Mechanism of Repression by the Sequence-Specific Mrna-Binding Protein Vts1p

Mechanism of Repression by the Sequence-Specific mRNA-Binding Protein Vts1p

Melissa Alice Bieman

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Biochemistry University of Toronto

Mechanism of Repression by the Sequence-Specific mRNA Binding Protein Vts1p

Melissa A. Bieman

Doctor of Philosophy

Department of Biochemistry University of Toronto

2014 Abstract

Vts1p is a member of the Smaug family of sequence-specific RNA binding proteins that interact with stem-loop sequences to degrade and/or translationally repress target transcripts. To identify the molecular mechanism by which Vts1p represses target transcripts, I have investigated the role of one of Vts1p’s conserved domains, the SSR1 domain, in Vts1p-mediated repression. In vitro SSR1 functions as a dimerization domain and I provide evidence that SSR1 mediates dimerization in vivo. I have created Vts1p mutants which abolish dimerization and found that dimerization is required for full Vts1p-mediated repression and robust destabilization of a reporter containing two SREs. My data supports a model whereby dimerization of Vts1p functions to enhance binding of Vts1p to a target transcript carrying two or more SREs. Analysis of putative Vts1p mRNA targets suggests that dimerization may be important for proper repression of many target transcripts since a majority contain multiple SREs.

I have also shown that Vts1p interacts with the Ccr4p/Pop2p/Not deadenylase in an RNA independent fashion. This is consistent with the model that Vts1p recruits the Ccr4p/Pop2p/Not deadenylase to target transcripts to induce deadenylation and subsequent degradation.

Finally, by tethering Vts1p to a reporter construct, I have shown that two separate regions of

Vts1p, the N-terminus and the C-terminus, are sufficient for repression, suggesting that Vts1p employs multiple mechanisms to regulate the expression of target mRNAs. I have also shown that the N-terminus contains a conserved motif that functions in Vts1p-mediated translational repression while playing no role in transcript decay. This is the first evidence that Vts1p mediates the translational repression of a target transcript.

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Acknowledgments

“every day is a journey, and the journey itself is home.” -Matsuo Basho

First, thank you to my supervisor, Craig Smibert, for allowing me to embark on this journey, and for his guidance and patience along the way. He has helped to shape the way I think about science.

Thank you also to my committee members Grant Brown and Howard Lipshitz for their insight and support throughout my projects.

Thank you to my lab mates Meryl Nelson, Heli Vari, Ben Pinder, Laura Rendl, Jason Dumelie, John Laver, Agata Orlowicz, Alex Marsolais, Matthew Cheng and Peter Sollazzo for their company, advice, ideas and entertainment.

My thanks go to the members of the Segall, Brown, Andrews and Boone lab for their scientific expertise and their generosity with reagents and strains.

Thank you to my family for their continual encouragement and interest along the way, even though they didn’t always understand the science. Finally, thank you to my traveling companion and husband, Albert Fuchigami, who has been unwavering in his encouragement and support during this leg of the journey and into the next one.

Table of Contents

Acknowledgments ...... iv

Table of Contents ...... iv

List of Figures ...... viii

List of Appendices ...... ix

List of Abbreviations ...... x

Chapter 1 Introduction ...... 1

1.1 Post-transcriptional Regulation ...... 2

1.2 Translational Regulation ...... 3

1.2.1 Cap-Dependent Translation Initiation ...... 3

1.2.2 Regulation of Translation Factors ...... 5

1.3 Mechanisms of mRNA Degradation ...... 6

1.3.1 Deadenylation-Dependent Decay ...... 7

1.3.1.1 Deadenylation Enzymes ...... 7

1.3.1.2 Decapping ...... 9

1.3.1.3 5’-3’ Exonucleolytic Decay ...... 10

1.3.1.4 3’-5’ Exonucleolytic Decay ...... 11

1.3.1.5 P Bodies ...... 11

1.3.2 Regulated mRNA Degradation ...... 13

1.3.2.1 Nonsense Mediated Decay (NMD) ...... 13

1.3.2.2 No-Go Decay ...... 14

1.3.2.3 Non-stop Decay ...... 15

1.4 mRNA Regulation by Sequence-Specific RNA Binding Proteins ...... 15

1.4.1 AU-Rich Binding Proteins ...... 15

1.4.2 Puf Family of Proteins ...... 16

1.4.3 Iron Homeostasis by Iron Regulatory Proteins ...... 17

1.4.4 Translational regulation by modulation of polyadenylation ...... 18

1.4.5 miRNA-Mediated Post-transcriptional Regulation ...... 19

1.5 The Smaug (Smg) Family of Proteins ...... 20

1.5.1 Drosophila Smg ...... 21

1.5.1.1 Smg-mediated post-transcriptional regulation of nos mRNA ...... 23

1.5.1.2 Smg-mediated mRNA degradation ...... 25

1.5.1.3 Smg is a major regulator of mRNA stability ...... 26

1.5.2 Mammalian Smg ...... 26

1.5.3 The S. cerevisiae homolog Vts1p ...... 27

1.6 Thesis Rationale ...... 28

Chapter 2 Materials and Mesthods ...... 29

2.1 Yeast Strains ...... 30

2.2 Plasmids ...... 30

2.3 Immunoprecipitations ...... 30

2.4 Flow Cytometry ...... 31

2.5 Transcriptional Pulse Chase ...... 31

2.6 SRE searching ...... 32

Chapter 3 The SSR1 domain is a dimerization domain involved in Vts1p-mediated repression . 33

3.1 Introduction ...... 34

3.2 Results ...... 36

3.2.1 Vts1 dimerizes in vivo though it’s SSR1 domain ...... 36

3.2.2 Dimerization mutants show defects in Vts1p-mediated repression and transcript decay ...... 38

3.2.3 Dimerization affects Vts1p function through a combination of mechanisms ...... 41

3.2.4 A large fraction of Vts1p target mRNAs possess two or more SREs...... 44

3.3 Discussion ...... 46

Chapter 4 The N-terminus of Vts1p mediates translational repression ...... 49

4.1 Introduction ...... 50

4.2 Results ...... 51

4.2.1 Vts1p interacts with Ccr4p/Pop2/Not deadenylase ...... 51

4.2.2 Establishing a tethering assay to study the mechanisms that underlie Vts1p- mediated repression...... 53

4.2.3 Vts1p carries two independent repression domains ...... 53

4.2.4 Vts1p 1-237 and Vts1p 170-523 mediate transcript degradation ...... 55

4.2.5 A conserved motif within the N-terminus is required for repression ...... 57

4.2.6 The N-terminal motif functions in Vts1p-mediated translational repression ...... 59

4.3 Discussion ...... 59

Chapter 5 Conclusions and Future Directions ...... 63

5.1 Conclusions ...... 64

5.2 Future Directions ...... 65

5.2.1 Characterization of Vts1p dimerization to mRNA targets ...... 65

5.2.2 Mapping the Vts1p/deadenylase interaction ...... 66

5.2.3 Characterization of mRNA degradation by Vts1p 1-237 ...... 67

5.2.4 Translational repression by the N-terminus ...... 69

5.2.5 Relative contributions of Vts1p-mediated mechanisms to repression ...... 70

References ...... 73

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List of Figures

Figure 1-1 Eukaryotic translation initiation ...... 4

Figure 1-2 Eukaryotic mRNA degradation ...... 8

Figure 1-3 Smaug family of proteins ...... 22

Figure 1-4 Mechanisms of Smg-mediated repression ...... 24

Figure 3-1 Protein alignment of D domains ...... 35

Figure 3-2 Vts1p dimerizes in vivo ...... 37

Figure 3-3 Mutations to SSR1 domain disrupt dimerization ...... 39

Figure 3-4 Dimerization mutants show defect in Vts1p-mediated repression ...... 40

