|0 0.984 RAF1_RPRP GLUD2 10200|10007 7|4 0.98|0.99 0|0|0|0|0|0|0 0.984 RAF1_RPRP SEC61A1 10200|10007 7|4 0.99|0.98 0|0|0|0|0|0|0 0.984 RAF1_RPRP DNAJA3 10200|10007 2|3 0.98|0.99 0|0|0|0|0|0|0 0.984 RAF1_RPRP TRIM28 10200|10007 5|2 0.98|0.98 0|0|0|0|0|0|0 0.983 RAF1_RPRP ATP5I 10200|10007 2|1 0.98|0.98 0|0|0|0|0|0|0 0.983 RAF1_RPRP EMD 10200|10007 16|16 0.98|0.98 0|0|2|0|0|0|0 0.98 RAF1_RPRP MSI1 10200|10007 1|2 0.97|0.99 0|0|0|0|0|0|0 0.98 RAF1_RPRP ATP5B 10200|10007 9|6 0.98|0.98 0|0|0|0|0|0|1 0.979 RAF1_RPRP CAND2 10200|10007 3|4 0.97|0.99 0|0|0|0|0|0|0 0.979 RAF1_RPRP CDK1 10200|10007 2|1 0.98|0.97 0|0|0|0|0|0|0 0.979 RAF1_RPRP FASTKD5 10200|10007 4|2 0.99|0.97 0|0|0|0|0|0|0 0.979 RAF1_RPRP CAD 10200|10007 6|8 0.97|0.98 0|0|1|0|0|0|0 0.978 RAF1_RPRP SCD 10200|10007 2|1 0.98|0.98 0|0|0|0|0|0|0 0.977 RAF1_RPRP TIMM8A 10200|10007 1|1 0.98|0.98 0|0|0|0|0|0|0 0.977 RAF1_RPRP TRAP1 10200|10007 7|5 0.98|0.97 0|0|1|0|0|0|0 0.973 RAF1_RPRP GCN1L1 10200|10007 23|13 0.98|0.96 0|0|0|2|0|0|0 0.973 RAF1_RPRP NACA 10200|10007 1|1 0.96|0.98 0|0|0|0|0|0|0 0.973 RAF1_RPRP HAUS5 10200|10007 2|3 0.96|0.99 0|0|0|0|0|0|0 0.973 RAF1_RPRP TBR1 10200|10007 2|1 0.98|0.97 0|0|0|0|0|0|0 0.972 RAF1_RPRP POLR2B 10200|10007 3|3 0.96|0.98 0|0|0|0|0|0|0 0.971 RAF1_RPRP ATP1A1 10200|10007 12|35 0.98|0.96 0|0|0|0|0|0|1 0.97 RAF1_RPRP ASPH 10200|10007 1|1 0.96|0.97 0|0|0|0|0|0|0 0.968 RAF1_RPRP TUBA1A 10200|10007 15|3 1.00|0.93 0|0|0|0|0|0|0 0.967 RAF1_RPRP EFHD2 10200|10007 2|1 0.97|0.97 0|0|0|0|0|0|0 0.967 RAF1_RPRP FANCD2 10200|10007 2|6 0.93|1.00 0|0|0|0|0|0|0 0.967 RAF1_RPRP BNIP3 10200|10007 1|1 0.96|0.97 0|0|0|0|0|0|0 0.967 RAF1_RPRP ATP5C1 10200|10007 1|1 0.97|0.96 0|0|0|0|0|0|0 0.966 RAF1_RPRP FANCI 10200|10007 2|2 0.96|0.98 0|0|0|0|0|0|0 0.966 RAF1_RPRP TDRKH 10200|10007 2|1 0.98|0.95 0|0|0|0|0|0|0 0.966 RAF1_RPRP CDC42EP1 10200|10007 2|1 0.97|0.96 0|0|0|0|0|0|0 0.966 RAF1_RPRP TMX3 10200|10007 2|1 0.98|0.95 0|0|0|0|0|0|0 0.966 RAF1_RPRP AIFM1 10200|10007 22|20 0.96|0.97 0|0|4|0|0|0|1 0.965 RAF1_RPRP DYNC1H1 10200|10007 10|8 0.96|0.96 0|0|4|0|0|0|0 0.964 RAF1_RPRP DCAF8 10200|10007 2|1 0.97|0.96 0|0|0|0|0|0|0 0.964 RAF1_RPRP NDUFAF3 10200|10007 1|1 0.96|0.97 0|0|0|0|0|0|0 0.964 RAF1_RPRP RBBP7 10200|10007 5|4 0.96|0.96 0|0|1|0|0|0|0 0.963 RAF1_RPRP ARPC1B 10200|10007 1|1 0.96|0.97 0|0|0|0|0|0|0 0.963 RAF1_RPRP SEC16A 10200|10007 2|4 0.94|0.99 0|0|0|0|0|0|0 0.962 RAF1_RPRP TM9SF1 10200|10007 1|3 0.94|0.98 0|0|0|0|0|0|0 0.962 RAF1_RPRP TRIP13 10200|10007 1|2 0.95|0.98 0|0|0|0|0|0|0 0.962 RAF1_RPRP MAGED2 10200|10007 1|2 0.94|0.98 0|0|0|0|0|0|0 0.96 RAF1_RPRP USP11 10200|10007 1|2 0.95|0.97 0|0|0|0|0|0|0 0.959 RAF1_RPRP YME1L1 10200|10007 2|1 0.97|0.95 0|0|0|0|0|0|0 0.959 RAF1_RPRP IRAK1 10200|10007 1|2 0.94|0.98 0|0|0|0|0|0|0 0.958 RAF1_RPRP FAR1 10200|10007 2|1 0.96|0.96 0|0|0|0|0|0|0 0.956 RAF1_RPRP GRAMD1A 10200|10007 1|4 0.92|0.99 0|0|0|0|0|0|0 0.956 RAF1_RPRP ARAF 10200|10007 1|3 0.93|0.98 0|0|0|0|0|0|0 0.954 RAF1_RPRP MTOR 10200|10007 3|2 0.96|0.94 0|0|0|0|0|0|0 0.953 RAF1_RPRP HAUS6 10200|10007 2|1 0.97|0.93 0|0|0|0|0|0|0 0.952 260

RAF1_RPRP RCN1 10200|10007 9|5 0.96|0.94 0|0|2|0|0|0|0 0.95 RAF1_RPRP HAUS7 10200|10007 1|1 0.94|0.96 0|0|0|0|0|0|0 0.95 RAF1_RPRP TRMT1 10200|10007 1|1 0.95|0.95 0|0|0|0|0|0|0 0.949 RAF1_RPRP FKBP8 10200|10007 1|1 0.94|0.95 0|0|0|0|0|0|0 0.947 RAF1_RPRP ZEB1 10200|10007 2|1 0.96|0.93 0|0|0|0|0|0|0 0.946 RAF1_RPRP TUBGCP2 10200|10007 2|1 0.96|0.93 0|0|0|0|0|0|0 0.945 RAF1_RPRP CALU 10200|10007 6|3 0.97|0.92 0|0|0|0|0|0|1 0.944 RAF1_RPRP ATR 10200|10007 2|2 0.93|0.95 0|0|0|0|0|0|0 0.943 RAF1_RPRP NUP107 10200|10007 3|1 0.98|0.91 0|0|0|0|0|0|0 0.942 RAF1_RPRP ABR 10200|10007 1|1 0.93|0.95 0|0|0|0|0|0|0 0.942 RAF1_RPRP KRT18 10200|10007 1|4 0.92|0.96 0|0|0|0|0|0|0 0.941 RAF1_RPRP MCM4 10200|10007 1|1 0.94|0.94 0|0|0|0|0|0|0 0.94 RAF1_RPRP NOLC1 10200|10007 1|1 0.93|0.95 0|0|0|0|0|0|0 0.939 RAF1_RPRP STT3A 10200|10007 1|1 0.92|0.96 0|0|0|0|0|0|0 0.939 RAF1_RPRP TFRC 10200|10007 1|1 0.92|0.95 0|0|0|0|0|0|0 0.938 RAF1_RPRP DNAJC7 10200|10007 1|1 0.94|0.93 0|0|0|0|0|0|0 0.938 RAF1_RPRP CTNNA2 10200|10007 1|3 0.89|0.97 0|0|0|0|0|0|0 0.932 RAF1_RPRP SDF4 10200|10007 6|2 0.96|0.90 0|0|1|0|0|0|0 0.931 RAF1_RPRP ATG9A 10200|10007 1|1 0.92|0.94 0|0|0|0|0|0|0 0.931 RAF1_RPRP RPS27L 10200|10007 3|4 0.90|0.96 0|0|1|0|0|0|0 0.929 RAF1_RPRP TUBB4 10200|10007 24|9 0.97|0.88 2|2|0|0|0|0|0 0.927 RAF1_RPRP LETM1 10200|10007 4|2 0.95|0.89 0|0|1|0|0|0|0 0.922 RAF1_RPRP SMC3 10200|10007 2|1 0.94|0.90 0|0|0|0|0|0|0 0.921 RAF1_RPRP EIF2S1 10200|10007 2|4 0.85|0.95 0|0|1|0|0|0|0 0.903 RAF1_RPRP PTPRK 10200|10007 1|1 0.91|0.90 0|0|0|0|0|0|0 0.903 RAF1_RPRP MTDH 10200|10007 2|3 0.87|0.93 0|0|1|0|0|0|0 0.902 RAF1_RPRP POLA1 10200|10007 1|1 0.89|0.91 0|0|0|0|0|0|0 0.901 RAF1_RPRP BCAP31 10200|10007 2|4 0.85|0.94 0|0|1|0|0|0|0 0.893 RAF1_RPRP LPHN2 10200|10007 1|1 0.87|0.90 0|0|0|0|0|0|0 0.887 RAF1_RPRP ALDH3A2 10200|10007 3|5 0.82|0.94 0|0|2|0|0|0|0 0.883 RAF1_RPRP HSPD1 10200|10007 12|11 0.84|0.88 0|0|2|0|0|3|0 0.858 RAF1_RPRP RPL9 10200|10007 2|2 0.83|0.88 0|0|0|0|1|0|0 0.856 RAF1_RPRP PIK3C2A 10200|10007 1|1 0.85|0.86 0|0|0|0|0|0|0 0.852 RAF1_RPRP RPS10 10200|10007 2|1 0.87|0.81 0|0|0|0|0|1|0 0.839 RAF1_RPRP PGAM5 10200|10007 3|4 0.79|0.87 0|0|3|0|0|0|0 0.829 