Proteomic Analysis of Protein Arginine Methyltransferases 5 and 7 Using Bioid Method

Proteomic analysis of protein arginine methyltransferases 5 and 7 using BioID method

Arthur Jacob

Faculty of Medicine

Division of Experimental Medicine

McGill University, Montreal, Quebec, Canada

August, 2016

A Thesis Submitted to McGill University in Partial Fulfillment of the Requirements for the Degree of Master of Science

Abstract

Protein arginine methylation is a common post-translational modification catalyzed by a family of 9 protein arginine methyltransferases (PRMTs). The PRMTs are implicated in a variety of biological processes including epigenetic regulation, splicing, and DNA damage response pathways. Although the proteome of

PRMT1 is well-characterized, PRMT5 and in particular PRMT7 proteomes are less known. Almost none of the PRMT7 binding partners and substrates are known. Here I perform the first BioID screen for PRMT5 and PRMT7, and identify > 30 candidate interactors and substrates for each. Among them some were already known interactors of PRMT5, validating the assay. eIF2α was identified as a key candidate for PRMT7. eIF2α is involved in mRNA translation initiation and regulates this process in response to stress through a phosphorylation event at serine 51. I confirmed the interaction and established that eIF2α is methylated by PRMT7 in vitro and in vivo. In addition my findings show that PRMT7 is required for proper eIF2α phosphorylation in response to endoplasmic reticulum (ER) stress.

Résumé

La methylation de l’arginine est une modification post-traductionnelle catalysée par une famille de 9 methyltransferases d’arginine (PRMTs). Les PRMTs sont impliquées dans de nombreux processus biologiques tells que la regulation épigénétique, le “splicing”, et la réponse aux dommages à l’ADN. Bien que le proteome de PRMT1 est bien caractérisé, le proteome de PRMT5 et PRMT7 est moins connu – presque aucun partenaire d’intéraction ou substrat de PRMT7 n’a encore été identifié. Nous avons realisé le premier crible BioID pour PRMT5 et PRMT7 et avons identifié environ 50 candidats pour chacun. Parmi eux certains étaient connus pour intéragir avec PRMT5, ce qui valide le BioID. eIF2α a été identifié comme étant le meilleur candidat pour intéragir avec PRMT7. eIF2α a un role important dans l’initiation de la traduction des proteins et dans la regulation de ce processus en réponse au stress via la phosphorylation de sa serine 51. Nous avons confirmé l’intéraction et avons établi que PRMT7 methyle eIF2α in vitro et in vivo. De plus nos résultats indiquent que PRMT7 est requis pour une phosphorylation normale de eIF2α en réponse au stress du reticulum endoplasmique.

Preface

This Master of Science thesis was written in accordance with the Guidelines for Thesis Preparation from the Faculty of Graduate Studies and Research of McGill University.

The experiments and studies were conducted by Arthur Jacob under the supervision of Dr. Stéphane

Richard. As well, each contributor who has collaborated in this research is acknowledged in the following section and throughout the remainder of the text.

The mass spectrometry analysis discussed in this thesis was performed by Dr. François-Michel Boisvert

(Université de Sherbrooke) (Figure 6). The 293 cell line expressing stably tet-ON gene and the purified

PRMT7 enzyme were given genereously by Dr. Jean-Yves Masson (Université Laval).

Acknowledgments

I would first like to thank my thesis advisor Pr. Stéphane Richard of the Department of Oncology and

Medicine at McGill University. The door to Prof. Richard office was always open whenever I ran into a trouble spot or had a question about my research or writing. He consistently allowed this paper to be my own work, but steered me in the right direction whenever he thought I needed it.

I would like to thank Dr. François-Michel Boisvert (Université de Sherbrooke) who performed the mass spectrometry experiments.

I would like to thank Dr. Antonis Koromilas and Jothi Krishnamoorthy for cells, reagents and helpful discussion.

I would also like to thank the experts who were involved in the validation survey for this research project:

Dr. Zhenbao Yu, Gillian Vogel, and Roméo Blanc. Without their participation and input, the validation survey could not have been successfully conducted.

I would also like to acknowledge Dr. Sara Calabretta as the reader of this thesis, and I am gratefully indebted to her for her very valuable comments on this thesis.

Finally, I must express my very profound gratitude to my parents, my aunt Catherine and to my girlfriend Estelle for providing me with unfailing support and continuous encouragement throughout my years of study and through the process of researching and writing this thesis. This accomplishment would not have been possible without them. Thank you.

Table of Content

ABSTRACT …………………………………………………….……………………….…. Page 2

RÉSUMÉ ……………………………………………………………….……………….…. Page 3

PREFACE …………………………………………………………………………………. Page 4

AKNOWLEDGEMENTS ………………………………………………………………… Page 5

TABLE OF CONTENT ……………………………….…………………………...….….. Page 6

LIST OF FIGURES …………………………………...…………………………….….… Page 7

LIST OF ABBREVIATIONS ……………………………………………………………. Page 8

INTRODUCTION …………………………………………………………….………...... Page 11

Part I: Post-translational modifications and arginine methylation ...... Page 11

Part II: Proteomic methods for the study of PRMTs’ interacting partners

and substrates ………………………………………………………………………...… Page 18

Part III : the ER stress response and eIF2α ……………………………………….….… Page 21

EXPERIMENTAL PROCEDURES ………………………………...…………..…..…… Page 25

RESULTS …………….………………………………………………………….…....…… Page 30

Establishment of the BioID method to identify PRMT interactors …...... Page 30

BioID screen confirms PRMT5-known complexes and identifies new targets ………. Page 30

BioID screen identifies new PRMT7 interacting complexes ……………..…………... Page 31

eIF2α binds to PRMT7 and is released upon ER stress ……………………………..... Page 32

PRMT7 accumulates foci in response to ER stress ………………………..….…….... Page 33

PRMT7 methylates eIF2α in vitro and in vivo ……………...…………...... …....…… Page 34

DISCUSSION ……………………………………………………………….……………. Page 36

REFERENCES ……………………………..…………………………….……………… Page 47 6

List of tables and figures

Table 1: List of best candidates for PRMT5 bioID identified by mass spectrometry

Table 2: List of best candidates for PRMT7 bioID identified by mass spectrometry

Figure 1: Arginine Methylation in mammalian cells

Figure 2: BioID method

Figure 3 : Phosphorylation of eIF2α impairs translation initiation

Figure 4: The UPR triggers the eIF2α pathway

Figure 5: Establishment of BioID method in HEK293 cells

Figure 6: Mass spectrometry of BioID

Figure 7: PRMT7 interacts with eIF2α and dissociates under ER stress to form stress foci

Figure 8: PRMT7 methylates eIF2α in vitro and in vivo

List of abbreviations

ADMA: asymmetric di-methyl arginine

AR: androgen receptor

ATF4: activating transcription factor 4

BSA: bovine serum albumine

Co-IP: co-immunoprecipitation

COPR5: cooperator of PRMT5

Coum: coumermycin

CTCFL: CCCTC-binding factor-like

DDX23: ATP-dependent RNA helicase

DNA: desoxyribonucleic acid eIF2α : eukaryotic initiation factor 2 alpha eIF2B: eukaryotic initiation factor 2 B

ER: endoplasmic reticulum

ES: embryonic stem

FBS: fetal bovine serum

FOXO1: forkhead box protein 01

G3BP1: Ras GTPase activating protein-binding protein 1

G9a: Euchromatic histone-lysine N-methyltransferase 2

GCN2: general control nonderepressible 2

GDP: guanosine diphosphate

GLP: Ras GTPase activating protein-binding protein 2

GTP: guanosine triphosphate

H2AR3: histone 2A arginine 4

H2B: histone 2 B

H3R2: histone 3 arginine 2

H3R8: histone 3 arginine 8

H4R3: histone 4 arginine 3

H4R3me2a: histone 4 arginine 3 dimethyl asymmetric

HNRNPK: heterogeneous nuclear ribonucleoprotein K

HNRNPU: heterogeneous nuclear ribonucleoprotein U

HRI: Eukaryotic translation initiation factor 2-alpha kinase 1

IB : immunoblot

IF: immunofluorescence

IgG: immunoglobuline G

IP: immunoprecipitation

IPA: ingenuity pathway analysis

IPTG: isopropyl β-D-1-thiogalactopyranoside

JAK2: janus kinase 2

KHDRBS1: KH domain-containing, RNA-binding, signal transduction-associated protein 1

KO: knock out

MEP50: methylosome protein 50

MMA: mono methyl arginine mRNA: messenger RNA

MS: mass spectrometry

NPC: neural stem/progenitor cells

OSBPL: oxysterol-binding protein

PBS: phosphate-buffered saline

PCCB: propionyl-CoA carboxylase beta chain, mitochondrial

PCR: polymerase chain reaction

PERK: protein kinase R-like endoplasmic reticulum kinase

PICln: chloride channel nucleotide sensitive 1A

PKR: protein kinase R

POLD1: polymerase D1

PRMT: protein arginine methyltransferase

PTM: post transcriptional modification

POI: protein of interest

PPI: protein-protein interaction

RER: rough endoplasmic reticulum

RNA: ribonucleic acid

RIOK1: rio domain-containing protein

RPL: 60S ribosomal protein

RPS: 40S ribosomal protein

RT: room temperature

RT-PCR: reverse transcriptase polymerase chain reaction

SAM: S-adenosyl methionine

SDMA: symmetric dimethyl arginine

SDS: sodium dodecyl sulphate

PAGE: Polyacrylamide gel electrophoresis

SMN: survival of motor neurone

SRSF7: serine/arginine-rich splicing factor 7

THW: threonine-histidine-tryptophane tRNA: transfer RNA

Met tRNAi : methionyl-transfer RNA

UPR: unfolded protein response

WDR77: WD repeat protein 77

WT: wild-type

Introduction

This project aims to identify binding partners and non-histone substrates of the protein arginine methyltransferases (PRMTs) 5 and 7 to characterize new roles for these enzymes in biological processes and signalling pathways.

Part I: Post-translational modifications and arginine methylation

It has emerged in the last few decades that the human proteome is far more complex than the human genome. Although it is estimated that only 20,000 to 25,000 genes are encoded by the human genome (International Human Genome Sequencing, 2004), the human proteome comprises over 100,000 proteins (Jensen, 2004). These data indicate that a single gene of the human genome encodes for an average of at least five different proteins. The complexity of the human proteome results from multiple mechanisms that increase the number of proteins that are encoded by one single gene, including alternative splicing, translation initiation at alternative promoters, and genomic recombination (Ayoubi & Van De

Ven, 1996). In addition, post-translational modifications (PTM) greatly enhance this complexity. Post- translational modifications play a key role in the modulation of protein function, be it in terms of protein activity, half-life, interactions, or cellular localization. Proteins’ PTMs are highly dynamic processes that occur in response to a wide range of stimuli, thereby representing an adaptive response to the environment.

As chemical modifications of proteins, PTMs are carried by several enzymes during or after protein biosynthesis. Enzymes represent approximately 5% of the human proteome and are able to catalyze over

200 different types of PTMs (Jensen, 2006). Kinases, phosphatases, ligases, and methyltransferases are among the best known enzymes that add or remove functional groups to the side chains of amino acids.

Protein methylation is one of the most frequently occurring PTM which can regulate multiple cellular processes, such as messenger RNA (mRNA) translation, splicing, and DNA damage. Furthermore, it plays a key role in the epigenetic regulation of gene expression, as the balance between histone methylation and demethylation regulates the availability of DNA for transcription (Cheung & Lau, 2005).