Figure 3-5 Transcriptional pulse chase of Vts1p dimerization mutants ...... 42

Figure 3-6 Models of dimerization-mediated repression of Vts1p ...... 43

Figure 3-7 Dimerization of Vts1p stabilizes its interaction with mRNA ...... 45

Figure 4-1 Vts1p-interacts with Pop2p ...... 52

Figure 4-2 Vts1p functions through two separate regions ...... 54

Figure 4-3 Transcriptional pulse chase of Vts1p-Tat fusion proteins ...... 56

Figure 4-4 A 10 amino acid motif is important for Vts1p function ...... 58

Figure 4-5 The N-terminal motif of Vts1p mediates translational repression ...... 62, 61

Figure 5-1 Protein alignment of Vts1p 1-186 ...... 68

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List of Appendices

Table A-1 List of Vts1p targets and their SREs from immuniprecipitation/microarray data ...... 89

List of Abbreviations

4E-BP eIF4E binding proteins

ACT1 actin 1 Ago Argonaute

ARE AU-rich element bcd bicoid

BIV bovine immunodeficiency virus

CPE cytoplasmic polyadenylation element

CPEB cytoplasmic polyadenylation element binding

CPSF cleavage and polyadenylation specificy factor

CsrA carbon storage regulator DDT dithiothreitol eIF eukaryotic initiation factor

EJC exon junction complex

ELAV embryonic lethal abnomal vision

GFP green fluorescent protein

GO gene ontology hb hunchback

Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Hsp83 Heat shock protein 83

HuR Hu-antigen R IRE iron responsive element

IRES Internal ribosomal entry sites

IRP iron regulatory protein

KH hnRNP K homology m7G 7-methylguanosine miRISC microRNA-induced silencing complex miRNA microRNA mRNA messenger ribonucleic acid mRNP messenger ribonucleoprotein mTOR mammalian target of rapamycin

N asparagine

NMD nonsense mediated decay nos nanos

ORF open reading frame

P body processing body

Pabp poly(A) binding protein

PAGE polyacrylamide gel electrophoresis PARN poly(A) ribonuclease

PIC preinitation complex piRNA piwi-assoiated RNAs

PTC premature termination codon

PUF pumilio and FBF

Puf-HD PUF-homology domain

Q Glutamine

RBD RNA binding domain RNA ribonucleic acid

RNP Ribonucleoprotein

RRM RNA recognition motif

RT-qPCR real-time quantitative polymerase chain reaction

SAM sterile alpha motif SCR1 small cytoplasmic RNA 1 SDS sodium dodecyl sulfate SILAC stable-isotope labelling by amino acids in cell culture

Smg Smaug

SMG suppressor with morphogenetic effect on genitalia

SRE Smaug recognition element

SSR Smaug similarity region

STAR signal transducer and activator of RNA

TfR transferrin receptor TOM translocase of the mitochondrial outer membrane

Tris tris (hydroxymethyl) aminomethane TTP Tristetraprolin uORF upstream open reading frame UTR untranslated region

VICKZ Vg1RBP/Vera, IMP, CRD-BP, KOC, ZBP-1 VTS1 VTI1-2 suppressor 1

xi 1

Chapter 1

Introduction

1 Introduction 1.1 Post-transcriptional Regulation

Post-transcriptional regulation can occur at all stages in an mRNA’s lifetime. The processes involved encompass a wide variety of mechanisms that can control when and where that protein is produced as well as its sequence. Genome-wide studies have highlighted the prevalence of these events. High-throughput sequencing and microarrays have revealed that many transcripts undergo regulatory processes, such as alternative splicing, which allows multiple mRNAs to be expressed from the same gene locus, and is thought to provide additional complexity to higher eukaryotes (Graveley et al., 2011; Wang et al., 2008b). These same methods have also been employed to measure transcript levels in the genomes of many organisms. These levels reflect the rate of transcription as well as the rate of mRNA decay. In an effort to separate these processes, temperature sensitive alleles of RNA polymerase II or transcription inhibitors have been combined with microarrays to monitor mRNA decay globally in yeast. These studies revealed that the rate of transcript degradation occurs over a large range of half-lives for transcripts and that these half-lives were comparable for mRNAs that encode proteins within the same complexes. Also, these microarrays revealed that deadenylation had a major impact on mRNA decay (Grigull et al., 2004; Wang et al., 2002). In addition, protein levels have been measured globally through stable-isotope labeling by amino acids in cell culture (SILAC) followed by mass spectrometry (de Godoy et al., 2008). Similar to the microarray data, these proteome experiments also reflect multiple processes: the rate of translation as well as protein stability. More recently, ribosome profiling has been conducted which measures ribosome density on transcripts through high-throughput sequencing and solely reflects only the rate of protein synthesis (Ingolia et al., 2009). Interestingly, when the levels of mRNAs and proteins are compared from these types of experiments, there is very little correlation between transcript and protein levels [reviewed in (de Sousa Abreu et al., 2009)]. This phenomenon, which suggests a major role for regulation of translation and/or protein stability in regulating protein levels, is conserved from bacteria to humans.

Post-transcriptional regulation is mediated through RNA-binding proteins and non-coding RNAs that interact with the transcript in complexes termed ribonucleoprotein (RNP) particles. mRNAs are initially packaged into RNPs as they are being transcribed and over the course of an mRNA’s

3 lifetime the mRNPs composition changes to coordinate all the post-transcriptional events that control an mRNA’s expression. In the nucleus, nascent pre-mRNAs are packaged into RNPs and have a 7-methylguanosine cap (m 7G) cap added to their 5’ end, have their introns removed through splicing, and for most mRNAs have a 3’ poly(A) tail added. These modifications to the transcript are required for the formation of an export-competent messenger RNP (mRNP) that transports the mRNA to the cytoplasm (Muller-McNicoll and Neugebauer, 2013). Once in the cytoplasm mRNAs can be subject to several fates. They can be translated through recruitment of the translation initiation machinery that promotes efficient production of proteins. Alternatively, transcripts can be translationally repressed until environmental or developmental cues induce their translation. Transcripts can also be localized to discrete regions within the cell to promote localized protein production. Finally, transcripts are subject to degradation through one of a number of pathways. The regulatory mechanisms that control mRNA translation and stability will be subject of this Introduction.

1.2 Translational Regulation

Translation of mRNA into protein represents the final step of gene expression and its regulation can allow for the rapid expression of proteins. Translation is divided into initiation, elongation and termination, where most of the regulatory mechanisms defined to date function during the initiation phase, which is described below.