RAF1_RPRP DYNC1I2 10200|10007 1|3 0.75|0.91 0|0|0|0|0|0|1 0.828 RAF1_RPRP WNK1 10200|10007 2|2 0.81|0.84 0|0|0|0|1|0|0 0.826 RAF1_RPRP KRT8 10200|10007 18|11 0.85|0.75 0|0|9|0|0|4|0 0.804 RAF1_RPRP WDR18 10200|10007 1|2 0.73|0.88 0|0|1|0|0|0|0 0.804 RAF1_RPRP DYRK1B 10200|10007 7|13 0.61|0.96 0|0|0|0|0|0|17 0.788 RAF1_RPRP TNRC18 10200|10007 1|1 0.71|0.81 0|0|0|0|0|0|0 0.761 RAF1_RPRP RUVBL1 10200|10007 1|1 0.75|0.77 0|0|1|0|0|0|0 0.757 RAF1_RPRP GEMIN4 10200|10007 5|2 0.83|0.66 1|0|1|0|0|0|0 0.747 RAF1_RPRP NEXN 10200|10007 1|1 0.72|0.77 0|0|1|0|0|0|0 0.744 RAF1_RPRP KRT19 10200|10007 5|13 0.53|0.91 0|0|3|0|0|2|0 0.718 RAF1_RPRP ATAD3A 10200|10007 4|6 0.61|0.78 0|0|3|0|0|0|1 0.696 RAF1_RPRP RPS27 10200|10007 6|2 0.86|0.49 0|0|2|0|0|1|0 0.675 RAF1_RPRP SNRPF 10200|10007 6|3 0.78|0.49 0|1|2|0|0|0|1 0.637 RAF1_RPRP HSPA8 10200|10007 39|39 0.57|0.70 4|2|9|7|0|8|4 0.635 RAF1_RPRP RPL23 10200|10007 2|10 0.33|0.86 1|0|1|1|0|0|0 0.596 RAF1_RPRP ZRANB2 10200|10007 7|1 0.64|0.34 0|0|2|0|0|0|1 0.489 RAF1_RPRP SLC25A3 10200|10007 1|2 0.35|0.62 0|0|1|0|0|0|1 0.488 RAF1_RPRP ATP5A1 10200|10007 11|8 0.50|0.42 0|2|3|0|0|3|2 0.461 RAF1_RPRP RPS12 10200|10007 2|1 0.50|0.38 0|0|1|0|0|0|1 0.444 261

RAF1_RPRP RPS14 10200|10007 5|8 0.11|0.58 0|0|4|2|0|3|2 0.348 RAF1_RPRP QARS 10200|10007 2|2 0.32|0.38 0|0|2|0|0|2|0 0.346 RAF1_RPRP HSPA1B 10200|10007 39|62 0.03|0.65 17|6|9|0|4|16|6 0.342 RAF1_RPRP HSPA9 10200|10007 8|13 0.11|0.54 0|4|2|1|0|4|2 0.329 RAF1_RPRP RPL27A 10200|10007 3|4 0.22|0.43 1|0|1|0|0|2|1 0.325 RAF1_RPRP TUBB2C 10200|10007 4|8 0.12|0.52 0|0|4|0|0|3|2 0.32 RAF1_RPRP IPO8 10200|10007 3|2 0.34|0.25 0|0|3|0|0|0|3 0.298 RAF1_RPRP TUBB2B 10200|10007 2|3 0.21|0.38 0|0|3|3|0|0|0 0.295 RAF1_RPRP EFTUD2 10200|10007 1|1 0.26|0.33 0|0|1|0|0|0|1 0.294 RAF1_RPRP VIM 10200|10007 6|8 0.16|0.41 2|0|5|0|0|1|2 0.284 RAF1_RPRP SLC25A5 10200|10007 5|5 0.19|0.25 2|1|1|0|1|3|0 0.218 RAF1_RPRP MATR3 10200|10007 1|2 0.12|0.29 0|0|3|0|0|0|2 0.203 RAF1_RPRP RPL19 10200|10007 3|6 0.02|0.35 1|0|1|3|0|1|2 0.187 RAF1_RPRP XRCC6 10200|10007 2|1 0.16|0.11 0|0|0|0|0|2|1 0.137 RAF1_RPRP SNRNP200 10200|10007 3|2 0.14|0.12 0|0|1|0|0|0|1 0.127 RAF1_RPRP RPS18 10200|10007 7|15 0.00|0.24 2|0|9|0|1|7|6 0.12 RAF1_RPRP YBX1 10200|10007 6|4 0.12|0.07 0|0|5|1|2|0|2 0.096 RAF1_RPRP ARL6IP4 10200|10007 1|1 0.06|0.08 0|0|3|0|0|2|0 0.071 RAF1_RPRP HNRNPA1 10200|10007 2|7 0.00|0.13 1|3|3|0|0|3|2 0.064 RAF1_RPRP RPS11 10200|10007 2|2 0.05|0.08 0|0|3|0|0|3|1 0.061 RAF1_RPRP RPS8 10200|10007 5|10 0.00|0.11 3|0|6|0|4|5|3 0.054 RAF1_RPRP RPS6 10200|10007 4|3 0.06|0.04 2|1|4|0|0|0|3 0.046 RAF1_RPRP DSP 10200|10007 7|10 0.01|0.08 0|17|4|0|0|0|2 0.045 RAF1_RPRP RPL27 10200|10007 3|2 0.05|0.02 0|0|1|0|1|2|3 0.037 RAF1_RPRP RPS3 10200|10007 5|11 0.00|0.05 3|0|6|2|1|9|3 0.025 RAF1_RPRP RPS9 10200|10007 6|6 0.01|0.03 2|0|4|0|2|6|3 0.018 RAF1_RPRP HNRNPD 10200|10007 2|1 0.03|0.00 1|1|2|0|0|1|1 0.017 RAF1_RPRP RPL36 10200|10007 1|1 0.01|0.02 0|0|2|0|1|2|0 0.017 RAF1_RPRP RPL35A 10200|10007 3|2 0.02|0.01 0|1|4|0|0|3|2 0.014 RAF1_RPRP HIST1H1C 10200|10007 11|13 0.00|0.02 8|0|8|0|4|7|0 0.013 RAF1_RPRP RPL12 10200|10007 2|2 0.01|0.01 1|0|3|0|0|1|2 0.011 RAF1_RPRP TUBB 10200|10007 16|21 0.00|0.01 25|1|1|0|3|8|3 0.007 RAF1_RPRP RPL24 10200|10007 1|1 0.01|0.01 2|0|0|0|1|4|0 0.007 RAF1_RPRP RPL3 10200|10007 4|5 0.00|0.00 0|0|7|0|0|8|4 0.001 RAF1_RPRP NPM1 10200|10007 14|13 0.00|0.00 14|8|15|6|1|3|6 0 RAF1_RPRP XRCC5 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP WDR77 10200|10007 4|5 0.00|0.00 20|11|14|0|11|14|6 0 RAF1_RPRP KIF11 10200|10007 0|4 0.00|0.00 100|19|78|36|19|43|38 0 RAF1_RPRP HNRNPK 10200|10007 2|0 0.00|0.00 0|1|4|0|0|2|0 0 RAF1_RPRP DDB1 10200|10007 4|4 0.00|0.00 0|1|2|0|0|2|0 0 RAF1_RPRP TRIM21 10200|10007 3|0 0.00|0.00 0|0|6|0|1|0|2 0 RAF1_RPRP RPLP0 10200|10007 1|3 0.00|0.00 4|2|3|0|1|4|0 0 RAF1_RPRP PRMT5 10200|10007 7|13 0.00|0.00 59|15|55|14|17|43|30 0 RAF1_RPRP STK38L 10200|10007 1|1 0.00|0.00 3|1|4|0|0|4|1 0 RAF1_RPRP IVNS1ABP 10200|10007 0|1 0.00|0.00 5|8|13|4|0|9|7 0 RAF1_RPRP SKP1 10200|10007 1|0 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RPRP CLNS1A 10200|10007 1|5 0.00|0.00 8|6|19|6|5|15|8 0 RAF1_RPRP RPLP2 10200|10007 9|0 0.00|0.00 0|0|1|0|0|4|2 0 RAF1_RPRP SF3B3 10200|10007 0|2 0.00|0.00 3|3|9|0|2|6|4 0 RAF1_RPRP NCL 10200|10007 11|14 0.00|0.00 4|1|14|16|6|12|6 0 RAF1_RPRP STK38 10200|10007 6|15 0.00|0.00 32|10|32|11|3|25|12 0 RAF1_RPRP DHX15 10200|10007 4|2 0.00|0.00 0|2|13|1|1|5|5 0 RAF1_RPRP TUBA1C 10200|10007 16|4 0.00|0.00 42|0|10|8|5|16|7 0 RAF1_RPRP PPM1B 10200|10007 3|13 0.00|0.00 21|33|2|31|2|40|28 0 262