Protein methylation is characterized by the transfer of methyl groups to nitrogen or to oxygen of specific amino-acid side chains (N- and O-methylation respectively), thereby resulting in an increase of protein hydrophobicity. A single or multiple methyl groups can be added to the target proteins. Methylation is carried out by specific enzymes that are called methyltransferases, which transfer the methyl group to the proteins, mainly though the primary donor group S-adenosyl methionine (SAM) to lysine or arginine residues. These two types of methylations are catalyzed by different types of enzymes: lysine methyltransferase, and protein arginine methyltransferases respectively.

This master thesis focuses on the PRMT family of enzymes. Arginine methylation is a common

PTM in eukaryotic cells, and is facilitated by the unique hydrogen-bonding potential of the arginines guanidine group (Bedford & Clarke, 2009; Gary & Clarke, 1998). Arginine methylation impacts cell signalling, DNA repair, RNA maturation and nucleocytoplasmic transport, protein stability, ribosomal assembly, and regulation of gene expression (Bedford & Clarke, 2009; Bedford & Richard, 2005). Nine

PRMTs (PRMT1-9) have been identified in mammals. All of the PRMTs carry a highly conserved methyltransferase-catalytic domain (Figure 1. A), which is known to oligomerize into a ring-like structure

(Weiss et al., 2000; Zhang & Cheng, 2003), as well as a threonine-histidine-tryptophan (THW) loop known to interact with the methyl-accepting substrate arginine and mediate substrate specificity (Jain et al., 2016).

However, they differ regarding the presence of additional protein domains, cellular localization, and tissue expression. Despite PRMTs share the same catalytic domain, it has been demonstrated that there is no major redundancy within the activity of PRMTs, since mouse knockouts for different PRMT generally result in different and sometimes dramatic phenotypes (Bedford & Clarke, 2009). Protein arginine methyltransferases are ranked into three categories (Atkinson & Murray, 1967; Bedford & Clarke, 2009).

Type I PRMTs form ω-NG, NG-asymmetric dimethylarginine (ADMA) via the addition of two methyl groups to the same terminal nitrogen of the arginine residue. This category also contains PRMT1, PRMT3,

PRMT4 (also known as CARM1), PRMT6, and PRMT8. Type II PRMTs catalyze mainly ω-NG’, NG- symmetric dimethylarginine (SDMA), by the addition of the second methyl group to the second nitrogen of

12 the arginine residue. Protein arginine methyltransferases 5 and 9 (PRMT5 and PRMT9) figure into this category (Bedford, 2007). Finally, type III enzymes catalyze the ω-NG-monomethylarginine (MMA) reaction, and to date, only PRMT7 has been established as a member of this category (Zurita-Lopez,

Sandberg, Kelly, & Clarke, 2012) (Figure 1. B).

catalytic domain THW loop A PRMT1*

PRMT2*

PRMT3*

CARM1

PRMT5*

PRMT6*

PRMT7**

Myristoyl- PRMT8*

PRMT9*

Preference for: Legend: • * RGG/RG motifs • Type I (ADMA) • ** RXR motifs • Type II (SDMA) • Type III (MMA)

Figure 1: Arginine Methylation in mammalian cells. A) The nine members of the protein arginine Figure 1 methyltransferases family. B) The three flavours of protein arginine methyltransferases. Adapted from

"The role of arginine methylation in the DNA damage response" by Y. Auclair and S. Richard, 2013, DNA

PRMT1

PRMT1 is the major type I PRMT in mammalian cells, which contributes to at least 85% of the entire arginine methyltransferase activity in eukaryotic cells (Tang, Kao, & Herschman, 2000). It was the first

PRMT characterized. Protein arginine methyltransferases 1 null mice (PRMT1-/-) die during embryogenesis at embryonic day 6.5 (E6.5), underlying PRMT1’s essential function for mouse development (Pawlak,

Scherer, Chen, Roshon, & Ruley, 2000). PRMT1 is present in both the cytoplasm and the nucleus and has substrates in both compartments. PRMT1 deposits a dimethyl mark on histone H4 at arginine 3

(H4R3me2a) (Strahl et al., 2001), and this mark is associated with transcriptional activation.

PRMT1 is already well-characterized and has been shown to methylate several non-histone proteins.

PRMT1-/- embryonic stem (ES) cells display hypomethylation of several proteins, such as MRE11

(Boisvert, Hendzel, Masson, & Richard, 2005), Sam68 (KHDRBS1) (Cote, Boisvert, Boulanger, Bedford,

& Richard, 2003), and hnRNPK (Ostareck-Lederer et al., 2006). This activity confers to PRMT1 an important role in DNA damage, mRNA splicing and protein localization. PRMT1 is also responsible for the regulation of Akt signaling pathway: it was shown that PRMT1-mediated arginine methylation blocks

Akt phosphorylation. In particular this was shown for the forkhead box protein O1 (FOXO1), which was methylated on R248 and R250, thus inhibiting the phosphorylation by Akt on S253, leading to regulation of apoptosis, cell cycle regulation and overall cell survival (Yamagata et al., 2008). Several additional

PRMT1 substrates have been identified, allowing for the characterization of the sites of methylation: preferably arginines within a region that is rich RGG/RG motifs (Thandapani et al., 2013).

PRMT5

PRMT5 is the predominant type II methyltransferase in mammals (Pawlak et al., 2000).

Similarly to PRMT1, it is an essential enzyme, since PRMT5 null mice (PRMT5 -/-) display early embryonic lethality between 3.5 and 6.5 embryonic days (Tee et al., 2010). PRMT5 symmetrically dimethylates histones in vivo, generating the repressive marks H2AR3, H4R3, H3R2, and H3R8, which diminish DNA transcription (Ancelin et al., 2006; Fabbrizio et al., 2002; Xu, Hoang, Mayo, & Bekiranov,

2010). Examples of genes that are regulated epigenetically by PRMT5 include cyclin E1 (Fabbrizio et al.,

2002), retinoblastoma protein (Wang, Pal, & Sif, 2008), and ribosomal genes (Majumder et al., 2010).

PRMT5 form two dimers in the head-to-tail arrangement that is typical of PRMTs. Directly binding

PRMT5, MEP50 greatly enhances its histone methyltransferase ability, primarily through increased affinity for protein substrate. The PRMT5–MEP50, or methylosome complex, has a higher level of methyltransferase activity compared to PRMT5 alone (Antonysamy et al., 2012). The methylosome is formed as a hetero-octomeric complex that is composed of four PRMT5 proteins and four MEP50 proteins

(Ho et al., 2013).

PRMT5 and splicing

The PRMT5–MEP50 complex is a key regulator of pre-mRNA splicing by regulating the assamblay of snRNPs (Gonsalvez et al., 2007). The Sm proteins D1, D3, and B/B’ are symmetrically dimethylated by the methylosome, a 20S methyltransferase complex, formed by PRMT5-MEP50 in complex with chloride channel nucleotide sensitive 1A (pICln) (Brahms, Meheus, de Brabandere, Fischer,

& Luhrmann, 2001). The pICln binds the Sm domain and assembles the complex formed with the methylosome (Friesen, Paushkin, et al., 2001; Grimm et al., 2013). The sDMA of Sm D1, D3, and B/B’ enable the binding of these three proteins to the Tudor domain-containing protein SMN (survival of motor neuron), which is the product of the spinal muscular atrophy gene (Brahms et al., 2001; Friesen, Massenet,

Paushkin, Wyce, & Dreyfuss, 2001), thereby promoting the assembly of the seven Sm proteins onto the pre-mRNA (Meister & Fischer, 2002). Accordingly, a conditional PRMT5 knockout in mouse neural stem/progenitor cells (NPCs) shows that PRMT5 is required for a proper splicing reaction: the absence of

PRMT5 leads to retention of introns and skipping of exons with weak 5’ donor sites (Bezzi et al., 2013).

Binding partner regulation of PRMT5

The activity and localization of PRMT5 is regulated in multiple ways, including the association with different binding partners. PRMT5 binds to pICln or the Rio domain-containing protein (RioK1) in a mutually exclusive manner in order to modulate the choice of the substrates: the recruitement of RioK1 by 16

PRMT5 favors methylation of nucleolin, whereas pICln binding allows for the methylation of spliceosomal machinery components (Guderian et al., 2011). Another interactor is cooperator of PRMT5 (COPR5), which is associated with histones in the nucleus and recruits PRMT5 to nucleosomes (Lacroix et al., 2008).

It acts as a chromatin adaptor for PRMT5 and promotes histone H4 (R3) methylation versus the others histone methylation marks (Lacroix et al., 2008). Moreover, the kinase JAK2 binds and phosphorylates

PRMT5. Nimer and coworkers showed that the tyrosine phosphorylation of PRMT5 by a constitutive

JAK2 impairs the ability of PRMT5 to methylate its histone substrates, thus contributing to regulate gene expression and chromatin organization (Liu et al., 2011). Through these examples we can conclude that interacting partners of PRMT5, and of the PRMT family in general, are very important to regulate their activity. Defining these interactors is important because they can impact multiple biological processes.

PRMT7

PRMT7 was first identified from a genetic-suppressor element screen in Chinese hamster cells, in which the loss of PRMT7 results in enhanced cell sensitivity to a broad range of DNA‐damaging agents

(Gros et al., 2003). In this study, PRMT7 is described as a gene that encodes two proteins, namely p77 and p82, which were later renamed as PRMT7α and β due to significant sequence homology with the PRMT enzymes family (Gros et al., 2003). PRMT7 has been reported to play a role in drug resistance (Zheng et al., 2005), and has been shown to repress the expression of several genes that are involved in DNA repair

(Hu, Sif, & Imbalzano, 2013).

Although PRMT7 was initially classified as a type II PRMT, recent findings show that it is a type III enzyme, due to its MMA specificity (Feng, Hadjikyriacou, & Clarke, 2014). Specifically, it methylates arginines of the RXR motif, whereas the other PRMTs favor RGG/RG motifs (Thandapani,

O'Connor, Bailey, & Richard, 2013). PRMT7 is present both in the cytoplasm and in the cytosol (Lee et al.,

2005). PRMT7 is known to methylate recombinant histone H2B in vitro and is able to mono-methylate in

17 vivo histone H4R3 at gene promoters, which leads to the modulation of their expression, as shown for the

Bcl6 gene in the germinal centers (Ying et al., 2015) and for the polymerase δ catalytic subunit gene,

POLD1 (Hu et al., 2013).

PRMT7 is involved in multiple functions in vivo. PRMT7 is notably implicated in male germline imprinted-gene methylation through its interaction with CCCTC-binding factor-like (CTCFL)

(Jelinic, Stehle, & Shaw, 2006). As a proposed oncogene (Martin-Kleiner, 2012), CTCFL’s association with PRMT7 may be important for its transforming activity. Furthermore our laboratory recently defined a role for PRMT7 in muscle regeneration, as well as in the maintenance of muscle stem cell regenerative capacity through the regulation of cellular senescence (Blanc, Vogel, Chen, Crist, & Richard, 2016).

However, to our knowledge no PRMT7 methylation complexes or PRMT7 non-histone substrates have yet been identified. That is why my project aims to identify the proteome of PRMT7, and potentially to identify non-histone substrates of PRMT7.