1.2.1 Cap-Dependent Translation Initiation

Translation initiation is a complex process that consists of a number of steps mediated by over 30 polypeptides that include thirteen eukaryotic initiation factors (eIFs) (Fig1-1) [reviewed in (Jackson et al., 2010)]. Initiation begins with the recruitment of the eIF4F complex to the 5’ end of the mRNA. This complex consists of eIF4E, the cap-binding protein, a DEAD box helicase eIF4A, and eIFG, a scaffold protein that interacts with eIF4E, eIF4A, eIF3 and poly(A) binding protein (Pabp). The interaction between eIFG and eIF4E increases eIF4E’s affinity for the cap(Volpon et al., 2006), and the eIF3 interaction is responsible for recruitment of the 43S preinitiation complex (PIC). The 43S PIC consists of eIF3, the 40S ribosome, eIF1, eIF1A, eIF5 Met and the eIF2-GTP-Met-tRNA i ternary complex. Recruitment of the 43S to the mRNA results in formation of the 48S complex.

Figure 1-1 Eukaryotic translation initiation The cap binding complex eIF4F recruits the 43S Pre-initiation complex (PIC) through the eIF4G/eIF3 interaction. This results in the recruitment of the 40S ribosome to the RNA that can identify the AUG start codon through scanning the mRNAs 5’UTR in the 5’-3’ direction in a process that requires the helicase eIF4A to remove secondary structure that would disrupt scanning. Upon identification of the start codon GTP hydrolysis and eIF5B promote the release of the initiation factors and recruitment of the 60S ribosome to form the translation competent 80S ribosome. Poly(A) binding protein (Pabp) interacts with eIFG and the poly(A) tail, which circularizes the mRNA and enhances translation.

Following binding of the 43S PIC, the AUG initiation codon is identified through a scanning mechanism which requires eIF1 and eIF1A for movement of the 40S ribosome along the 5’ untranslated region (UTR). This process also requires eIF4A whose helicase activity removes secondary structure that could impede scanning (Svitkin et al., 2001). Fidelity of translation through recognition of the proper AUG initiation codon is maintained by eIF1 where codon/anti- codon base pairing at the initiation codon results in a conformational change and release of eIF1, thereby halting 40S ribosome scanning at the start codon for translation (Maag et al., 2005). In addition, upon start codon recognition, eIF5, an eIF2-specific GTPase activating protein (GAP) promotes GTPase activity of eIF2. The resulting GTP hydrolysis reduces eIF2’s affinity for Met- Met tRNA i and results in the release of eIF2-GDP from the ribosome. Once the initiation codon has been selected, there is release of the initiation factors and the 60S ribosomal subunit joins the 40S subunit, creating the 80S translationally competent ribosome. 60S ribosomal subunit joining is mediated by eIF5B. Finally, eIF5B’s release from the initiation complex requires its GTPase activity and this release is required to create a translationally competent ribosome (Shin et al., 2002).

In addition to the initiation factors, Pabp bound to an mRNA’s poly(A) tail has also been shown to have a stimulatory effect on the translation of the bound mRNA. This effect is due to the interaction between eIF4G and Pabp’s second RRM domain, which results in bridging the 5’ and 3’ ends of the mRNA together (Imataka et al., 1998; Kessler and Sachs, 1998). This creates a “closed loop” conformation that is thought to promote recycling of ribosomes. Alternatively, the interaction between eIF4G and Pabp effectively tethers the eIF4F complex to the mRNA even when it is dissociated from the 5’ end. This would allow for efficient re-initiation as the local concentration of eIF4F would be much higher (Amrani et al., 2008; Kahvejian et al., 2005).

1.2.2 Regulation of Translation Factors

Given the complexity of translation initiation, regulation of initiation can occur at multiple steps and through a variety of mechanism. Regulation can be mediated through sequence-specific RNA-binding proteins, as described in Section 1.4, or through general mechanisms that regulate initiation factor function. One such example is through the phosphorylation of eIF2. During amino acid starvation in yeast, Gcn2p phosphorylates eIF2 at Serine 51 during nutrient starvation. This results in increased affinity for eIF2B, a guanine nucleotide exchange factor, for

6 eIF2-GDP thereby inhibiting eIF2B’s activity (Hinnebusch, 2005). Thus there is decreased eIF2 ternary complex and a general decrease of translation. Paradoxically, translation at some genes is stimulated upon eIF2 phosphorylation. GCN4 mRNA contains four upstream open reading frames (uORFs) in its 5’ UTR. When levels of eIF2 ternary complex are high, uORF1 is translated and translation reinitiates at a downstream uORF, which are refractory to reinitiation. Thus, in this situation initiation at the ORF encoding Gcn4p is inhibited. In contrast, under conditions of amino acid starvation when there are low levels of eIF2 ternary complex, following translation of uORF1, the ribosome bypasses the other uORFs and allowing for translation at the GCN4 ORF, which in turn promotes amino acid synthesis (Hinnebusch, 2005).

Translation can also be regulated through mechanisms that regulate the activity of eIF4F. This regulation can be mediated by eIF4E binding proteins (4E-BPs) that interact with eIF4E through a sequence known as an eIF4E-binding motif, with the consensus YXXXXL Φ (where Φ denotes any hydrophobic amino acid) (Marcotrigiano et al., 1999). This is the same motif that is used by eIF4G to interact with eIF4E and thus 4E-BPs block the eIF4E/eIF4G interaction thereby inhibiting the cap-dependent formation of the initiation complex on mRNAs. The binding of 4E- BPs is regulated by phosphorylation where the hypophosphorylated 4E-BPs interact strongly with eIF4E thereby decreasing translation. 4E-BP phosphorylation is mediated by mammalian target of rapamycin (mTOR) who’s activity is influenced by external stimuli, nutrient availability and the energy status of the cell and serves to promote translation (Gingras et al., 1999). While the above model would suggest that 4E-BPs would have a similar effect on the translation of all mRNAs, some transcripts are more dependent on eIF4E for their translation than are others. For example, mRNAs with extensive secondary structure in their 5’UTRs are particular dependent upon eIF4E and therefore particularly sensitive to translational repression mediated by 4E-BPs (Koromilas et al., 1992).

1.3 Mechanisms of mRNA Degradation

All transcripts are ultimately degraded through a number of different pathways. In eukaryotes, these pathways include deadenylation-dependent mechanisms and specialized quality control mechanisms that detect and destroy aberrant mRNAs (Fig1-2).

1.3.1 Deadenylation-Dependent Decay

There are two general mechanisms of deadenylation-dependent mRNA degradation. Following deadenylation, transcripts can be decapped and degraded in a 5’-3’ manner. Alternatively, following deadenylation transcripts are degraded in a 3’-5 direction by a complex of exonucleases known as the exosome. The details of both of these sets of pathways have been well worked out in yeast as outlined below.