RAF1_RPRP KRT1 10200|10007 25|2 0.00|0.00 192|250|68|43|29|31|51 0 RAF1_RPRP HNRNPM 10200|10007 3|7 0.00|0.00 2|5|7|0|0|5|6 0 RAF1_RPRP MAP1B 10200|10007 7|7 0.00|0.00 0|0|10|1|0|7|6 0 RAF1_RPRP RPL18 10200|10007 6|1 0.00|0.00 4|1|5|1|2|3|3 0 RAF1_RPRP QPCT 10200|10007 0|4 0.00|0.00 9|5|15|10|0|11|4 0 RAF1_RPRP ACTA2 10200|10007 0|7 0.00|0.00 21|0|1|0|0|0|0 0 RAF1_RPRP SPTBN1 10200|10007 1|0 0.00|0.00 0|3|25|3|0|25|10 0 RAF1_RPRP LARS 10200|10007 2|1 0.00|0.00 0|3|7|0|0|3|1 0 RAF1_RPRP HNRNPA2B1 10200|10007 2|1 0.00|0.00 2|0|5|0|1|6|1 0 RAF1_RPRP RPL10A 10200|10007 2|0 0.00|0.00 1|0|2|0|0|2|1 0 RAF1_RPRP HNRNPR 10200|10007 0|1 0.00|0.00 2|0|3|0|0|3|1 0 RAF1_RPRP RPS4X 10200|10007 2|1 0.00|0.00 1|2|5|0|0|4|0 0 RAF1_RPRP RPL10 10200|10007 2|0 0.00|0.00 0|0|0|0|1|2|1 0 RAF1_RPRP RPS15A 10200|10007 2|0 0.00|0.00 0|0|1|0|0|2|0 0 RAF1_RPRP RPS16 10200|10007 0|1 0.00|0.00 4|1|2|0|0|6|0 0 RAF1_RPRP RPLP1 10200|10007 0|1 0.00|0.00 0|1|0|0|0|1|0 0 RAF1_RPRP UBB 10200|10007 1|2 0.00|0.00 2|0|2|2|0|2|1 0 RAF1_RPRP RPS19 10200|10007 2|0 0.00|0.00 0|0|2|0|0|4|0 0 RAF1_RPRP TXN 10200|10007 2|0 0.00|0.00 0|0|4|0|0|2|0 0 RAF1_RPRP SUB1 10200|10007 2|1 0.00|0.00 5|0|15|6|1|8|3 0 RAF1_RPRP HNRNPAB 10200|10007 0|3 0.00|0.00 1|0|2|0|0|1|3 0 RAF1_RPRP LOC731751 10200|10007 0|17 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RPL14 10200|10007 2|0 0.00|0.00 3|0|0|0|0|1|1 0 RAF1_RPRP C1QBP 10200|10007 2|0 0.00|0.00 1|0|0|0|0|2|0 0 RAF1_RPRP HNRNPL 10200|10007 0|1 0.00|0.00 1|0|1|0|0|0|0 0 RAF1_RPRP IQGAP3 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP EEF1A1 10200|10007 6|7 0.00|0.00 3|3|5|0|1|7|4 0 RAF1_RPRP RPL7A 10200|10007 4|1 0.00|0.00 4|0|5|0|1|9|4 0 RAF1_RPRP PABPC4 10200|10007 0|1 0.00|0.00 0|0|2|0|0|0|0 0 RAF1_RPRP ACTB 10200|10007 14|0 0.00|0.00 65|33|48|59|23|15|47 0 RAF1_RPRP DHX9 10200|10007 1|0 0.00|0.00 0|0|6|0|0|2|0 0 RAF1_RPRP RCN2 10200|10007 4|0 0.00|0.00 0|0|2|0|0|3|0 0 RAF1_RPRP THOC4 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|1 0 RAF1_RPRP IKBKAP 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP PA2G4 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RPL11 10200|10007 1|0 0.00|0.00 1|0|1|0|1|0|1 0 RAF1_RPRP FLNA 10200|10007 4|2 0.00|0.00 0|6|23|0|0|12|11 0 RAF1_RPRP DDX17 10200|10007 0|1 0.00|0.00 0|1|3|0|1|0|2 0 RAF1_RPRP SPTAN1 10200|10007 4|0 0.00|0.00 0|3|31|1|2|20|11 0 RAF1_RPRP HSPA5 10200|10007 9|11 0.00|0.00 3|5|11|0|2|10|3 0 RAF1_RPRP RPL4 10200|10007 2|0 0.00|0.00 3|0|4|5|0|7|2 0 RAF1_RPRP KRT10 10200|10007 19|6 0.00|0.00 143|154|31|55|28|22|31 0 RAF1_RPRP KPNB1 10200|10007 0|1 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RPRP CMBL 10200|10007 0|2 0.00|0.00 15|5|9|5|2|8|5 0 RAF1_RPRP ILF3 10200|10007 3|0 0.00|0.00 0|0|7|0|0|2|2 0 RAF1_RPRP ILF2 10200|10007 1|0 0.00|0.00 0|0|2|0|0|1|0 0 RAF1_RPRP EIF3B 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP HNRNPA3 10200|10007 2|0 0.00|0.00 0|0|4|0|0|0|1 0 RAF1_RPRP RPS28 10200|10007 1|0 0.00|0.00 0|0|1|0|0|1|1 0 RAF1_RPRP KRT9 10200|10007 6|11 0.00|0.00 89|168|64|12|11|20|31 0 RAF1_RPRP SLC30A9 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RPL30 10200|10007 1|0 0.00|0.00 0|0|0|0|0|1|0 0 RAF1_RPRP IARS 10200|10007 2|0 0.00|0.00 0|1|3|0|0|2|4 0 RAF1_RPRP KRT14 10200|10007 2|0 0.00|0.00 6|37|4|3|6|0|7 0 263

RAF1_RPRP RBBP4 10200|10007 5|0 0.00|0.00 2|0|2|0|0|0|0 0 RAF1_RPRP LTBP1 10200|10007 1|1 0.00|0.00 3|1|19|1|1|10|5 0 RAF1_RPRP RPL17 10200|10007 2|0 0.00|0.00 1|0|5|2|0|0|0 0 RAF1_RPRP PRKDC 10200|10007 44|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RPL13 10200|10007 7|5 0.00|0.00 4|0|7|3|2|6|2 0 RAF1_RPRP CCT7 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP THRAP3 10200|10007 0|1 0.00|0.00 16|0|9|7|2|7|4 0 RAF1_RPRP PRMT1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP STAU1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP CCT2 10200|10007 4|0 0.00|0.00 0|0|0|3|0|0|0 0 RAF1_RPRP RPL7 10200|10007 0|3 0.00|0.00 4|2|10|2|0|5|3 0 RAF1_RPRP RPL6 10200|10007 8|6 0.00|0.00 8|0|11|2|0|8|5 0 RAF1_RPRP EPRS 10200|10007 4|5 0.00|0.00 0|3|17|3|1|9|5 0 RAF1_RPRP RPS20 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP CKAP5 10200|10007 2|0 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RPRP PSMD11 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RARS 10200|10007 5|1 0.00|0.00 0|3|6|0|0|7|3 0 RAF1_RPRP CCT5 10200|10007 0|4 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RPRP KRT2 10200|10007 7|5 0.00|0.00 93|150|29|45|9|17|39 0 RAF1_RPRP EIF2S2 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP HNRNPU 10200|10007 1|0 0.00|0.00 7|3|7|0|1|9|4 0 RAF1_RPRP EIF3I 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RPS2 10200|10007 1|0 0.00|0.00 4|0|1|1|2|3|3 0 RAF1_RPRP DNAJB11 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP DARS 10200|10007 6|1 0.00|0.00 0|2|9|0|0|9|5 0 RAF1_RPRP TPM3 10200|10007 1|0 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RPRP PRKAR1A 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP HEATR1 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RPL23A 10200|10007 6|2 0.00|0.00 2|0|8|0|0|5|3 0 RAF1_RPRP MYCBP 10200|10007 1|0 0.00|0.00 0|0|1|0|0|1|0 0 RAF1_RPRP NAP1L1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|1|0 0 RAF1_RPRP RYR3 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP STOML2 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RPL5 10200|10007 2|0 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RPRP KIAA1967 10200|10007 1|0 0.00|0.00 0|0|7|0|0|2|0 0 RAF1_RPRP KRT16 10200|10007 3|0 0.00|0.00 7|40|0|5|0|0|2 0 RAF1_RPRP MARCKS 10200|10007 3|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RPL22 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP PRPF6 10200|10007 2|0 0.00|0.00 0|0|5|0|0|0|0 0 RAF1_RPRP KRT6C 10200|10007 3|0 0.00|0.00 0|0|0|3|0|0|0 0 RAF1_RPRP DRG1 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ZNF638 10200|10007 22|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP MRPS2 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TTN 10200|10007 1|0 0.00|0.00 1|0|1|0|0|2|0 0 RAF1_RPRP TPM1 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RPL8 10200|10007 4|3 0.00|0.00 1|0|7|5|1|5|3 0 RAF1_RPRP NFKB1 10200|10007 0|3 0.00|0.00 0|0|0|0|4|0|19 0 RAF1_RPRP PRSS1 10200|10007 2|0 0.00|0.00 3|0|7|0|2|5|6 0 RAF1_RPRP MYBBP1A 10200|10007 1|0 0.00|0.00 0|0|9|0|0|6|2 0 RAF1_RPRP HOOK1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP MAN2A1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SUN2 10200|10007 0|1 0.00|0.00 1|0|1|0|0|0|0 0 RAF1_RPRP KIAA1671 10200|10007 0|3 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP HIST1H4J 10200|10007 1|1 0.00|0.00 4|1|3|2|0|4|2 0 264