Part II: Proteomic methods for the study of PRMT interacting proteins and substrates

Multiple types of transient or stable protein-protein interactions (PPIs) have been depicted.

Stable interactions are associated with proteins that are purified as multiprotein complexes, while by definition, transient interactions are temporary.

PPIs are modulated by multiple events such as the post-translational modifications (i.e. phosphorylation, methylation) that influence protein-conformational changes or localization to specific areas of the cell.

PPIs occur mainly via protein domains, the nature that determines the strength and reversibility of the association: interactions can be either strong or weak. Protein interaction is usually the result of a combination of covalent links, hydrophobic interactions, salt bridges, and van der Waals forces between interacting proteins.

BioID: a new method based on proximity-dependent biotin labeling for the analysis of PPI

The most commonly used techniques to study PPI are co-immunoprecipitation (co-IP) and pull- down assays. These methods allow for the detection of stable interactions and the formation of multiprotein complexes, whereas the transient interactions are not easily detectable because they may disassemble rapidly or because the denaturing and stringent conditions that are used can cause PPI’s destruction. However, transient interactions are expected to represent most of the interaction of PRMTs, including their substrates. Indeed, the great majority of PRMTs’ substrates does not form a stable complex but interacts with its PRMT temporarily. Aiming to identify PRMT interacting partners and to define new substrates in this study, co-IP and classic pull-down assay were not the most accurate techniques with which to perform a high throughput screening of PRMT interactions.

Recently, a new technique for the screening of PPI based on proximity-dependent biotin labeling has been elaborated by Roux et al., called BioID (Roux, Kim, & Burke, 2013). This method relies on the generation of a fusion protein through the joining of the Escherichia coli biotin ligase BirA to the protein target of the current interaction study. BirA is a 35-kD DNA-binding biotin protein ligase in that biotinylates Acetyl-CoA carboxylase (Chapman-Smith & Cronan, 1999). As the wild type BirA has a stringent selectivity for its endogenous substrate, Roux et al. used a BirA mutant instead (R118G, called

BirA*) that biotinylates proteins on lysine residues without any substrate specificity in a proximity- dependent manner. Once it is expressed in the cells, the fusion protein BirA*-protein of interest (POI) is expected to maintain the same localization and activity as the endogenous POI, so that every protein that interacts with POI, even temporally, will be biotinylated. Cells are then lysed and the biotinylated proteins are pull-downed by a biotin-affinity capture. Finally, the biotinylated proteins are identified by mass- spectrometry analysis (MS) (Fig.2).

Figure 2: BioID method. The biotin ligase BirA is fused to the protein of interest. BirA biotinylates every proteins on lysines that is close to the BirA-fusion protein. After 24h cells are lysed and the protein are SDS-denatured, allowing their biotin affinity capture using streptavidin beads. Biotinylated proteins can then be identified by mass spectrometry. Adapted from « BioID: a screen for protein- protein interactions”, by K. J. Roux, D. I. Kim, & B. Burke, 2013, Curr Protoc Protein Sci, 74, Unit 19

Adapted with permission.

Using BioID as a method by which to screen for PPIs, this study intends to characterize the interacting proteome of PRMT5 and PRMT7 in order to identify new binding partners, as well as new substrates of these two enzymes. Interestingly, PRMT7 best candidate screened by the BioID assay was the

α subunit of the eukaryotic initiation factor 2 (eIf2α). As eIF2α is an important protein playing an essential role in the control of translation initiation and has potential biological relevance in ER stress, cancer

(through the control of cell growth) and a variety of major biological processes, we decided to direct our project on the characterization of interaction between PRMT7 and eIF2α.

Part III: the ER stress response via eIF2α phosphorylation

The endoplasmic reticulum (ER) is an organelle present in eukaryotic cells and bound to the nuclear membrane. The rough endoplasmic reticulum (RER) assembles and transports proteins destined for membranes and secretion. A few minutes after protein biosynthesis, most join the Golgi apparatus, to be expressed at the plasmic membrane or to be secreted. In the RER proteins can be modified, folded and quality "controlled". Protein folding and quality control in the ER is crucial for protein function and viability. It is performed in the ER by molecular chaperones.

Protein folding and quality control may be deficient especially in cells that express large amount of proteins such as the secreting cells, leading to the occurence of misfolded proteins in the ER. ER stress is characterized by the accumulation of unfolded or misfolded proteins in the lumen of the ER and triggers the unfolded protein response (UPR). In response to cellular stresses, cells need to adapt their metabolism to maintain energy homeostasis. The UPR aims to restore the normal cell conditions by stopping protein syntesis, degrading unfolded proteins and to promote the expression of molecular chaperone involved in protein folding. If these objectives are not reached in a certain amount of time or if the disruption remains, the UPR trigger apoptotic pathways leading to cell death.

One of the first events following ER stress is the inhibition of most of mRNA translation.

Protein synthesis is the production peptide chains by the ribosomes from mRNAs. These amino acid chains later fold into active and functional proteins in the ER. Protein synthesis basically occurs in three steps: the initiation part where the ribosomes assemble around the targeted mRNA, followed by the elongation part where a transfer RNA (tRNA) carrying a specific amino acid recognize the codon and add the amino acid to the amino acid chain, thus lengthening the amino acid chain. Finally during the termination phase a stop codon is reached and the amino acid chain is released.

The major regulatory step of mRNA translation is the initiation phase. The mechanism involves rapid eIF2α phosphorylation on its α subunit on serine 51 by four different kinases that act as a result of stress, such as amino acid deprivation (GCN2 kinase), ER stress (PERK kinase), the presence of dsRNA (PKR

21 kinase), or Heme deficiency (HRI kinase). In the case of ER stress, PERK dimerize at the surface of the

ER, thereby inducing its activation and phosphorylates its substrate eIF2α (serine 51) in the cytoplasm. eIF2α is one of the subunits of the heterotrimeric complex eIF2. eIF2 is required for the initiation of translation. After being activated by the guanine nucleotide-exchange factor eIF2B that exchanges a GTP

Met from GDP, the binary complex eIF2-GTP binds to the initial methionyl-transfer RNA (tRNAi ) that forms a ternary complex that then associates with the small (40S) ribosomal subunit, thereby proceeding to carrying the initial methionine to the start-codon AUG in a GTP-dependent manner. Phosphorylation on serine 51 by one of the four eIF2α kinases impairs the transfer of GTP on eIF2apha by the GTPase eIF2B, leading to attenuation of global translation initiation (Fig.3). eIF2α phosphorylation inhibits global protein synthesis, but allows the translation of ATF4 mRNA, which encodes a transcription factor controlling the transcription of genes involved in autophagy, apoptosis, amino acid metabolism and protein folding

(Fig.4).

We hypothesize that PRMT7 interacts with eIF2α, and regulates translation initiation in response to ER stress through the methylation of eIF2α. This project aims to validate the PRMT7/eIF2α association and to assess the function of this interaction in response to ER stress and under normal conditions.

Met Figure 3 : Phosphorylation of eIF2α impairs translation initiation. eIF2α-GTP binds to tRNAi and load it on the start codon inside the 40S ribosomal subunit. The GTP is hydrolyzed into GDP, and eIF2α-

GDP is recycled into eIF2α-GTP by eIF2B. Phosphorylation of eIF2α by the eIF2α kinases inhibit recycling of eIF2α and lead to overall attenuation of mRNA translation. Adapted from “Biochemical mechanisms for translational regulation in synaptic plasticity”, by E. Klann and T.E. Dever, 2004, Nat Rev

Cytoplasm

Figure 4: The UPR triggers the eIF2α pathway. Following ER stress PERK dimerization at the ER membrane triggers its kinase activity and lead to eIF2α phosphorylation. This results in overall inhibition of translation initiation, and the expression of the transcription factor ATF4 which activates expression of genes involved autophagy, apoptosis and protein folding. Reprinted from « The unfolded protein response: controlling cell fate decisions under ER stress and beyond” by C. Hetz, 2012, Nat Rev Mol Cell Biol 13(2):

Experimental Procedures

Construct and stable-cell line generation pcDNA 3.1 mycBioID, pRetroX-Tight PURO and pRetroX tetON Advanced vectors were purchased from

Addgene. Fusion proteins mycBirA*-PRMT5 and mycBirA*-PRMT7 were generated by subcloning the cDNA of human PRMT5 and PRMT7 into the pcDNA3.1 mycBioID next to BirA*, and in frame with the initiation codon (fig. 5 A and B). Subsequently, the fusion proteins were subcloned into a pRetroX-Tight-

PUR vector in order to control its expression by using the tetracycline-inducible pTight promoter, because the constitutive expression of the fusion proteins could result in loss of cell viability.

Stable cell lines of 293T cells expressing both pRetroX-Tight-PUR expressing BirA* fusion proteins and pRetroX-tetON-Advanced were then generated in order to create a system in which mycBirA*-PRMT5 and mycBirA*-PRMT7 expression is inducible by tetracycline.

Stable cell lines were generated by retrovirus infection. Viruses were produced by transfecting Phoenix cells with pTight plasmid expressing BirA-PRMT5 and BirA PRMT7. Two days after the transfection of

Phoenix cells, Phoenix supernatant was filtered through a 0.45 µm syringe filter (EMD Millipore,

#SLHV033RS) and 4 µg/ml polybrene was added to the 293T cells. Twenty-four hours after, the infection cells were selected with puromycin (1 µg/ml). A few single clones were selected via western-blotting analysis of stably transfected cells. The experiment was done in duplicates for each generated stable cell line.

Cell culture, transfection and drug treatments

293T and U2OS were purchased from the American Type Culture Collection (ATTC, Manassas, VA).

HT1080 cells stably expressing GyrB-PERK were the generous gift of Dr. Koromilas lab and were generated as in (Krishnamoorthy et al., 2014). All of the cells were cultured in Dulbecco’s modified Eagle 25 medium (DMEM; GE Healthcare, #SH30081.01) and supplemented with 10% fetal bovine serum (FBS;

BioSera), 1 mM sodium pyruvate and penicillin-streptomycin. Plasmids were transfected with

Lipofectamine 2000 (Invitrogen, #11668019) and siRNAs were transfected with Lipofectamine

RNAiMAX (Invitrogen, #13778150) according to the manufacturer’s instructions. siRNA were all used at

100nM. Cells were analyzed either 48h or 72h after transfection. Thapsigargin was purchased from Sigma

Aldrich (T9033) and used at 750ng/mL concentration. Coumermycin was purchased from Sigma Aldrich

(C9270) and was used at 100ng/mL for 6h.

Antibodies, immunoprecipitations and immunoblotting

PRMT5, PRMT7 and HA tag antibodies were purchased from Millipore Inc (#07-405 and #07-639). eIF2α total and Monomethyl (Me-R4-100) antibodies were from Cell Signaling (#9722 and #8015S). ATF4 and eIf2α pSer51 antibodies were purchased from Santa Cruz (sc-200 and sc-12412). Tubulin antibody was obtained from Abcam (ab6046). Protein-A-Sepharose beads were purchased from Sigma-Aldricht (P3391).

Species-specific immunoglobulin G (IgG) control antibodies were procured from Invitrogen (#10500C).

Immunoprecipitations (IPs) and immunoblotting (IB) were performed as previously described (Boisvert et al., 2002). Briefly, cells were lysed in lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl) that was supplemented with EDTA-free protease inhibitor cocktail (Roche) and 1% Triton X-100 on ice for 15 min.