1.3.1.1 Deadenylation Enzymes

Poly(A) tail shortening is often the beginning of mRNA degradation. In yeast, there are two complexes that mediate deadenylation: the Ccr4p/Pop2p/Not deadenylase and Pan2p/Pan3p complex. The Ccr4p/Pop2p/Not deadenylase is a nine protein complex consisting of Ccr4p, Pop2p, Not1p-Not5p, Caf130p and Caf40p (Bai et al., 1999; Chen et al., 2001). Ccr4p is a member of the ExoIII nuclease family (Dlakic, 2000; Dupressoir et al., 1999) and is thought to be the catalytic components of this complex since mutations in the exonuclease domain block deadenylation in vitro and in vivo (Chen et al., 2002; Tucker et al., 2002). Pop2p is a member of the RNaseD family although it contains 2 non-canonical residues within its active site (Thore et al., 2003) suggesting it is catalytically inactive. Consistent with this is the fact that Pop2p does not retain in vitro deadenylase activity in the absence of Ccr4p. The fact that Pop2p mutants show deadenylation defects in vivo is thought to reflect a requirement for the direct interaction of Pop2p with Ccr4p for its recruitment to the deadenylase complex (Bai et al., 1999; Ohn et al., 2007; Tucker et al., 2001). The role of the Not proteins as well as Caf130p and Caf40p remain undefined, although Not1 is considered to be a scaffolding protein that serves to bring the various deadenylases components into the complex. Co-immunoprecipitations with various deadenylase components revealed that the interactions between Ccr4p, Pop2p and Not1p were independent of Not2p, Not3p and Not5p, and that Pop2p is required for the interaction between Ccr4p and Not1p. Likewise, the presence of Not2p, Not4p and Not5p in the deadenylase was independent of Pop2p. Finally, Not2p and Not5p formed a strong interaction and could interact with other deadenylase components independent of Not3p and Not4p. These data suggested that Not1p interacts with two separate complexes to form the deadenylase: Ccr4p/Pop2p, and Not2p- Not5p (Bai et al., 1999).

Figure 1-2 Eukaryotic mRNA degradation (A) General deadenylation-dependent mRNA decay. After poly(A) tail shortening, transcripts are decapped and degraded in a 5’-3’ direction. This pathway in known as the major decay pathway in yeast. Alternatively, following deadenylation the transcript is degraded in a 3’-5’ by the exosome. (B) Aberrant mRNAs are recognized and degraded through specialized quality control mechanisms such as: deadenylation-independent 5’-3’ decay (NMD), 3’-5’ degradation by the exosome (nonstop decay), or endonucleolytic cleavage (no-go decay).

A second deadenylase complex consists of Pan2p and Pan3p, where Pan2p is a member of the RNAseD family of nucleases. (Boeck et al., 1996; Brown et al., 1996). Both the Ccr4p/Pop2p/Not and Pan2p/Pan3p complexes represent the major deadenylases in yeast since a ccr4 ∆/pan2 ∆ double mutant retains no deadenylase activity (Tucker et al., 2001). Interestingly, Pan2p activity is stimulated by the presence of Pab1p (Boeck et al., 1996), while the activity of Ccr4p is inhibited by Pab1p (Tucker et al., 2002). This suggests that the presence of Pab1p on an mRNA may determine which complex is involved in its deadenylation.

These two deadenylation complexes appear to act on mRNAs in a temporal manner, where Pan2p/Pan3p trims the poly(A) tail. Mutations to either Pan2p or Pan3p resulted in mRNAs with poly(A) tails that were over 200 nucleotides long, as compared to wild-type poly(A) tails that were 60-80 nucleotides. This suggests that Pan2p/Pan3p is involved in trimming the poly(A) tail following synthesis of the poly(A) tail (Brown and Sachs, 1998). Cells lacking Ccr4p however, are still able to deadenylate transcripts, although at a much slower rate, which suggests that Pan2p can also mediate some deadenylation of mRNA transcripts (Tucker et al., 2001). In mammalian cells, Ccr4-Caf1 (Pop2) and Pan2/Pan3 also represent the major deadenylases for mRNA degradation and deadenylation of transcripts occurs through a biphasic mechanism. First there is an initial shortening of the poly(A) tail where the poly(A) tail length is synchronous and the transcripts are not degraded. In the second phase of deadenylation, the poly(A) tail length of the transcripts becomes more heterogeneous and mRNA degradation starts to occur. Overexpression of Pan2 or Ccr4 mutants has shown that Pan2 is responsible for the first phase of deadenylation and Ccr4 carries out the second phase (Yamashita et al., 2005). This suggests a model where Pan2 is involved in initial poly(A) tail trimming, which may destabilize the interaction of Pab1p with the poly(A) tail. Deadenylation of the transcript would then be passed to Ccr4 which then carries out the majority of the deadenylation.

1.3.1.2 Decapping mRNA decapping is carried out by two enzymes, Dcp1p and Dcp2p and is influenced by a number of other proteins. Dcp2p is the catalytic subunit and member of the Nudix family of pyrophosphatases (Steiger et al., 2003; van Dijk et al., 2002) that cleaves the m 7GTP cap structure to release m 7GDP and a 5’ monophosphate mRNA (LaGrandeur and Parker, 1998).

Dcp1p promotes the catalytic activity of Dcp2p through a direct interaction. This interaction is thought to result in a conformational change in Dcp2p to a catalytically active form (Deshmukh et al., 2008; She et al., 2008).

A number of other proteins, known as decapping enhancers, have been identified that serve to increase the rate of decapping. These proteins include: Dhh1p, Pat1p, Edc1p, Edc2p, Edc3p, Scd6p, and the Lsm1p-Lsm7p complex. They promote decapping through a direct activation of decapping activity and/or through inhibition of translation (Nissan et al., 2010). The process of translation stabilizes eIF4E’s interaction with the 5’ cap thereby protecting the transcript from decapping. Thus, translational inhibitors, such as Dhh1p and Scd6p, indirectly enhance transcript decapping (Nissan et al., 2010; Rajyaguru et al., 2012; Sweet et al., 2012). Edc1p- Edc3p however, show no effect on translation, but interact with Dcp2p and increases its catalytic activity (Dunckley et al., 2001; Kshirsagar and Parker, 2004; Nissan et al., 2010). Pat1p is unique in that it inhibits translation and can also activate the decapping activity of Dcp2p. In yeast, Pat1p interacts with components of the translation initiation machinery such as eIF4G, eIF3, and Pab1p in an RNA dependent manner, as well as directly interacting with translational repressors such Dhh1p, Scd6p, the decapping machinery Dcp1p/Dcp2p, and the mRNA degradation machinery Xrn1p (Nissan et al., 2010; Tharun and Parker, 2001). Human Pat1b has also been shown to interact with the Ccr4/Pop2/Not deadenylase (Ozgur et al., 2010), therefore Pat1 could serve as a central scaffold to coordinate the mRNA degradation process and connect translational repression to decapping and 5’-3’ mRNA decay. The heptameric ring complex of Lsm1p-Lsm7p stimulates decapping by associating with the remaining few A residues of the tail that are left after the bulk of the mRNA’s poly(A) tail have been removed. The Lsm1p-Lsm7p complex then promotes the interaction of Dcp1p/Dcp2p with the transcript although the mechanism by which this occurs is not fully understood (Chowdhury et al., 2007; Tharun and Parker, 2001).