RAF1_RPRP KRT5 10200|10007 8|1 0.00|0.00 24|57|9|3|3|2|9 0 RAF1_RPRP KRT6B 10200|10007 1|0 0.00|0.00 4|9|0|0|0|0|4 0 RAF1_RPRP RPL38 10200|10007 1|0 0.00|0.00 1|0|0|0|0|0|0 0 RAF1_RPRP RPS25 10200|10007 0|1 0.00|0.00 1|0|0|0|0|1|1 0 RAF1_RPRP KRT6A 10200|10007 5|0 0.00|0.00 9|12|2|0|0|0|3 0 RAF1_RPRP LOC100292021 10200|10007 0|2 0.00|0.00 0|0|0|0|1|0|0 0 RAF1_RPRP FLG2 10200|10007 1|0 0.00|0.00 1|12|0|0|0|0|1 0 RAF1_RPRP RIF1 10200|10007 1|0 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RPRP FAU 10200|10007 1|0 0.00|0.00 2|0|0|0|0|1|0 0 RAF1_RPRP RPL35 10200|10007 1|0 0.00|0.00 0|0|4|2|0|0|0 0 RAF1_RPRP LRPPRC 10200|10007 2|0 0.00|0.00 0|0|4|0|0|0|0 0 RAF1_RPRP PTPLAD1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|1 0 RAF1_RPRP DDX21 10200|10007 4|1 0.00|0.00 0|0|15|0|0|9|2 0 RAF1_RPRP SLC6A15 10200|10007 3|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SEC61B 10200|10007 0|4 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP CDK9 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP CHD4 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ATP6V1H 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RPL13AP25 10200|10007 2|0 0.00|0.00 3|0|2|0|0|0|2 0 RAF1_RPRP TMPO 10200|10007 2|0 0.00|0.00 1|0|3|0|0|0|2 0 RAF1_RPRP DCD 10200|10007 1|0 0.00|0.00 1|11|4|2|0|2|0 0 RAF1_RPRP DSG2 10200|10007 1|0 0.00|0.00 3|0|0|0|0|0|0 0 RAF1_RPRP ZBTB33 10200|10007 0|1 0.00|0.00 7|0|0|0|0|0|0 0 RAF1_RPRP ATP2A2 10200|10007 0|12 0.00|0.00 0|0|2|0|0|0|0 0 RAF1_RPRP CLASP2 10200|10007 1|0 0.00|0.00 0|0|8|0|0|1|3 0 RAF1_RPRP TCOF1 10200|10007 3|0 0.00|0.00 0|0|2|0|0|0|0 0 RAF1_RPRP LOC646804 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP USP25 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP MGA 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|1 0 RAF1_RPRP RBM4B 10200|10007 0|4 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP CLPB 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP NOMO2 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP DNAH2 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SCAPER 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP THADA 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP AFG3L2 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SRSF8 10200|10007 2|0 0.00|0.00 0|0|2|1|0|2|1 0 RAF1_RPRP PRDM5 10200|10007 0|4 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RBM39 10200|10007 0|3 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP CYP26B1 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP WBP11 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP P4HB 10200|10007 0|2 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP EWSR1 10200|10007 1|0 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RPRP MKI67 10200|10007 1|0 0.00|0.00 0|0|2|0|0|0|0 0 RAF1_RPRP XPOT 10200|10007 3|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ATP2A1 10200|10007 4|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP DBT 10200|10007 1|0 0.00|0.00 0|1|1|0|0|2|1 0 RAF1_RPRP NUP205 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP NAT10 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP CCT3 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ATAD5 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP BAG2 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LMNB2 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP MRPS23 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 265

RAF1_RPRP LDHA 10200|10007 0|2 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LOC100128370 10200|10007 0|4 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SMC4 10200|10007 2|0 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RPRP MYL12B 10200|10007 1|0 0.00|0.00 0|0|2|0|0|0|0 0 RAF1_RPRP DDX46 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|2 0 RAF1_RPRP RANGAP1 10200|10007 3|0 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RPRP RNF219 10200|10007 1|0 0.00|0.00 0|0|4|0|1|3|3 0 RAF1_RPRP MDN1 10200|10007 6|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RAD50 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SMC2 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP EEF2 10200|10007 1|0 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RPRP PSMD6 10200|10007 3|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP EPB41L3 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|1 0 RAF1_RPRP SERBP1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP PRPF19 10200|10007 1|0 0.00|0.00 0|0|1|0|0|2|0 0 RAF1_RPRP LMNB1 10200|10007 