After removal of the Triton insoluble phase by centrifugation, the supernatant was incubated for 2h on ice for with the indicated antibodies. The bound proteins were immunopurified by using protein A Sepharose beads that rotated at 4 ̊C for 1h and which were separated by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with the indicated antibodies as previously described (Boisvert et al.,

2002).

Proximity biotinylation coupled to mass spectrometry

The procedure described by Lambert et al. was followed (Lambert, Tucholska, Go, Knight, & Gingras,

2015). Four 150mm plates were used for each biological replicate of the BioID-MS. After reaching 70% confluency, cells were induced with 1µg/ml tetracycline and culture media was supplemented with 12uM biotin (Sigma Aldrich, B4501) for 24h. Confluent cells were then harvested by scraping in 1ml of ice-cold

Phosphate-buffered saline (PBS), after medium removal and two washes with 5ml PBS per plate.

Pellets were then thawed in 1.5 mL ice-cold RIPA buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1%

NP-40, 1 mM EDTA, 1 mM EGTA, 0.1% SDS and 0.5% sodium deoxcycholate). PMSF (1 mM), DTT (1 mM) and Sigma protease inhibitor cocktail (P8340, 1:500) were added immediately before use. The lysates were sonicated, supplemented with 250U of benzonase (Sigma aldricht, E1014) and centrifuged. For each sample, 100 µL of streptavidin-sepharose bead slurry (GE Healthcare, #17-5113-01) were pre-washed three times with 1 mL of lysis buffer and used to capture biotinylated proteins, thereby incubating protein extract for 3h at 4°C under rotation. The beads were then gently pelleted and washed three times with 1 mL of

RIPA buffer and three times with 1mL of 50 mM ammonium bicarbonate (pH 8.0).

Mass-spectrometry analysis

Mass spectrometry was performed by Dr. François-Michel Boisvert in Sherbrooke University, following the procedure developed by Lambert et al. (Lambert et al., 2015).

IPA software was used to analyze the mass-spectrometry data and to generate the figures.

Immunofluorescence

Wild-type (WT) Mouse Embryonic Fibroblast (PRMT7+/+ MEFs) and PRMT7 knock out (KO) MEFs

(PRMT7-/-) were cultured on coverslips until they reached 40% confluence. Cells were then washed once with PBS and fixed with PFA 4% for 15 min. After another PBS wash, cells were permeabilized with PBS

0,1% triton for 10 min. at room temperature (RT), and then washed in PBS. Cells were incubated with

27 primary antibody diluted in PBS 3% BSA for 2h at RT and then washed three times in PBS. Finally, cells were incubated in secondary antibody diluted in PBS 1% BSA and washed three times with PBS.

Coverslips were mounted with ImmunoMOUNT supplemented with DAPI. Images were acquired with a

Zeiss Axio Imager 2 microscope and analyzed with Axio imager software, and the same settings for all image sets were used.

In vitro methylation assay

In vitro methylation of eIF2α by PRMT7 was performed by using recombinant proteins. Positive control was performed with recombinant H2B (substrate of PRMT7) from Sigma (SRP0407). Recombinant substrates were incubated with GST-PRMT7 and 0.55 µCi of [methyl-3H] S-adenosyl-L-methionine

(PerkinElmer, #NET155V250UC) in the presence of 25 mM Tris- HCl at pH 7.4 for 1h in a final total volume of 30 µl. The reactions were stopped by adding 30 µl of 2X Laemmli buffer (2% SDS, 10% glycerol, 50mM Tris-Cl (pH 6.8), 2,5% Beta-Mercaptoethanol, 0,01% bromophenol blue) and by boiling the samples for 10 min. The methylation of eIF2α was assessed by polyacrylamide gel electrophoresis, stained with Coomassie Blue. The destained gel was soaked in EN3HANCE (PerkinElmer, #6NE9701) for

1h, and followed by a gentle agitation in cold water for 30 min. The washed gel was dried at 60 ̊C for 1.5h and visualized by fluorography, as described in (Cote et al., 2003).

GST purification

Bacteria were placed overnight at 37°C in 20mL of LB medium. Then 10mL of the overnight culture were added to 90mL 2x YT medium and was incubated at 37°C for 1h. One hour later, isopropyl β-D-1- thiogalactopyranoside (IPTG) was added for 3h to induce expression of the recombinant protein. Bacteria were pelleted, resuspended in 1mL 1X PBS, sonicated 3 times 15 seconds, and centrifuged in microfuge

4°C for 5 minutes. The supernantant was kept and 100 ul 10% Triton X 100 (1% final) with 100 ul of GST beads 50% slurry were added. After 1h of tumble end-over-end, beads were washed twice with 1X PBS;

1% Triton, then once with 1X PBS. Finally the beads were eluted with glutathione and the elution product was dialysed for 24h in purified nanopure H2O.

Results

Establishment of the BioID method to identify PRMT interactors

BioID in 293T cells was performed in order to identify new binding partners of PRMT5 and

PRMT7. Expression vectors encoding fusion proteins of mycBirA*-PRMT5 and mycBirA*-PRMT7 were generated by subcloning and these plasmids were stably transfected in 293 cells as indicated in the

“Experimental procedure” section. Expression of mycBirA-PRMT5 and mycBirA-PRMT7 fusion proteins were under tetracycline induction and their expression confirmed by Western blotting (Fig.5 A and B).

Once this system was established, BioID assay was performed by incubating the stable cell lines with tetracycline 1uM and biotin 12uM for 24h. The cells were lysed and the pull-down of biotinylated proteins was performed by biotin-affinity capture using streptavidin beads. The pull-down of biotin-labeled proteins was confirmed by Western blotting (Fig.5 C). It was possible to identify larger bands at 105kDA and

115kDa in the second and third lanes corresponding respectively to mycBirA*-PRMT5 and mycBirA*-

PRMT7 (Fig.5 C). This confirms the activity of BirA* that biotinylate proteins that are nearby, including the fusion protein itself. Beads were then sent to the mass spectrometry platform for high throughput identification of potential binding partners of PRMT5 and PRMT7. This experiment was done in triplicate.

BioID screen confirms PRMT5-known complexes and identifies new targets

Mass-spectrometry analysis identified around 2000 baited proteins for BirA*-PRMT5 fusion protein. The interactors were trimmed by comparing their peptide score with negative control BirA* alone, giving us a ratio. The only candidates that were kept were those for which the ratio was superior or equal to

3 in the three replicates, rendering a list of about 50 candidates (Table 1). Proteins included in the

CRAPome which correspond to proteins identified as contaminants in affinity purification-mass spectrometry data were removed (Mellacheruvu et al., 2013). Additionally, endogenously biotinylated proteins such as the propionyl-CoA carboxylase beta chain, mitochondrial (PCCB) were removed.

IPA software was used to lay out the network of interaction formed by the protein that was identified on the screen (Fig.6 A).

Among the significant interacting proteins of PRMT5, many proteins that were known to interact with PRMT5 were found, thereby validating the accuracy of the assay: MEP50 (also called

WDR77), RioK1, one of the top hits, and COPR5. Importantly, pICln and a protein complex corresponding to the spliceosomal machinery were also identified, including Sm proteins B, D1 and D2, as well as other

RNA-binding proteins that play a role in pre-mRNA processing and splicing, such as Heterogeneous nuclear ribonucleoprotein U (HNRNPU), Serine/arginine-rich splicing factor 7 (SRSF7), ATP-dependent

RNA helicase (DDX23) (Fig.6 A).

Interestingly, a large complex of ribosomal proteins was also identified, including the 40S ribosomal proteins RPS7, RPS14 and RPS20, as well as the 60S ribosomal proteins RPL9, RPL10, RPL11, RPL14,

RPL24, RPL27, and RPL28 (Fig.6 A).

In addition, Ras GTPase activating protein-binding protein 1 (G3BP1) was identified, which is known to be methylated by PRMT1 to regulate the Wnt/β-catenin signaling (Arginine methylation of G3BP1 in response to Wnt3a regulates b-catenin mRNA) (Fig.6 A).

Finally, the protein WIZ, which has an important role in regulating the G9a/GLP complex for gene repression through histone methylation was significantly pull- downed (Bian, Chen, & Yu, 2015)(Fig.6 A).

These results bring some strong potential interactors of PRMT5 involved in a wide variety of biological processes that could be starting point for future projects.

BioID screen identifies new PRMT7 interacting complexes

The PRMT7 interacting proteome remains largely unknown. The IPA analysis of significantly interacting proteins did not find any previously identified protein interaction among the 50 candidates

(Table 2). Three different complexes that interact with PRMT7 were identified (Fig.6 B).

Three members of the oxysterol binding protein-like family, including OSBPL9, OSBPL10 and OSBPL11, were strongly represented. The role of these proteins is poorly understood, even if some evidence indicates that they act as intracellular lipid receptors, and regulate Golgi structure and function (Raychaudhuri &

Prinz, 2010) (Fig.6 B).

As in the PRMT5 BioID, a complex of ribosomal proteins containing the four ribosomal proteins RPL10,

RPL12, RPL35A and RPS14 were identified (Fig.6 B). In addition, the PRMT7 BioID screen revealed eIF2α as a top hit (Fig.6 B). It was possible to detect two other components of the translation-initiation complex, namely eIF2A and eIF2B, despite the fact that their peptide amount did not prove to be significant among the replicates (Fig.6 B). Another protein that was abundantly pull-downed in the PRMT7 assay is the DNA/RNA-binding protein KIN17. This protein is involved in DNA replication and is recruited to DNA in response to DNA damage due to RecA domains-like (Miccoli et al., 2003) (Fig.6 B).

Sam68 appears to bind to PRMT7 and is an RNA binding protein that is known to be a substrate in vivo of

PRMT1 (Cote et al., 2003) (Fig.6 B).

eIF2α binds to PRMT7 and is released upon ER stress

eIF2α controls the initiation of mRNA translation, conferring to it important implications in the control of cell growth, in the response to ER stress and in response to virus infection. Given that such an important protein was one of the top hit of PRMT7 BioID and that PRMT7 interactors are very poorly characterized, we decided to validate eIF2α as a binding partner of PRMT7. The binding of PRMT7 to eIF2α was confirmed by performing an IP in HT1080 cells that express constitutively HA-tagged eIF2α. eIF2α was immunoprecipited with HA antibodies and it was possible to detect PRMT7 by Western blotting, thereby confirming the interaction (Fig.7 A, lane 1).

As we have seen in the introduction, phosphorylation on serine 51 leads to inhibition of overall translation initiation. Interestingly, the serine 51 is directly next to a RXRXR motif, which has been shown to be targeted for arginine monomethylation by PRMT7 in vitro and in vivo by Clarke et al. (Feng et al., 2014).

This is the only RXR site within the amino acid sequence of eIF2α. I subsequently hypothesized that phosphorylation of eIF2α could have an effect on the binding of PRMT7.

Aiming to determine whether the phosphorylation of eIF2α in vivo affects the interaction between PRMT7 and eIF2α, specific HT1080 cells were used that stably expressed both HA-eIF2α and the fusion protein

GyrB-PERK, and which were developed by Dr. Koromilas’ lab (see experimental procedure section).