1.3.1.3 5’-3’ Exonucleolytic Decay

In yeast, the major pathway of decay involves deadenylation stimulated decapping, followed by 5’-3’ exonucleolytic digestion by Xrn1p. Xrn1p is a member of a conserved family of exonucleases with an unknown catalytic mechanism (Parker and Song, 2004). Xrn1p preferentially degrades RNA with a 5’ monophosphate, the end product of decapping, and in

11 yeast, xrn1 ∆ cells show accumulation of deadenylated, decapped transcripts (Hsu and Stevens, 1993; Muhlrad et al., 1994; Stevens and Maupin, 1987). In Drosophila , a direct interaction between Xrn1p and Dcp1p has been identified (Braun et al., 2012) while in yeast, an interaction between Xrn1p and Dcp1p is mediated by Pat1p (Nissan et al., 2010). This suggests that decapping and exonucleolytic decay are tightly coordinated to efficiently degrade a transcript once it has been decapped. A second 5’-3’ exonuclease, Rat1p, also exists in yeast. While it normally functions in the nucleus, it can rescue degradation defects found in an xrn1 ∆ strain when mislocalized to the cytoplasm (Johnson, 1997).

1.3.1.4 3’-5’ Exonucleolytic Decay

Deadenylation represents the first step in decay of many transcripts in a variety of organisms (Decker and Parker, 1993). As an alternative to the degradation pathway outlined above, deadenylation can also be followed by 3’-5’ exonucleolytic degradation by the exosome. The exosome is a multi-subunit complex consisting of several 3’-5’ exonucleases that form a ring structure (Anderson and Parker, 1998; Liu et al., 2006; Mitchell et al., 1997). While several exonucleases are part of the exosome, Rrp44p has been shown to be the catalytic subunit for exonuclease digestion (Dziembowski et al., 2007). In addition to the exonuclease ring, the Ski complex that consists of Ski2p, Ski3p, Ski8p as well as Ski7p are required for nuclease activity (Anderson and Parker, 1998; Araki et al., 2001; Brown et al., 2000). Synthetic lethality in strains that lack components of both the 5’-3’ and 3’-5’ degradation machinery, such as in an xrn1 ∆, ski2 ∆ double mutant, indicate that the two pathways represent the general mRNA degradation mechanisms in yeast (Anderson and Parker, 1998).

1.3.1.5 P Bodies

The finding that a number of proteins involved in degradation, such as Dhhp1 and Pat1p, were also involved in translational repression brought about the idea that an mRNP complex may be involved in the translational repression, storage and eventual degradation of an mRNA. Consistent with this, a core group of proteins have been observed in cytoplasmic foci, known as processing (P) bodies. These foci contain many components that are conserved from yeast to mammals [reviewed in (Jain and Parker, 2013)]. The core components of P bodies include the decapping machinery, Dcp1p/Dcp2p, decapping activators Dhh1p, Pat1p, Edc3p and the Lsm

12 complex . Other decay components are also be found in P bodies such as Xrn1p and the Ccr4/Pop2/Not deadenylase that has been observed in mammalian P bodies (Brengues et al., 2005; Cougot et al., 2004; Eulalio et al., 2007; Ingelfinger et al., 2002; Sheth and Parker, 2003; Teixeira et al., 2005). In addition, RNA is also an important component of P bodies since RNase A treatment in vitro or in vivo results in P body disassembly (Eulalio et al., 2007; Teixeira et al., 2005).

P bodies are highly dynamic structures and their presence is inversely associated with the translational status of the mRNAs within a cell. For example, decreasing the non-translating pool of mRNAs through cycloheximide treatment, which stabilizes polysomes, results in the disappearance of P bodies (Cougot et al., 2004; Teixeira et al., 2005). In yeast, glucose deprivation, which results in global repression of translation, causes increased P body formation (Brengues et al., 2005; Teixeira et al., 2005). Finally, deletion of components of the mRNA decay machinery, such as the 5’-3’ exonuclease Xrn1p, also results in increased P body formation (Cougot et al., 2004; Sheth and Parker, 2003; Teixeira et al., 2005). This is most likely due to the accumulation of mRNA decay intermediates. Taken together these data suggest that mRNAs exist in two populations, one associated with polysomes and another associated with P bodies, and that mRNAs can be transition between these two populations. mRNAs localized to P bodies can undergo one of two fates: they can be degraded or return to the translating pool of mRNAs. Data consistent with transcripts being degraded in P-bodies comes from experiments where perturbation in mRNA decay result in larger and more abundant P bodies due to the accumulation of mRNA decay intermediates (Cougot et al., 2004; Sheth and Parker, 2003). As an alternative to degradation, mRNAs can also re-enter the pool of translated mRNAs. Reporter mRNAs localized to P bodies during glucose starvation were found to leave P bodies and become polysome associated in a translation initiation-dependent manner following glucose addition (Brengues et al., 2005). Similarly, when growth resumes after recovery from stationary phase, transcripts also leave P bodies and become polysome associated (Brengues et al., 2005).

The formation of P bodies is thought to occur through the following steps [reviewed in (Jain and Parker, 2013)]: following the release of an mRNA from polysomes, pre-assembled complexes that contain P body components are recruited to the mRNA and these mRNPs aggregate, forming

13 visible, cytoplasmic foci. The recruitment of pre-assembled complexes to an mRNA is inferred from work on decapping activators as they co-purify with one another (Fenger-Gron et al., 2005; Gavin et al., 2006; Teixeira and Parker, 2007; Tharun and Parker, 2001). In addition recruitment of certain factors to P bodies is dependent on other P body proteins. For example, Dcp1p requires Dcp2p for recruitment to P bodies and the Lsm1-7p requires Pat1p for its recruitment (Teixeira and Parker, 2007). Interactions between the Lsm complex and Pat1p at the 3’ end of an mRNA and the decapping machinery with the 5’ cap suggest that a “closed loop” complex could be created (Chowdhury and Tharun, 2009). Finally, aggregation of the mRNPs is accomplished through glutamine/asparagine (Q/N)-rich regions, which are prone to aggregation, that are found on a number of proteins involved in mRNA degradation. The self-interaction YjeF domain in Edc3p as well as the Q/N rich region of Lsm4p have been shown to be necessary for P body formation (Decker et al., 2007; Reijns et al., 2008). How the process of P body formation is regulated, as well as the mechanisms by which it is decided whether an mRNA is degraded or returns to the translating pool are two issues that remain to be resolved.

1.3.2 Regulated mRNA Degradation

Aberrant mRNAs can arise through a number of events during transcription and mRNA processing. Translation of these mRNAs can result in defects in translation and the production of deleterious protein products [reviewed in (Doma and Parker, 2007)]. Quality control mechanisms within the cytoplasm exist where these RNAs are recognized as defective rapidly degraded.

1.3.2.1 Nonsense Mediated Decay (NMD)

NMD recognizes and degrades mRNAs that contain premature termination (nonsense) codons (PTCs). Degradation of these mRNAs prevents the synthesis of truncated proteins that could have deleterious effects in a cell. The core NMD machinery consists of Upf1p, Upf2p and Upf3p. Upf1p is a superfamily I helicase and its maximal ATPase activity requires Upf2p and Upf3p (Chamieh et al., 2008). In addition, Upf1p overexpression can compensate for mutations in UPF2 or UPF3 , but not vice versa suggesting that Upf1p is the main effector of NMD and Upf2p and Upf3p regulate Upf1p function (Kadlec et al., 2004). Upf1p activity is also regulated by phosphorylation. The suppressor with morphogenetic effect on genitalia (SMG) proteins

SMG1, SMG5, SMG6, SMG7, SMG8 and SMG9 function in the phosphorylation and dephosphorylation of Upf1. Phosphorylation of Upf1 promotes interactions with Upf2p, and Upf3p, thereby triggering degradation of the mRNA (Grimson et al., 2004; Ohnishi et al., 2003; Yamashita et al., 2009). This degradation is achieved through accelerated deadenylation and deadenylation-independent decapping, which trigger 5’-3’ digestion by Xrn1p and 3’-5’ degradation by the exosome (Cao and Parker, 2003; Mitchell and Tollervey, 2003). In addition, translation of NMD targeted mRNAs is repressed (Muhlrad and Parker, 1999) and the nascent peptide is degraded in a Upf1-dependent manner (Kuroha et al., 2009).