3|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP PHB2 10200|10007 1|0 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RPRP PHB 10200|10007 0|1 0.00|0.00 0|0|2|0|0|0|0 0 RAF1_RPRP RSL1D1 10200|10007 2|0 0.00|0.00 0|0|2|0|0|0|0 0 RAF1_RPRP ZC3HAV1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP PSMB2 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP USMG5 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP MON2 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TUBG1 10200|10007 0|2 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP KTN1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP NME1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP EIF4G1 10200|10007 2|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP EIF5B 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP UPF1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP PURB 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP CSDA 10200|10007 1|0 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RPRP AKAP1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LRRFIP1 10200|10007 1|0 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RPRP KIF21B 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ABCC1 10200|10007 0|2 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RDBP 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP FIP1L1 10200|10007 0|1 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RPRP MYCL1 10200|10007 0|3 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP CAB39 10200|10007 5|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SUGP2 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP MRPL47 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP BVES 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SSBP1 10200|10007 1|0 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RPRP ATP5L 10200|10007 1|0 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RPRP ACTL6A 10200|10007 1|0 0.00|0.00 0|0|1|0|0|0|0 0 RAF1_RPRP MYH14 10200|10007 0|1 0.00|0.00 0|0|0|1|0|0|0 0 RAF1_RPRP CBX3 10200|10007 1|0 0.00|0.00 0|0|0|10|0|0|0 0 RAF1_RPRP TELO2 10200|10007 3|0 0.00|0.00 0|0|0|2|0|0|0 0 RAF1_RPRP ICAM1 10200|10007 0|1 0.00|0.00 0|0|0|1|0|0|0 0 RAF1_RPRP DCTN4 10200|10007 1|0 0.00|0.00 0|0|0|0|0|1|0 0 RAF1_RPRP ZNF814 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|1 0 RAF1_RPRP C8orf30B 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|1 0 RAF1_RPRP LOC100289343 10200|10007 0|6 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SRPK1 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 266

RAF1_RPRP PHF6 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ADAMTS12 10200|10007 0|8 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP MRPL24 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RANBP2 10200|10007 7|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP PHGDH 10200|10007 4|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TECR 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SSR4 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP NUDC 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP STIP1 10200|10007 11|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TMCO7 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP UBE3C 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RPAP1 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ESYT2 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP PPP6R3 10200|10007 3|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP PSMG1 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP COX4I1 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SLC25A11 10200|10007 4|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SNX1 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP BAG5 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP MAGED1 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SORT1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ZW10 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP FLII 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP MTHFD1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP NF1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP PPM1E 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RDH13 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TUBGCP6 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP CANX 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP FTSJ1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ASAH1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP GNAS 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP URB2 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP CCDC55 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP KIAA0564 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RCOR1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP CCDC56 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP HAUS1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TRIM65 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP IGBP1 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ARCN1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP EXOC4 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RAP1A 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SEC13 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP GANAB 10200|10007 3|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ANKRD27 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SALL2 10200|10007 3|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SND1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP BZW1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LUZP1 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP KIAA1274 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SERAC1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SLC1A3 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP AK2 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 267