GyrB is a bacterial domain that is placed in frame with PERK (at its N-terminal side) and which is able to dimerize in the presence of coumermycin (Coum). Dimerization of GyrB domains leads to PERK dimerization which triggers its kinase activity (Fig.7 B). Cells were treated with coumermycin 1µM for 6h and it was possible to detect the expected phosphorylation of eIF2α serine 51 by using a specific antibody

(Fig.7 A, lower panel). In these conditions, HA-eIF2α was immunoprecipitated and it was not possible to detect PRMT7 co-IP any longer, which suggests that eIF2α-PRMT7 interaction is lost when PERK is activated (Fig.7 A, lane 2).

This loss of interaction may be caused by either the phosphate group that was deposited on serine 51, or by the binding of PERK on eIF2α, for example by steric encumbrance. In order to test this hypothesis, the same HT1080 cells were used, but now expressing mutant HA-eIF2α R51S, which cannot be phosphorylated by the kinase. PRMT7 binding was detected in normal conditions, and the interaction was lost after coumermycin treatment, as previously occurred (Fig.7. A, lane C and D). These findings suggest that the phosphate residue that was deposited by PKR is not responsible for the detachment of the complex.

Then PRMT7/eIF2α dissociation could be explained by steric encumbrance generated by the binding of

PERK to the complex.

PRMT7 accumulates in dots and forms foci in response to ER stress

In order to understand what happens to the PRMT7-eIF2α association after the ER stress- mediated PERK kinase activation, we analyzed cellular localization by immunofluorescence. We used

MEF cells, depleted or not for PRMT7 (WT versus KO), and treated or not with thapsigargin, a drug which induces ER stress.

In the non-treated cells, eIF2α and PRMT7 were both localized in the cytoplasm as expected. Their localization was diffused and mainly in the cytoplasm. In contrast, in cells that were treated for 2h with thapsigargin, PRMT7 localization was aggregated in small dots in the cytoplasm, thereby forming foci, while eIF2α localization was not changed compared to the non-treated cells. This observation suggests that in response to ER stress, PRMT7 re-localize and accumulate in multiple dense foci in the cytoplasm, similarly to how stress granules assemble (Fig.7 C). At the bottom of the panel, PRMT7 KO MEFs was stained as a negative control. No PRMT7 staining was observed, thereby showing that the staining is specific to PRMT7. The eIF2α pattern was not affected by the loss of PRMT7 (Fig.7 C).

In light of these results, the model proposed here is that the activation of eIF2α kinase PERK disrupts the

PRMT7/eIF2α association. In thapsigargin-induced ER stress conditions, PRMT7 re-localize in dense cytoplasmic aggregations, independently of eIF2α phosphorylation events.

PRMT7 methylates eIF2α in vitro and in vivo

The following question was whether eIF2α is a substrate of PRMT7. The amino acid sequence of eIF2α shows the presence of the consensus-methylation motif for PRMT7 and RXRXR, which is predicted to be methylated in vivo by previous large-scale proteomic studies (Guo et al., 2014). In order to test this hypothesis, an in vitro methylation assay was performed by using free radioactive -3H to detect the methyl transfer on the substrate. First, both GST-eIF2α and PRMT7 were purified by GST purification, as explained in the experimental procedures section. Purified histone H2B obtained from Sigma was used as a positive control, which is known to be methylated in vitro by PRMT7 (Feng et al., 2014). An active recombinant PRMT7 enzyme was incubated for 1h, with recombinant H2B and eIF2α proteins respectively, as confirmed on the Coomassie Blue gel (respectively lanes 1 and 2) at the bottom of Fig.8.

A. As expected, PRMT7 bands were observed at around 78kDa in lanes 1 and 2. H2B band (lane 1) was detected at 15kDa and GST-eIF2α band (lane 2) was observed at around 63kDa, as the molecular weight of

GST and eIF2α is respectively 26kDa and 37kDa.

At the top of the panel, the autoradiography shows a large band at 15kDa of the first lane corresponding to the positive control H2B, validating the in vitro assay. At lane 2, a band was observed at around 63kDa, which corresponds exactly to the molecular weight of GST-eIF2α, thereby demonstrating the methylation of eIF2α by PRMT7 in vitro.

The subsequent aim was to investigate whether such methylation occurs in vivo in HT1080 cells. In order to test this, HA-eIF2α was immunoprecipitated by using the HA specific antibody, and immunoblotted with Monomethyl Me-R4-100 (Cell Signaling) to detect monomethylation marks that were deposited by PRMT7. A monomethyation band was observed at around 37 kDa (Fig.8 B, top panel), which corresponds to the molecular weight of HA-eIF2α that was pull-downed (Fig.8 B, lower panel). HT1080 cells expressing an empty vector instead of HA-eIF2α vector were used to control the experiment.

Interestingly, when the cells were treated with coumermycin and eIF2α phosphorylation was subsequently induced by PERK, no methylation band was observed, thereby suggesting that eIF2α is not methylated when it is being phosphorylated by PERK. These results are consistent with the co-IP showing that PRMT7 dissociates from eIF2α following PERK activation.

Discussion

The first goal of my project is to identify binding partners and/or substrates of PRMT5 and

PRMT7 to uncover new roles for the PRMTs and arginine methylation. Finding new PRMTs substrates is highly challenging due to the low affinity and transient interactions between the PRMTs and their targets.

The BioID method was performed for the first time on PRMT5 and PRMT7. The BioID method has many advantages over screening for PPI because it is not only expected to identify the stable complexes and interactions as IPs do, but it also allows for the identification of transient and temporary bindings to the protein of interest. The entire proteome of PRMT5 and PRMT7 was identified in 293 cells.

Importantly, most of the complexes that are known to interact with PRMT5 were identified, such as the methylosome complex and the spliceosomal machinery. Although this screen did not identified every

PRMT5 substrate, it did identify Sm proteins SmD1, SmD2, and SmB/B’, which are key substrates of

PRMT5 (Friesen, Paushkin, et al., 2001) and part of the spliceosomal complex. No other PRMT5 substrates were identified with the BioID screen.

The fact that the stable association was detected well, while the substrates were not, indicates that the screen was not optimally sensitive. One of the hypotheses by which to explain this lack of sensitivity is that the background that was generated by the negative control BirA was too high. With such high background, and the high peptide score for BirA alone, the low affinity and transient associations that are characterized by only few peptides which are detected by the mass spectrometry analysis did not contrast with the background. In order to reduce the background in the future, I propose to diminish the incubation time in the presence of free biotin, or to decrease the number of biotinylated peptides. Another way by which to reduce the background is to decrease the expression of BirA in the 293 cells.

Additionally, the BioID method has been improved in the last months with the discovery of a smaller biotin ligase, « BioID 2 », which enables more selective targeting of fusion proteins, requires less biotin supplementation, and exhibits an enhanced labeling of proximate proteins (Kim et al., 2016).

In the PRMT5 assay, a large number of ribosomal proteins from both the 40S and 60S subunits were identified, thereby strongly suggesting that PRMT5 binds to the ribosomes and may have a role in mRNA translation. It has been reported that PRMT5 methylates ribosomal protein S10 (RPS10) and is likely to regulate cell proliferation through the methylation of ribosomal proteins (Ren et al., 2010).

One of the most interesting targets obtained for PRMT5 from the BioID was protein WIZ. A recent study reports that WIZ binds to both histone methyltransferases GLP and GL9, and regulates their activity. They are transcription repressors and the WIZ is essential in guiding them to the chromatin region where it functions as a repressor (Bian et al., 2015). As PRMT5 is a transcription repressor through its histone methyltransferase activity, another mechanism would have to account for the involvement of PRMT5 in transcription regulation and repression. In addition, WIZ presents an RG/RGG motif that is predicted to be methylated in vivo, thereby reinforcing that WIZ is potentially targeted by PRMT5. This path demands to be explored in further studies.

Concerning the PRMT7 BioID assay, three members of the OSBPL family were observed as potential binding partners. The OSBPL proteins are a family of proteins that binds to lipids, and plays an important role in the Golgi organization and in vesicles trafficking. The screen in this study suggests an implication of PRMT7 in these processes, in coordination with the OSBPL family proteins, and appeals to further investigation. In addition, kin17 (KIN gene) was significantly pull-downed, which is involved in

DNA replication and is recruited to DNA in response to DNA damage, thanks to domains that are similar to recA protein domains (Cloutier, Lavallee-Adam, Faubert, Blanchette, & Coulombe, 2014; Miccoli et al.,

2003). It would be interesting to study the association between PRMT7 and kin17 to show a role for

PRMT7 in the DNA damage response. Indeed, it has been reported several times that PRMT7-deficient cells are more sensitive to DNA damage (Gros et al., 2003), although the molecular mechanism is still not elucidated. eIF2α was the second best hit after OSPL proteins in the PRMT7 assay. This target is very interesting because of its crucial importance in the initiation of mRNA translation and regulation. In stress conditions (i.e. ER stress), eIF2α is phosphorylated on serine 51, thereby inducing a global inhibition of

37 protein synthesis, except for some particular genes implicated in the stress response (i.e. apoptosis, autophagy, and protein folding) which become induced.

In the first instance, the screen was confirmed by showing by IP that PRMT7 interacts with eIF2α.

Interestingly, following the activation of eIF2α kinase PERK, the interaction dissociates and the IF experiments show an accumulation of PRMT7 proteins in dense cytoplasmic foci, resembling the stress granules. It would be interesting to test by IF whether PRMT7 relocalizes under different stress stimuli, such as replicative stress (hydroxyurea), and oxidative stress (arsenic), as well as to stain the stress granule marker TIA-1 to investigate whether PRMT7 accumulation corresponds to the stress granules’ assemblies.

It was subsequently demonstrated that PRMT7 methylates eIF2α in vitro and in vivo. This finding is important because it tentatively constitutes the only validated non-histone PRMT7 substrate. As eIF2α amino-acid sequence include only one consensus target RXRXR motif for PRMT7 (amino acids 52 to 56), we strongly hypothesize that the methylation occurs on one of these three arginines. It would be necessary to generate mutants where arginines are substituted by a lysine or an alanine to precisely determine the methylation site.

Interestingly the RXRXR motif is located alongside the phosphorylated serine 51. Given the amino-acid sequence of eIF2α and previous proteomic studies (Guo et al., 2014), eIF2α is predicted to be arginine methylated at R54 and R55, which correspond to an Akt consensus site RXRXXS/T. It was questioned whether methylation and phosphorylation could influence each other, either positively or negatively. Recently, several papers show a competition between phosphorylation and arginine methylation in which they are mutually exclusive (Scaramuzzino et al., 2015; Yamagata et al., 2008) and this triggers opposed signaling pathways. As we have seen in the introduction, PRMT1 is able to counteract phosphorylation by Akt. In particular Akt phosphorylates S253 in FOXO1 regulating apoptosis, cell cycle and overall cell survival and PRMT1 methylates FOXO1 on R248 and R250. These arginine methylation marks block FOXO1-S253 phosphorylation, regulating nuclear export and proteasome degradation. The increase of arginine methylation of FOXO1 by PRMT1 results in nuclear export arrest and an enhancement

38 of the transcription of FOXO1 target genes (Yamagata et al., 2008). Moreover, PRMT6 catalyze arginine methylation of androgen receptor (AR) at Akt consensus site motif, which is mutually exclusive with serine phosphorylation by Akt. The expression of a mutant AR that enhances the interaction with PRMT6 leads to neurodegeneration in mouse and fly models (Scaramuzzino et al., 2015). Then I assume that there is likely a crosstalk between eIF2α arginine methylation by PRMT7 at R54 and R55 and phosphorylation at S51. I expect that PRMT7-dependant arginine methylation and PERK phosphorylation of eIF2α are mutually exclusive which means that PRMT-dependent methylation blocks phosphorylation like in the previous examples.