It is still contentious how transcripts bearing PTCs are recognized as substrates for NMD. In one model, the location of the exon-junction complex (EJC) determines whether a stop codon is in an appropriate location. The EJC consists of a number of proteins including eIF4AIII and Upf3p and is deposited on an mRNA just upstream of exon-exon junctions during splicing. Interactions between the termination complex, which contains Upf1p, and a downstream EJC result in activation of Upf1p-mediated NMD. A second model suggests that length of the 3’ UTR influences whether a transcript will be an NMD substrate. In yeast, deleting regions between the PTC and the poly(A) tail to create a 3’ UTR of average length results in stabilization of the RNA and also, mRNAs with extended 3’UTRs become substrates for NMD (Amrani et al., 2004; Kebaara and Atkin, 2009).

1.3.2.2 No-Go Decay

Strong stalls in translation elongation result in degradation of the mRNA by the process known as No-go decay. Such stalls trigger endonucleolytic cleavage of the mRNA followed by exonucleolytic degradation of the resulting mRNAs fragments. Xrn1p degrades the 3’ cleavage product in the 5’-3’ direction while the exosome degrades the 5’ cleavage product in the 3’-5’ direction (Doma and Parker, 2006). This process requires translation to the stall site as mRNA structures that prevent scanning to the AUG initiation codon inhibit cleavage. This process is brought about by Dom34p and Hbs1p, paralogs of the translation termination factors eRF1 and eRF3 (Doma and Parker, 2006). Dom34p and Hbs1p may also function in a similar fashion as the termination factors since they can bind empty A-site within ribosomes and thereby terminate translation (Becker et al., 2011). The endonuclease involved in No-go decay has yet to be identified.

1.3.2.3 Non-stop Decay

Non-stop decay targets transcripts lacking a translation termination codon for 3’-5’ degradation by the exosome, which first removes the transcript poly(A) tail and then degrades the body of the transcript (Frischmeyer et al., 2002; van Hoof et al., 2002). This process requires the GTPase domain of Ski7p, which is similar to the translation elongation factor EF1A. It is thought that Ski7p interacts with the ribosome at the empty A site when it reaches the 3’ end of the RNA and recruits the Ski complex Ski2p, Ski3p and Ski8p. This would then allow recruitment of the exosome and degradation of the transcript. This degradation is different than that of conventional degradation by the exosome since Rrp44p can utilize either its exonuclease or endonuclease activity on a non-stop decay substrate whereas normal mRNA targets are solely targeted by the Rrp44p exonuclease (Schaeffer and van Hoof, 2011).

1.4 mRNA Regulation by Sequence-Specific RNA Binding Proteins

Specific regulation of mRNA function can also be achieved by sequence-specific RNA-binding proteins. These proteins recognize cis -elements within target transcripts and can function to regulate the translation and/or the stability of target mRNAs.

1.4.1 AU-Rich Binding Proteins

In mammalian cells, a number of cytokines, growth factors and oncogenes are rapidly degraded through AU-rich element-mediated decay. These transcripts contain AU-rich elements (AREs) in their 3’ UTRs that promote their degradation. AREs are typically 50-100 nucleotide long sequences rich in adenosine and uracil residues and often contain one or more AUUUA pentamers (Chen and Shyu, 1995; Zubiaga et al., 1995). Many proteins interact with AREs to influence mRNA stability, including the two best characterized proteins Tristetraprolin (TTP) and HuR.

TTP recognizes AREs through two CCCH zinc fingers (Lai et al., 2000) and interacts with a number of components of the decay machinery to induce deadenylation and degradation of target transcripts (Lai et al., 1999; Lykke-Andersen and Wagner, 2005). These interactions with the decay machinery are likely to occur in P bodies and TTP is thought to mediate localization of an

ARE-containing mRNAs to P bodies. This localization is dependent on TTP since depletion of TTP or a dominant negative mutant of TTP result in reduced localization of a target transcript to P bodies and their stabilization. In addition, ARE-containing mRNAs accumulate in P bodies when components of the decay machinery are depleted (Franks and Lykke-Andersen, 2007). Regulation of TTP by phosphorylation has been described although the consequences of TTP phosphorylation and dephosphorylation remain to be identified [reviewed in (von Roretz et al., 2011)].

In contrast to TTP-mediated degradation of ARE-containing mRNAs, HuR, an embryonic lethal abnormal vision (ELAV) family member, has been shown to stabilize target transcripts, although the mechanism involved is unclear. While of HuR can stabilize an ARE-containing mRNA, it does not affect the rate of transcript deadenylation, suggesting an alternative mechanism of stabilization (Peng et al., 1998). In contrast, a neuronal homolog HuD can delay the deadenylation of a target transcript (Beckel-Mitchener et al., 2002). Regulation of HuR also occurs through phosphorylation at multiple sites. This can affect the nucleocytoplasmic localization of HuR as well as its binding to target mRNAs (von Roretz et al., 2011).

1.4.2 Puf Family of Proteins

The Pumilio and FBF (PUF) family of proteins mediate post-transcriptional regulation on a diverse group of transcripts. PUF proteins bind mRNAs through their Pumilio Homology Domains (Pum-HD) that typically consist of 8 Puf repeat domains (Edwards et al., 2001; Wang et al., 2001). Puf proteins bind mRNA elements found within the 3’ UTR of target transcripts. The sequence of these elements varies for each family member, but typically contains a UGUR tetranucleotide sequence followed by the nucleotides UA spaced 2, 3, or 4 nucleotides apart (Gerber et al., 2004).

In yeast, there are six Puf family members (Puf1p- Puf6p) that interact with different sets of mRNAs and have different roles in post-transcriptional regulation. Large-scale identification of mRNA targets revealed that Puf1p and Puf2p target mRNAs are enriched for transcripts mRNAs encoding membrane associated proteins, Puf3p target mRNAs are enriched for transcripts that encode mitochondrially localized proteins that are encoded by the nucleus, and finally Puf4p and Puf5p interact with mRNAs that are enriched for transcripts that encode nuclear components (Gerber et al., 2004).

The Puf proteins in yeast mediate post-transcriptional regulation by different mechanisms. Puf1p, Puf4p and Puf5p mediate degradation of target transcripts. Puf4p and Puf5p each interact with the Ccr4p/Pop2p/Not deadenylase to recruit it to target transcripts, thus inducing deadenylation and transcript decay (Goldstrohm et al., 2006; Goldstrohm et al., 2007; Hook et al., 2007). Multiple Puf proteins can also act on one mRNA. For example Puf4p and Puf5p have been shown to regulate the HO mRNA and both proteins are required for full repression and deadenylation of the HO transcript (Hook et al., 2007). Puf1p and Puf5p have also been show to act together to mediated degradation of HXK1 and TIF1 mRNAs, although the mechanism of Puf1p-mediated repression has not been identified (Ulbricht and Olivas, 2008).