RAF1_RPRP FASN 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP BUB1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP FKBP4 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP C5orf15 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP POLR1B 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP PANK4 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TRIM32 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP AGPAT6 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP AP3D1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP EXOC7 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP CXorf26 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP DNAJB6 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ACTR2 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP GALC 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP GLS 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ARF4 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP DPM1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LOC730429 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LOC651610 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LOC389217 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP CUL7 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP BCAS3 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TMEM161B 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SLC27A2 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TES 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP CDC73 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LEPREL4 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP VAMP8 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ZNF174 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP HPS6 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP HUWE1 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP USP9X 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ACSL3 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP CEPT1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LOC100288068 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP C8orf33 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP CLASP1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP NUP133 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TUBA4A 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SRPRB 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP DYNC1LI2 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP GGT7 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ATP5D 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ANAPC5 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP AGPAT2 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TUBB2A 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP BSG 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP EDEM3 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ZSCAN18 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP MLLT11 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP GTPBP3 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP CERCAM 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP HOOK3 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP DNAH8 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 268

RAF1_RPRP EXOC6 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LOC100133737 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP EXT2 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LTV1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP GSTCD 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP CACNA1H 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP PDLIM5 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP IPO7 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP EHD4 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP DDOST 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP NELL2 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TMEM201 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP NLRX1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP COPG2 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TBC1D9B 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SLC25A10 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP C1R 10200|10007 3|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP NCAPH 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP COPB1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RNF126 