We recently generated a PRMT7-deficient U2OS cell line by using CRISP-Cas9 technology to perform some future experiment in this project. To test the methylation-phosphorylation crosstalk hypothesis, we will induce eIF2α phosphorylation either by treating HT1080 cells with Coumermycin, or by inducing ER stress with thapsigargin in U2OS cells and compare the eIF2α phosphorylation induction by Western blot in control cells versus PRMT7-depleted cells. This experiment would be important to determine whether

PRMT7 affects eIF2α phosphorylation.

An other major objective in the future would be to determine how the absence of PRMT7 affect the eIF2α phosphorylation pathway and more generally the response to ER stress. In this context it would also be interesting to assess the level of ATF4 in the PRMT7-deficient cells in order to observe the impact of the loss of PRMT7 downstream eIF2α phosphorylation.

Under ER stress, the transcription factor ATF4 is expressed and triggers the expression of many genes that are involved in stress response, such as apoptosis, autophagy, and protein folding (Hetz, 2012; Yorimitsu,

Nair, Yang, & Klionsky, 2006). I propose to assess the activation of these genes in PRMT7-deficient cells, either at the RNA level (RT-PCR) or at the protein level (western blot), to test the effect of PRMT7 on eIF2α pathway in response to ER stress.

More generally, to further determinate the effect of PRMT7 on general translation initiation, a further experiment should include ribosome profiling assays on PRMT7-/- cells, with or without ER stress. This would determine which mRNAs are being actively translated and whether PRMT7 deficit affects the

39 regulation of mRNA translation. Interestingly, the BioID screen identified a complex of ribosomal proteins containing the four ribosomal proteins RPL10, RPL12, RPL35A and RPS14, suggesting that PRMT7 directly binds to the ribosome and modulates translation.

In conclusion, this project contributes to characterize the proteome of two members of the protein arginine methyltransferase family PRMT5 and PRMT7 using a new PPI screening method called

BioID. In addition we determined eIF2α as a new substrate of PRMT7 in vitro and in vivo with possible implications in translation regulation. The loss of eIF2α/PRMT7 interaction and the re-localization of

PRMT7 in stress foci in response to ER stress suggest a role in ER stress response.

Peptides Peptides Protein names Gene names BirA_Ctl BirA_PRMT5 Fold change Protein arginine N-methyltransferase 5 PRMT5 5 31 6,2 Probable E3 ubiquitin-protein ligase HERC1 HERC1 0 27 #DIV/0! Serine/threonine-protein kinase RIO1 RIOK1 5 24 4,8 Protein Wiz WIZ 0 21 #DIV/0! MAP7 domain-containing protein 3 MAP7D3 4 12 3 Ras GTPase-activating protein-binding protein 1 G3BP1 3 10 3,333333333 Exocyst complex component 4 EXOC4 1 9 9 Eukaryotic translation initiation factor 4E type 2 EIF4E2 2 9 4,5 Methylosome protein 50 WDR77 3 9 3 Protein PAT1 homolog 1 PATL1 1 7 7 Tight junction protein ZO-2 TJP2 2 7 3,5 Suppressor of SWI4 1 homolog PPAN 0 7 #DIV/0! Transcription initiation factor TFIID subunit 7 TAF7 1 6 6 RUN and FYVE domain-containing protein 1 RUFY1 1 6 6 Ras GTPase-activating protein-binding protein 2 G3BP2 1 6 6

WD repeat domain phosphoinositide-interacting protein 2 WIPI2 1 6 6 Coilin COIL 2 6 3 E3 ubiquitin-protein ligase CBL CBL 0 6 #DIV/0! TGF-beta-activated kinase 1 and MAP3K7-binding protein 1 TAB1 1 5 5 Serologically defined colon cancer antigen 3 SDCCAG3 1 5 5 Methylosome subunit pICln CLNS1A 0 5 #DIV/0! Probable E3 ubiquitin-protein ligase MYCBP2 MYCBP2 0 5 #DIV/0! Coordinator of PRMT5 and differentiation stimulator COPRS 0 5 #DIV/0! Small nuclear ribonucleoprotein F SNRPF 1 4 4 Transformer-2 protein homolog beta TRA2B 1 4 4 KN motif and ankyrin repeat domain-containing protein 2 KANK2 1 4 4 Coiled-coil domain-containing protein 43 CCDC43 1 4 4 La-related protein 4B LARP4B 1 4 4 Transcription termination factor 2 TTF2 1 4 4 Ribonuclease H2 subunit A RNASEH2A 0 4 - Serine/threonine-protein kinase PAK 4 PAK4 0 4 - AP2-associated protein kinase 1 AAK1 0 4 - Calmodulin-regulated spectrin-associated protein 3 CAMSAP3 0 4 - Luc7-like protein 3 LUC7L3 1 3 3 28S ribosomal protein S34, mitochondrial MRPS34 1 3 3

Calcium/calmodulin-dependent protein kinase type II subunit delta CAMK2D 1 3 3 Survival motor neuron protein SMN1;SMN2 1 3 3 Mitochondrial glutamate carrier 1;Mitochondrial glutamate carrier 2 SLC25A22;SLC25A18 1 3 3 Serine/threonine-protein kinase WNK1 WNK1 1 3 3 28S ribosomal protein S17, mitochondrial MRPS17 1 3 3 Gem-associated protein 2 GEMIN2 1 3 3

Mitochondrial import inner membrane translocase subunit Tim23;Putative mitochondrial import inner membrane translocase subunit Tim23B TIMM23;TIMM23B 1 3 3 DnaJ homolog subfamily A member 2 DNAJA2 1 3 3 Serine/threonine-protein kinase OSR1 OXSR1 1 3 3 cAMP-dependent protein kinase catalytic subunit alpha PRKACA 1 3 3 Ribonucleoside-diphosphate reductase large subunit;Ribonucleoside- diphosphate reductase RRM1 1 3 3 Dual specificity protein kinase TTK TTK 1 3 3 F-actin-capping protein subunit alpha-2 CAPZA2 1 3 3 Dual specificity mitogen-activated protein kinase kinase 1 MAP2K1 1 3 3 Protein kinase C delta type;Protein kinase C delta type regulatory subunit;Protein kinase C delta type catalytic subunit PRKCD 1 3 3 Mitotic spindle assembly checkpoint protein MAD2A MAD2L1 1 3 3 Centrosomal protein of 55 kDa CEP55 1 3 3 PAB-dependent poly(A)-specific ribonuclease subunit 3 PAN3 1 3 3 Calmodulin-regulated spectrin-associated protein 1 CAMSAP1 1 3 3 Dehydrogenase/reductase SDR family member 7B DHRS7B 1 3 3 SURP and G-patch domain-containing protein 1 SUGP1 1 3 3 ER membrane protein complex subunit 1 EMC1 1 3 3 DnaJ homolog subfamily A member 3, mitochondrial DNAJA3 1 3 3 Exosome complex component RRP41 EXOSC4 1 3 3 General transcription factor 3C polypeptide 4 GTF3C4 1 3 3 Probable ATP-dependent RNA helicase DDX52 DDX52 1 3 3 U7 snRNA-associated Sm-like protein LSm10 LSM10 0 3 - Eukaryotic translation initiation factor 2A EIF2A 0 3 -

Table 1: List of best candidates for PRMT5 bioID identified by mass spectrometry

Peptides Peptides Protein names Gene names BirA_Ctl BirA_PRMT7 Fold change Protein arginine N-methyltransferase 7 PRMT7 1 20 20

Oxysterol-binding protein-related protein 9;Oxysterol-binding protein OSBPL9 6 20 3,333333333 Oxysterol-binding protein-related protein 11 OSBPL11 2 19 9,5 Eukaryotic translation initiation factor 2 subunit 1 EIF2S1 2 18 9 MAP7 domain-containing protein 3 MAP7D3 4 17 4,25 DNA/RNA-binding protein KIN17 KIN 0 11 Myosin-9 MYH9 3 10 3,333333333 Transgelin-2 TAGLN2 2 8 4 Protein transport protein Sec24A SEC24A 2 7 3,5 Signal recognition particle receptor subunit alpha SRPR 2 7 3,5 Tight junction protein ZO-2 TJP2 2 7 3,5 Copine-3 CPNE3 2 6 3 Methyltransferase-like protein 2B METTL2B 0 6 - Transformer-2 protein homolog beta TRA2B 1 5 5

TGF-beta-activated kinase 1 and MAP3K7-binding protein 1 TAB1 1 5 5 Elongation factor G, mitochondrial GFM1 1 5 5 Transcription termination factor 2 TTF2 1 5 5 Protein arginine N-methyltransferase 6 PRMT6 0 5 - Eukaryotic translation initiation factor 2A EIF2A 0 5 - 28S ribosomal protein S17, mitochondrial MRPS17 1 4 4 Sentrin-specific protease 3 SENP3 1 4 4

Dual specificity mitogen-activated protein kinase kinase 1 MAP2K1 1 4 4 Oxysterol-binding protein-related protein 10;Oxysterol-binding protein OSBPL10 1 4 4

WD repeat domain phosphoinositide-interacting protein 2 WIPI2 1 4 4 UPF0609 protein C4orf27 C4orf27 0 4 -

Calmodulin-regulated spectrin-associated protein 3 CAMSAP3 0 4 - Hematological and neurological expressed 1-like protein HN1L 1 3 3 Coiled-coil domain-containing protein 85C CCDC85C 1 3 3 Serine/arginine repetitive matrix protein 1 SRRM1 1 3 3

Isocitrate dehydrogenase [NADP];Isocitrate dehydrogenase [NADP], mitochondrial IDH2 1 3 3 Luc7-like protein 3 LUC7L3 1 3 3 Charged multivesicular body protein 2b CHMP2B 1 3 3 Calcium/calmodulin-dependent protein kinase type II subunit delta CAMK2D 1 3 3 Heat shock 70 kDa protein 4L HSPA4L 1 3 3 Unconventional myosin-VI MYO6 1 3 3 Caldesmon CALD1 1 3 3 28S ribosomal protein S22, mitochondrial MRPS22 1 3 3 Anamorsin CIAPIN1 1 3 3 Thioredoxin PDIA3 1 3 3 Ribosomal protein L19;60S ribosomal protein L19 RPL19 1 3 3

Mitochondrial import inner membrane translocase subunit Tim23;Putative mitochondrial import inner membrane translocase subunit Tim23B TIMM23;TIMM23B 1 3 3 Cyclin-G-associated kinase GAK 1 3 3 Phosphoglycerate kinase 1;Phosphoglycerate kinase;Phosphoglycerate kinase 2 PGK1;PGK2 1 3 3 Eukaryotic translation initiation factor 2 subunit 2 EIF2S2 1 3 3 14-3-3 protein beta/alpha;14-3-3 protein beta/alpha, N-terminally processed YWHAB 1 3 3 Dual specificity protein kinase TTK TTK 1 3 3 Serine/arginine-rich splicing factor 2 SRSF2;SFRS2 0 3 - Cell division control protein 45 homolog CDC45 0 3 - Serine/threonine-protein kinase PAK 4 PAK4 0 3 - Fumarate hydratase, mitochondrial FH 0 3 - Origin recognition complex subunit 2 ORC2 0 3 - Phosphatidylinositol-binding clathrin assembly protein PICALM 0 3 -

Table 2: List of best candidates for PRMT7 bioID identified by mass spectrometry

42 A A

Tet-On A Tet-On B

myc BirA* PRMT7 myc BirA* PRMT5

293T 293T Doxy, 24h - + Doxy, 24h - + - 150

Myc - 100 PRMT7PRMT5 - 100 - 75 - 75 - 50 Actin - 50 Actin

IP: Strep-beads WCL IP WCL IP WCL IP

- 250 - 150 * * - 100 IB: Strep-HRP - 75

- 50

- 37

- 25

* BirA-PRMT5 and BirA-PRMT7

Figure 2 Figure 5: Establishment of BioID method in HEK293 cells. A, B) The fusion protein myc-BirA-PRMT5 and PRMT7 were generated by subcloning PRMT5 and PRMT7 cDNA into a myc-BirA pcDNA3.1; the

BirA fusion proteins were put under a doxycycline-dependent promotor. After 24h doxycycline induction, the fusion proteins myc-birA-PRMT5 and myc-birA-PRMT7 (indicated by an arrow) were expressed at respectively 107kDa and 115kDa. C) An immunoblot against biotinylated proteins was done to confirm protein biotinylation and immunoprecipitation.