Puf3p has been implicated in mitochondrial localization of its mRNA targets. A puf3 ∆ strain results in the mislocalization of many mitochondrially-localized transcripts. Also, deletion of the Puf3p binding site within the BCS1 mRNA results in its mislocalization (Saint-Georges et al., 2008). This localization is thought to involve the translocase of the mitochondrial outer membrane (TOM) complex since negative genetic interactions were seen in a tom20 ∆, puf3 ∆ double mutant (Eliyahu et al., 2010).

Finally, Puf6p has been shown to repress the translation of ASH1 mRNA. Puf6p interacts with the general translation initiation factor eIF5B and inhibits assembly of the 80S ribosome. Translational repression is relieved by phosphorylation of Puf6p by the casein kinase CK2. This phosphorylation event promotes translation of ASH1 by reducing the ability of Puf6p to bind mRNA (Deng et al., 2008; Gu et al., 2004).

1.4.3 Iron Homeostasis by Iron Regulatory Proteins

Iron is required as a cofactor for many biological processes, however excess iron can be toxic since it can readily generate free radicals. Thus, iron concentrations are tightly regulated within the cell. Transferrin binds to iron and delivers it to tissues through binding to the transferrin receptor (TfR). Within the cell, ferritin stores excess iron. The expression of both TfR and ferritin are coordinately and reciprocally regulated at the post-transcriptional level by iron regulatory proteins, IRP1 and IRP2. IRPs interact with stem-loop structure known as iron responsive elements (IREs) that have an unpaired C in the stem and a loop with the sequence CAGUGN, where the first and fifth residues form a base pair (Pantopoulos, 2004).

In iron-starved cells, IRP1 and IRP2 bind to IREs within the 5’ and 3’ UTRs of the ferritin and TfR mRNAs, respectively. Ferritin has one IRE in its 5’ UTR and binding of IRP results in translational repression of ferritin. This interaction occurs close to the cap and blocks 40S ribosome subunit loading onto the mRNA, although eIF4F can still bind to the cap (Gray and Hentze, 1994; Muckenthaler et al., 1998). TfR contains five IREs in its 3’ UTR and binding of the IRPs to these sites stabilizes the mRNA. This binding blocks an endonucleolytic cleavage event that occurs downstream from the third IRE (Binder et al., 1994; Mullner et al., 1989). The result of these regulatory events is an increase in the levels of free intracellular iron through: 1) increased TfR expression enhancing the ability of the cell to take up iron and 2) a decrease in the levels of ferritin, thereby freeing intracellular iron stores.

IRP1 is a bifunctional protein and is regulated by an iron-sulfur switch. In cases of high iron concentrations, a 4Fe-4S cluster forms within IRP1 and inhibits its ability to bind RNA. In this form, IRP1 acts as a cellular aconitase and is involved in the generation of NADPH in the Kreb’s cycle. During low iron concentrations, the iron-sulfur cluster disassembles and the apoIRP1 is then able to bind RNA and increase intracellular free iron levels (Gray et al., 1993; Haile et al., 1992). IRP2 is also regulated in response to iron supply where IRP2 is transcribed during iron starvation. When iron supplies normalize, IRP2 is targeted for proteosomal degradation by an E3 ubiquitin ligase that is also regulated by iron levels (Salahudeen et al., 2009; Vashisht et al., 2009). The relative contribution of IRP1 and IRP2 to post-transcriptional regulation of TfR and ferritin is still unclear.

1.4.4 Translational regulation by modulation of polyadenylation

Due to the stimulatory effects of the poly(A) tail on translation initiation, the removal and addition of a transcript’s poly(A) tail can regulate its translation. The best-characterized example of such a regulatory mechanism is mediated by cytoplasmic polyadenylation element binding (CPEB) protein that binds to cytoplasmic polyadenylation elements (CPEs) with the sequence UUUUUA 1-3U in the 3’UTR of mRNAs. CPEB was first identified and characterized in Xenopus oocytes.

When Xenopus oocytes are arrested in Prophase I, CPEB forms a complex with cleavage and polyadenylation specificity factor (CPSF), symplekin, a scaffolding protein, the poly(A) polymerase Gld-2, and poly(A) ribonuclease (PARN). Gld-2 and PARN play antagonistic roles

19 and compete to add and remove a transcript’s poly(A) tail. PARN however is more active and serves to keep the poly(A) tail short. During maturation, CPEB is phosphorylated resulting in the expulsion of PARN from the complex, which allows Gld-2 to polyadenylate the mRNA, thus activating its translation (Kim and Richter, 2006).

CPEB has also been observed in another complex with the protein Maskin. Maskin is a 4E-BP and as such it interacts with eIF4E through its eIF4E-binding motif (see above). This blocks the eIF4E/eIF4G interaction and recruitment of the 40S ribosomal subunit to an mRNA (Stebbins- Boaz et al., 1999). Polyadenylation is required to relieve Maskin-mediated translational repression. The increase in length of the poly(A) tail is thought to recruit eIF4G to the mRNA, which disrupts the Maskin/eIF4E interaction, thereby allowing translation initiation (Cao and Richter, 2002). The interplay between these two mechanisms is still unclear, and it remains to be determined whether Maskin and PARN can simultaneously regulate the same mRNA.

1.4.5 miRNA-Mediated Post-transcriptional Regulation

Transcripts can also be regulated in a sequence-specific manner by microRNAs (miRNAs). These are small (~21 nucleotides long), nuclearly encoded, non-coding RNAs that base pair to mRNAs with partial complementarity to regulate their expression. miRNAs are loaded into a complex termed the miRNA-induced silencing complex (miRISC) through an interaction with an Argonaute (Ago) family protein. These proteins are core RISC components that induce translational repression and/or transcript degradation when they function in the cytoplasm [reviewed in (Fabian and Sonenberg, 2012)]. Another component of miRISC is GW182. This protein is essential for repression since depletion of GW182 or blocking the GW182/Ago interaction in Drosophila S2 cells disrupts miRNA-mediated silencing (Eulalio et al., 2008). In addition, tethering GW182 to an mRNA induces repression, even in the absence of Ago (Behm- Ansmant et al., 2006), suggesting that GW182 is the downstream effector of miRNA-induced silencing. miRNA-mediated recruitment of miRISC results in translational repression and/or transcript degradation of the target transcript. However, it is worth noting that many of the details of the mechanisms involved remain very controversial. One prominent mechanism involved is this regulation is miRISC-induced deadenylation. This requires GW182 which serves as a platform to recruit the Ccr4/Pop2/Not deadenylase through a direct interaction between GW182 and Not1

(Braun et al., 2011; Chekulaeva et al., 2011; Fabian et al., 2011). Given the essential role of the poly(A) tail in both translation and transcript stability, this deadenylation could trigger both translational repression and induce degradation of target mRNAs.