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SVEP1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP MCCC2 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LOC100290530 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP PWP1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP P4HA2 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SAP30BP 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP CBWD1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP COPA 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP FKBP9 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TMEM109 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP COPE 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP POLE 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ANKRD58 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP PLOD1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP DCAF5 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP DDHD2 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TRIM14 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP VPS11 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RNASEH2C 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LOC100133775 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LOC652614 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TMOD4 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TMEM87A 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP JUB 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SUCLG1 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SPNS1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP NUP93 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TSPYL6 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TRAFD1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP XPO6 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP MAN1B1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RUFY3 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP BCCIP 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SOX8 10200|10007 8|0 0.00|0.00 0|0|0|0|0|0|0 0 269

RAF1_RPRP ARMC8 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP DHRS3 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TUFT1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP NET1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP MAP3K7 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP PPP6R1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LIN37 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP EPS8L2 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ARFGEF2 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SH3D19 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP HARBI1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ENTPD7 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP C18orf55 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP FAM71E2 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ARID4B 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LOC642776 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP CHD1L 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ATXN10 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SLC4A5 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP KRT35 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP MAD2L1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP DHRS4L1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RABGAP1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ZMYM2 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LOC100289566 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LTN1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP FRMD7 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP MADD 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP C19orf70 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP KIAA1524 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP DACT1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LOC100132588 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LOC100130274 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP GINS3 10200|10007 6|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SCO1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP NEK2 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP R3HCC1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LOC100294305 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP PCDH7 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP C11orf16 10200|10007 3|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP EFNB1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP CLPX 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RINT1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RPN2 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP COL4A3 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP DMXL2 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LOC100133673 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP EME2 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SIPA1L1 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP NPPC 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TIMM13 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SPG7 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP KDM5C 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP GJA8 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 270