Methylosome and A spliceosomal proteins

Ribosomal proteins

Ribosomal proteins and initiation control of translation

OSBPL protein family

Figure 3 Figure 6: Mass spectrometry of BioID. The best candidates of the BioID screen were sorted and

analysed. Ingenuity Pathway Analysis Software was used to generate a network of interactions

among best candidates for A) PRMT5 and B) PRMT7.

A HA - WT HA - S51A Coum, 6h - + - + IP HA-eIF2α 75 - IB : PRMT7 WCL 75 -

IP HA-eIF2α 37 - IB : HA WCL 37 -

WCL37 - IB : Phospho eIF2α

C Untreated Thapsi (ER stress)

PRMT7 / eIF2α PRMT7 / eIF2α

WT MEFS

Zoom

PRMT7 / eIF2α PRMT7 / eIF2α

PRMT7 -/-

Figure 7: PRMT7 interact with eIF2α and dissociate under ER stress to form stress foci. A) HA- eIF2α IP was performed in HT1080 cells and shows an interaction with PRMT7. Addition of

Coumermycin (Coum) in the media for 6h triggers eIF2α phosphorylation and disrupts PRMT7 interaction.

MutationFigure 4 of phosphorylation site (S51A) does not affect the association nor the disruption after Coum treatment. B) GyrB-PKR construct allows PKR dimerization and activation of PKR under Coum treatment.

Adapted from “Evidence for eIF2α phosphorylation-independent effects of GSK2656157, a novel catalytic

45 inhibitor of PERK with clinical implications” by J. Krishnamoorthy, et al., 2014, Cell Cycle 13(5): 801-

+ purified A PRMT7

250 - 150 - 100 - 75 - 50 - GST-eIF2 alpha 3H autoradiography 37 -

25 - 20 -

15 - H2B

PRMT7 GST-eIF2 alpha Comassie blue

H2B

Empty vector HA-eIF2α Coum - + - + IP HA-eIF2α 37 - IB : Monomethyl (Me-R4-100) WCL 37 -

IP HA-eIF2α 37 - Figure 8: PRMT7 methylates eIF2α in vitro and in vivo. A)IB : HAIn vitro methylation assay showing WCL 37 - methylation of recombinant GST-eIF2α by PRMT7. B) HA-eIF2α was immunoprecipited. The IP was loaded on a gel and imunoblotted for Monomethyl marks with a specific antibody. A band at the same molecular weight as HA-eIF2α (around 38kDa) was observed. After Coumermycin treatment, methylation band was not observable. Figure 5 46

References

Ancelin, K., Lange, U. C., Hajkova, P., Schneider, R., Bannister, A. J., Kouzarides, T., & Surani, M. A. (2006). Blimp1 associates with Prmt5 and directs histone arginine methylation in mouse germ cells. Nat Cell Biol, 8(6), 623-630. doi:10.1038/ncb1413 Antonysamy, S., Bonday, Z., Campbell, R. M., Doyle, B., Druzina, Z., Gheyi, T., . . . Emtage, S. (2012). Crystal structure of the human PRMT5:MEP50 complex. Proc Natl Acad Sci U S A, 109(44), 17960-17965. doi:10.1073/pnas.1209814109 Atkinson, M. R., & Murray, A. W. (1967). Regulation of adenylate deaminase from Ehrlich ascites- tumour cells by a phosphonate analogue of adenosine triphosphate. Biochem J, 104(1), 10C-12C. Auclair, Y., & Richard, S. (2013). The role of arginine methylation in the DNA damage response. DNA Repair (Amst), 12(7), 459-465. doi:10.1016/j.dnarep.2013.04.006 Ayoubi, T. A., & Van De Ven, W. J. (1996). Regulation of gene expression by alternative promoters. FASEB J, 10(4), 453-460. Bedford, M. T. (2007). Arginine methylation at a glance. J Cell Sci, 120(Pt 24), 4243-4246. doi:10.1242/jcs.019885 Bedford, M. T., & Clarke, S. G. (2009). Protein arginine methylation in mammals: who, what, and why. Mol Cell, 33(1), 1-13. doi:10.1016/j.molcel.2008.12.013 Bedford, M. T., & Richard, S. (2005). Arginine methylation an emerging regulator of protein function. Mol Cell, 18(3), 263-272. doi:10.1016/j.molcel.2005.04.003 Bezzi, M., Teo, S. X., Muller, J., Mok, W. C., Sahu, S. K., Vardy, L. A., . . . Guccione, E. (2013). Regulation of constitutive and alternative splicing by PRMT5 reveals a role for Mdm4 pre-mRNA in sensing defects in the spliceosomal machinery. Genes Dev, 27(17), 1903-1916. doi:10.1101/gad.219899.113 Bian, C., Chen, Q., & Yu, X. (2015). The zinc finger proteins ZNF644 and WIZ regulate the G9a/GLP complex for gene repression. Elife, 4. doi:10.7554/eLife.05606 Blanc, R. S., Vogel, G., Chen, T., Crist, C., & Richard, S. (2016). PRMT7 Preserves Satellite Cell Regenerative Capacity. Cell Rep, 14(6), 1528-1539. doi:10.1016/j.celrep.2016.01.022 Boisvert, F. M., Cote, J., Boulanger, M. C., Cleroux, P., Bachand, F., Autexier, C., & Richard, S. (2002). Symmetrical dimethylarginine methylation is required for the localization of SMN in Cajal bodies and pre-mRNA splicing. J Cell Biol, 159(6), 957-969. doi:10.1083/jcb.200207028 Boisvert, F. M., Hendzel, M. J., Masson, J. Y., & Richard, S. (2005). Methylation of MRE11 regulates its nuclear compartmentalization. Cell Cycle, 4(7), 981-989. doi:10.4161/cc.4.7.1830 Brahms, H., Meheus, L., de Brabandere, V., Fischer, U., & Luhrmann, R. (2001). Symmetrical dimethylation of arginine residues in spliceosomal Sm protein B/B' and the Sm-like protein LSm4, and their interaction with the SMN protein. RNA, 7(11), 1531-1542. Chapman-Smith, A., & Cronan, J. E., Jr. (1999). In vivo enzymatic protein biotinylation. Biomol Eng, 16(1-4), 119-125. Cheung, P., & Lau, P. (2005). Epigenetic regulation by histone methylation and histone variants. Mol Endocrinol, 19(3), 563-573. doi:10.1210/me.2004-0496 Cloutier, P., Lavallee-Adam, M., Faubert, D., Blanchette, M., & Coulombe, B. (2014). Methylation of the DNA/RNA-binding protein Kin17 by METTL22 affects its association with chromatin. J Proteomics, 100, 115-124. doi:10.1016/j.jprot.2013.10.008 Cote, J., Boisvert, F. M., Boulanger, M. C., Bedford, M. T., & Richard, S. (2003). Sam68 RNA binding protein is an in vivo substrate for protein arginine N-methyltransferase 1. Mol Biol Cell, 14(1), 274-287. doi:10.1091/mbc.E02-08-0484 Fabbrizio, E., El Messaoudi, S., Polanowska, J., Paul, C., Cook, J. R., Lee, J. H., . . . Sardet, C. (2002). Negative regulation of transcription by the type II arginine methyltransferase PRMT5. EMBO Rep, 3(7), 641-645. doi:10.1093/embo-reports/kvf136

Feng, Y., Hadjikyriacou, A., & Clarke, S. G. (2014). Substrate specificity of human protein arginine methyltransferase 7 (PRMT7): the importance of acidic residues in the double E loop. J Biol Chem, 289(47), 32604-32616. doi:10.1074/jbc.M114.609271 Friesen, W. J., Massenet, S., Paushkin, S., Wyce, A., & Dreyfuss, G. (2001). SMN, the product of the spinal muscular atrophy gene, binds preferentially to dimethylarginine-containing protein targets. Mol Cell, 7(5), 1111-1117. Friesen, W. J., Paushkin, S., Wyce, A., Massenet, S., Pesiridis, G. S., Van Duyne, G., . . . Dreyfuss, G. (2001). The methylosome, a 20S complex containing JBP1 and pICln, produces dimethylarginine-modified Sm proteins. Mol Cell Biol, 21(24), 8289-8300. doi:10.1128/MCB.21.24.8289-8300.2001 Gary, J. D., & Clarke, S. (1998). RNA and protein interactions modulated by protein arginine methylation. Prog Nucleic Acid Res Mol Biol, 61, 65-131. Gonsalvez, G. B., Tian, L., Ospina, J. K., Boisvert, F. M., Lamond, A. I., & Matera, A. G. (2007). Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins. J Cell Biol, 178(5), 733-740. doi:10.1083/jcb.200702147 Grimm, C., Chari, A., Pelz, J. P., Kuper, J., Kisker, C., Diederichs, K., . . . Fischer, U. (2013). Structural basis of assembly chaperone- mediated snRNP formation. Mol Cell, 49(4), 692-703. doi:10.1016/j.molcel.2012.12.009 Gros, L., Delaporte, C., Frey, S., Decesse, J., de Saint-Vincent, B. R., Cavarec, L., . . . Jacquemin- Sablon, A. (2003). Identification of new drug sensitivity genes using genetic suppressor elements: protein arginine N-methyltransferase mediates cell sensitivity to DNA-damaging agents. Cancer Res, 63(1), 164- 171. Guderian, G., Peter, C., Wiesner, J., Sickmann, A., Schulze-Osthoff, K., Fischer, U., & Grimmler, M. (2011). RioK1, a new interactor of protein arginine methyltransferase 5 (PRMT5), competes with pICln for binding and modulates PRMT5 complex composition and substrate specificity. J Biol Chem, 286(3), 1976- 1986. doi:10.1074/jbc.M110.148486 Guo, A., Gu, H., Zhou, J., Mulhern, D., Wang, Y., Lee, K. A., . . . Comb, M. J. (2014). Immunoaffinity enrichment and mass spectrometry analysis of protein methylation. Mol Cell Proteomics, 13(1), 372-387. doi:10.1074/mcp.O113.027870 Hetz, C. (2012). The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol, 13(2), 89-102. doi:10.1038/nrm3270 Ho, M. C., Wilczek, C., Bonanno, J. B., Xing, L., Seznec, J., Matsui, T., . . . Shechter, D. (2013). Structure of the arginine methyltransferase PRMT5-MEP50 reveals a mechanism for substrate specificity. PLoS One, 8(2), e57008. doi:10.1371/journal.pone.0057008 Hu, Y. J., Sif, S., & Imbalzano, A. N. (2013). Prmt7 is dispensable in tissue culture models for adipogenic differentiation. F1000Res, 2, 279. doi:10.12688/f1000research.2-279.v1 International Human Genome Sequencing, C. (2004). Finishing the euchromatic sequence of the human genome. Nature, 431(7011), 931-945. doi:10.1038/nature03001 Jain, K., Warmack, R. A., Debler, E. W., Hadjikyriacou, A., Stavropoulos, P., & Clarke, S. G. (2016). Protein Arginine Methyltransferase Product Specificity Is Mediated by Distinct Active-site Architectures. J Biol Chem, 291(35), 18299-18308. doi:10.1074/jbc.M116.740399 Jelinic, P., Stehle, J. C., & Shaw, P. (2006). The testis-specific factor CTCFL cooperates with the protein methyltransferase PRMT7 in H19 imprinting control region methylation. PLoS Biol, 4(11), e355. doi:10.1371/journal.pbio.0040355 Jensen, O. N. (2004). Modification-specific proteomics: characterization of post-translational modifications by mass spectrometry. Curr Opin Chem Biol, 8(1), 33-41. doi:10.1016/j.cbpa.2003.12.009 Jensen, O. N. (2006). Interpreting the protein language using proteomics. Nat Rev Mol Cell Biol, 7(6), 391-403. doi:10.1038/nrm1939 Klann, E., & Dever, T. E. (2004). Biochemical mechanisms for translational regulation in synaptic plasticity. Nat Rev Neurosci, 5(12), 931-942. doi:10.1038/nrn1557 Kim, D. I., Jensen, S. C., Noble, K. A., Kc, B., Roux, K. H., Motamedchaboki, K., & Roux, K. J. (2016). An improved smaller biotin ligase for BioID proximity labeling. Mol Biol Cell, 27(8), 1188-1196. doi:10.1091/mbc.E15-12-0844