RAF1_RPRP UTP6 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LOC100288824 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP MMS19 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP FAM115C 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ARID2 10200|10007 2|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TRIM36 10200|10007 1|0 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP P4HA1 10200|10007 0|2 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP C1orf57 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP NR3C1 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TARS2 10200|10007 0|4 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP PNKD 10200|10007 0|5 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LGALS3BP 10200|10007 0|2 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SEC22B 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP NARS 10200|10007 0|2 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LONP1 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP PRKAA1 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP STK11IP 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP AKAP10 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SLC7A5 10200|10007 0|2 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP DENND4A 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SLC1A5 10200|10007 0|2 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LOC100287455 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP POLD1 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP NDUFS7 10200|10007 0|2 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LSR 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ABLIM2 10200|10007 0|9 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP CHD5 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP RNF40 10200|10007 0|2 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TAP2 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ATP5F1 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP MLF2 10200|10007 0|3 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP PRKAG2 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ENTPD6 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ZNF507 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP PKP2 10200|10007 0|2 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP PCNT 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ESRP2 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP PDS5B 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SDCCAG3 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP PI4KA 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP PIK3R2 10200|10007 0|3 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SHMT1 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LOC100131573 10200|10007 0|2 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP HCN2 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP PDPR 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP UGCG 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP EI24 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP UBA1 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ADAMTS4 10200|10007 0|2 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP NAV1 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ZNF3 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP CTSB 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ZNF317 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ATP2B1 10200|10007 0|2 0.00|0.00 0|0|0|0|0|0|0 0 271

RAF1_RPRP RAVER1 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ACPT 10200|10007 0|3 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP IL1RAPL1 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP VPS26B 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP BCL6 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP KIAA0090 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SETD8 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP HTR2B 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP DENND2C 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SCD5 10200|10007 0|2 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ALDH18A1 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SLC27A3 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP NOXO1 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SPAG1 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP NDUFA4 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LOC100287843 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ETS2 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP IFT80 10200|10007 0|4 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP STXBP5L 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TRPM5 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP SHMT2 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP ALG5 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP TMEM38A 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0 RAF1_RPRP LOC100130825 10200|10007 0|1 0.00|0.00 0|0|0|0|0|0|0 0