Krishnamoorthy, J., Rajesh, K., Mirzajani, F., Kesoglidou, P., Papadakis, A. I., & Koromilas, A. E. (2014). Evidence for eIF2α phosphorylation-independent effects of GSK2656157, a novel catalytic inhibitor of PERK with clinical implications. Cell Cycle, 13(5), 801-806. doi:10.4161/cc.27726 Lacroix, M., El Messaoudi, S., Rodier, G., Le Cam, A., Sardet, C., & Fabbrizio, E. (2008). The histone-binding protein COPR5 is required for nuclear functions of the protein arginine methyltransferase PRMT5. EMBO Rep, 9(5), 452-458. doi:10.1038/embor.2008.45 Lambert, J. P., Tucholska, M., Go, C., Knight, J. D., & Gingras, A. C. (2015). Proximity biotinylation and affinity purification are complementary approaches for the interactome mapping of chromatin- associated protein complexes. J Proteomics, 118, 81-94. doi:10.1016/j.jprot.2014.09.011 Lee, J. H., Cook, J. R., Yang, Z. H., Mirochnitchenko, O., Gunderson, S. I., Felix, A. M., . . . Pestka, S. (2005). PRMT7, a new protein arginine methyltransferase that synthesizes symmetric dimethylarginine. J Biol Chem, 280(5), 3656-3664. doi:10.1074/jbc.M405295200 Liu, F., Zhao, X., Perna, F., Wang, L., Koppikar, P., Abdel-Wahab, O., . . . Nimer, S. D. (2011). JAK2V617F-mediated phosphorylation of PRMT5 downregulates its methyltransferase activity and promotes myeloproliferation. Cancer Cell, 19(2), 283-294. doi:10.1016/j.ccr.2010.12.020 Majumder, S., Alinari, L., Roy, S., Miller, T., Datta, J., Sif, S., . . . Jacob, S. T. (2010). Methylation of histone H3 and H4 by PRMT5 regulates ribosomal RNA gene transcription. J Cell Biochem, 109(3), 553- 563. doi:10.1002/jcb.22432 Martin-Kleiner, I. (2012). BORIS in human cancers -- a review. Eur J Cancer, 48(6), 929-935. doi:10.1016/j.ejca.2011.09.009 Meister, G., & Fischer, U. (2002). Assisted RNP assembly: SMN and PRMT5 complexes cooperate in the formation of spliceosomal UsnRNPs. EMBO J, 21(21), 5853-5863. Mellacheruvu, D., Wright, Z., Couzens, A. L., Lambert, J. P., St-Denis, N. A., Li, T., . . . Nesvizhskii, A. I. (2013). The CRAPome: a contaminant repository for affinity purification-mass spectrometry data. Nat Methods, 10(8), 730-736. doi:10.1038/nmeth.2557 Miccoli, L., Biard, D. S., Frouin, I., Harper, F., Maga, G., & Angulo, J. F. (2003). Selective interactions of human kin17 and RPA proteins with chromatin and the nuclear matrix in a DNA damage- and cell cycle-regulated manner. Nucleic Acids Res, 31(14), 4162-4175. Ostareck-Lederer, A., Ostareck, D. H., Rucknagel, K. P., Schierhorn, A., Moritz, B., Huttelmaier, S., . . . Wahle, E. (2006). Asymmetric arginine dimethylation of heterogeneous nuclear ribonucleoprotein K by protein-arginine methyltransferase 1 inhibits its interaction with c-Src. J Biol Chem, 281(16), 11115-11125. doi:10.1074/jbc.M513053200 Pawlak, M. R., Scherer, C. A., Chen, J., Roshon, M. J., & Ruley, H. E. (2000). Arginine N- methyltransferase 1 is required for early postimplantation mouse development, but cells deficient in the enzyme are viable. Mol Cell Biol, 20(13), 4859-4869. Rajesh, K., Papadakis, A. I., Kazimierczak, U., Peidis, P., Wang, S., Ferbeyre, G., . . . Koromilas, A. E. (2013). eIF2α phosphorylation bypasses premature senescence caused by oxidative stress and pro- oxidant antitumor therapies. Aging (Albany NY), 5(12), 884-901. doi:10.18632/aging.100620 Raychaudhuri, S., & Prinz, W. A. (2010). The diverse functions of oxysterol-binding proteins. Annu Rev Cell Dev Biol, 26, 157-177. doi:10.1146/annurev.cellbio.042308.113334 Ren, J., Wang, Y., Liang, Y., Zhang, Y., Bao, S., & Xu, Z. (2010). Methylation of ribosomal protein S10 by protein-arginine methyltransferase 5 regulates ribosome biogenesis. J Biol Chem, 285(17), 12695- 12705. doi:10.1074/jbc.M110.103911 Roux, K. J., Kim, D. I., & Burke, B. (2013). BioID: a screen for protein-protein interactions. Curr Protoc Protein Sci, 74, Unit 19 23. doi:10.1002/0471140864.ps1923s74 Scaramuzzino, C., Casci, I., Parodi, S., Lievens, P. M., Polanco, M. J., Milioto, C., . . . Pennuto, M. (2015). Protein arginine methyltransferase 6 enhances polyglutamine-expanded androgen receptor function and toxicity in spinal and bulbar muscular atrophy. Neuron, 85(1), 88-100. doi:10.1016/j.neuron.2014.12.031 Strahl, B. D., Briggs, S. D., Brame, C. J., Caldwell, J. A., Koh, S. S., Ma, H., . . . Allis, C. D. (2001). Methylation of histone H4 at arginine 3 occurs in vivo and is mediated by the nuclear receptor coactivator PRMT1. Curr Biol, 11(12), 996-1000.

Tang, J., Kao, P. N., & Herschman, H. R. (2000). Protein-arginine methyltransferase I, the predominant protein-arginine methyltransferase in cells, interacts with and is regulated by interleukin enhancer-binding factor 3. J Biol Chem, 275(26), 19866-19876. doi:10.1074/jbc.M000023200 Tee, W. W., Pardo, M., Theunissen, T. W., Yu, L., Choudhary, J. S., Hajkova, P., & Surani, M. A. (2010). Prmt5 is essential for early mouse development and acts in the cytoplasm to maintain ES cell pluripotency. Genes Dev, 24(24), 2772-2777. doi:10.1101/gad.606110 Thandapani, P., O'Connor, T. R., Bailey, T. L., & Richard, S. (2013). Defining the RGG/RG motif. Mol Cell, 50(5), 613-623. doi:10.1016/j.molcel.2013.05.021 Wang, L., Pal, S., & Sif, S. (2008). Protein arginine methyltransferase 5 suppresses the transcription of the RB family of tumor suppressors in leukemia and lymphoma cells. Mol Cell Biol, 28(20), 6262-6277. doi:10.1128/MCB.00923-08 Weiss, V. H., McBride, A. E., Soriano, M. A., Filman, D. J., Silver, P. A., & Hogle, J. M. (2000). The structure and oligomerization of the yeast arginine methyltransferase, Hmt1. Nat Struct Biol, 7(12), 1165- 1171. doi:10.1038/82028 Xu, X., Hoang, S., Mayo, M. W., & Bekiranov, S. (2010). Application of machine learning methods to histone methylation ChIP-Seq data reveals H4R3me2 globally represses gene expression. BMC Bioinformatics, 11, 396. doi:10.1186/1471-2105-11-396 Yamagata, K., Daitoku, H., Takahashi, Y., Namiki, K., Hisatake, K., Kako, K., . . . Fukamizu, A. (2008). Arginine methylation of FOXO transcription factors inhibits their phosphorylation by Akt. Mol Cell, 32(2), 221-231. doi:10.1016/j.molcel.2008.09.013 Ying, Z., Mei, M., Zhang, P., Liu, C., He, H., Gao, F., & Bao, S. (2015). Histone Arginine Methylation by PRMT7 Controls Germinal Center Formation via Regulating Bcl6 Transcription. J Immunol, 195(4), 1538-1547. doi:10.4049/jimmunol.1500224 Yorimitsu, T., Nair, U., Yang, Z., & Klionsky, D. J. (2006). Endoplasmic reticulum stress triggers autophagy. J Biol Chem, 281(40), 30299-30304. doi:10.1074/jbc.M607007200 Zhang, X., & Cheng, X. (2003). Structure of the predominant protein arginine methyltransferase PRMT1 and analysis of its binding to substrate peptides. Structure, 11(5), 509-520. Zheng, Z., Schmidt-Ott, K. M., Chua, S., Foster, K. A., Frankel, R. Z., Pavlidis, P., . . . Gharavi, A. G. (2005). A Mendelian locus on chromosome 16 determines susceptibility to doxorubicin nephropathy in the mouse. Proc Natl Acad Sci U S A, 102(7), 2502-2507. doi:10.1073/pnas.0409786102 Zurita-Lopez, C. I., Sandberg, T., Kelly, R., & Clarke, S. G. (2012). Human protein arginine methyltransferase 7 (PRMT7) is a type III enzyme forming omega-NG-monomethylated arginine residues. J Biol Chem, 287(11), 7859-7870. doi:10.1074/jbc.M111.336271