Other mechanisms of miRNA-mediated repression have been proposed and there is evidence that miRISC can repress translation at both initiation and post-initiation steps. Studies have shown that an m 7G cap is necessary for miRNA-mediated repression and that usage of an internal ribosomal entry site (IRES) or non-functional ApppN caps blocked translational repression by miRNAs (Mathonnet et al., 2007; Thermann and Hentze, 2007; Wakiyama et al., 2007). Furthermore, addition of purified eIF4F complex inhibited miRNA-mediated repression in a dose-dependent manner (Mathonnet et al., 2007), suggesting that miRISC may be interfering with the cap/eIF4F interaction. Polysome analysis has also shown decreased 80S formation, suggesting that miRISC may block 60S subunit joining, although how this is brought about is still controversial (Thermann and Hentze, 2007; Wang et al., 2008a).

Translational repression at steps post-initiation is even less clear with no clear molecular mechanism identified. Early work in C. elegans and work with mammalian cells has shown that mRNAs repressed by miRNAs were still associated with polysomes (Nottrott et al., 2006; Olsen and Ambros, 1999; Petersen et al., 2006). Also, in contrast to the experiments above, translation initiated at IRES elements that employ minimal translation initiation machinery were also sensitive to miRNAs (Petersen et al., 2006).

1.5 The Smaug (Smg) Family of Proteins

The Smg family of proteins is conserved from yeast to humans and regulates gene expression of its targets post-transcriptionally (Fig 1-3) (Aviv et al., 2003; Baez and Boccaccio, 2005; Dahanukar et al., 1999; Pinder and Smibert, 2013; Semotok et al., 2005; Smibert et al., 1999; Smibert et al., 1996; Tadros et al., 2007). Smg binds RNA through its conserved sterile alpha motif (SAM) domain and recognizes stem-loop structures term Smaug Recognition Elements (SREs) (Aviv et al., 2003; Dahanukar et al., 1999; Green et al., 2003; Smibert et al., 1999; Smibert et al., 1996). Smaug contains two other conserved domains, known as Smaug Similarity Regions (SSR) 1 and 2 that are found in many homologs (Aviv et al., 2003; Smibert et al., 1999).

The SSR1 domain of the S. cerevisiae homolog Vts1p has been shown to dimerize in vitro (Tang et al., 2007), while the SSR2 domain is uncharacterized.

Traditionally, SAM domains are protein-protein interaction domains that can mediate a variety of interactions (Qiao and Bowie, 2005). The SAM domain of Smg family members represents a subclass of these domains that can bind to RNA. The SAM domains of Drosophila Smg and the S. cereivisiae homolog, Vts1p, both form the canonical 5 helices fold similar to other SAM domains (Aviv et al., 2006a; Aviv et al., 2003; Edwards et al., 2006; Green et al., 2003; Oberstrass et al., 2006). Unique to the Smg class of SAM domains is a conserved cluster of basic residues that form an mRNA binding surface (Aviv et al., 2006a; Aviv et al., 2006b; Edwards et al., 2006).

The consensus sequence of an SRE binding site has been defined as a non-specific stem with the

loop region with the sequence CNGGN 0-3 (Aviv et al., 2006b). From the crystal structure, sequence specificity is conferred by the G at position 3 of the loop. Base-pairing between the first and fourth residues in the loop creates a unique fold that is recognized by the SAM domain (Aviv et al., 2006b; Johnson and Donaldson, 2006; Oberstrass et al., 2006). Basic residues also make contact with phosphates in the 5’ portion of both the loop and stem. In addition, the SAM domain does not make contact with nucleotides 3’ of the CNGG loop nucleotides, thus explaining the flexibility in the number of nucleotides found in the 3’ end of the loop sequence. Based on the absolute conservation of the amino acid residues that contact RNA, it is expected that all Smg homologs will bind RNA with the same specificity. This has been confirmed by studies in Drosophila , yeast and mammals (Aviv et al., 2003; Baez and Boccaccio, 2005).

1.5.1 Drosophila Smg

Post-transcriptional regulation of two transcripts, nanos (nos ) and Heat shock protein 83 (Hsp83 ), by Smg has been well characterized and Smg has been shown to repress the expression of these transcripts through three mechanisms.

Figure 1-3 Smaug family of proteins Cladogram representing domain architecture of the Smg homologs. Zif represents a CCHC zinc- finger domain. Species abbreviations: dm, Drosophila melanogaster ; ag, Anopheles gambiae ; hs, Homo sapiens ; mm, Mus musculus ; ce, Caenorhabditis elegans ; ca, Candida albicans ; sp, Schizosaccharomyces pombe ; sc, Saccharomyces cerevisiae . Adapted from Aviv et al (2003).

1.5.1.1 Smg-mediated post-transcriptional regulation of nos mRNA

Nos is required for posterior development in Drosophila embryos and acts to repress the expression of hunchback (hb ) specifically at the posterior of the embryo (Wang and Lehmann, 1991; Wharton and Struhl, 1991). Nos protein is expressed exclusively at the posterior of the embryo and ectopic expression results in lethal head defects (Wharton and Struhl, 1991). The posterior expression of Nos protein is due to translation of nos mRNA localized to the posterior (Wang and Lehmann, 1991). Localization of nos mRNA is inefficient, and only ~4% of the mRNA is localized to the posterior (Bergsten and Gavis, 1999). Thus, translational repression in the bulk of the embryo is an important mechanism to ensure posterior localization of Nos protein. Both translational repression and mRNA localization of nos are controlled through sequences in the nos mRNA’s 3’ UTR and Smg interacts with two SREs within nos 3’ UTR to repress the nos translation in the bulk of the embryo (Dahanukar et al., 1999; Smibert et al., 1999; Smibert et al., 1996).

Smg represses translation through its ability to recruit the eIF4E binding protein Cup to a target mRNA (Fig1-4A) (Nelson et al., 2004). Through biochemical experiments, Cup was shown to interact with Smg and mediate an indirect interaction between Smg and eIF4E. The Cup/eIF4E interaction is mediated by consensus eIF4E binding site (YXXXXL Φ, where Φ denotes any hydrophobic amino acid) identified within Cup. The interaction between Smg and Cup blocks the eIF4E/eIF4G interaction (Nelson et al., 2004) and therefore Smg-mediated recruitment of Cup to an mRNA inhibits the formation of the translation initiation complex and represses the translation.

Interestingly, translationally repressed nos mRNA remains polysome associated in early embryos suggesting an additional mechanism acts co-translationally on nos transcripts (Clark et al., 2000). Recently, another interaction was identified between Smg and Ago1 (Pinder and Smibert, 2013) which is the Drosophila Ago involved in the miRNA-RISC complex described above. Ago1 mutants showed ectopic Nos protein in early embryos, suggesting a role for Ago1 in nos regulation. This defect in repression was at the level of translation since degradation of nos transcripts was normal in Ago1 mutant embryos. Ago1 was unable to interact with nos mRNA in the absence of Smg suggesting that Smg directly recruits Ago to nos mRNA (Fig1-4B) and

Figure 1-4 Mechanisms of Smg-mediated repression (A) Smg binds to SREs and recruits Cup which mediates an indirect interaction between Smg and eIF4E. The Smg/Cup/eIF4E complex blocks binding of eIF4G and represses mRNA translation. (B) Smg-mediated recruitment of Ago1 to a target transcript mediates translational repression through a miRNA-independent mechanism. (C) Smg recruitment of the consistent with this model, a mutant Ago protein that is defective for miRNA binding is still able to interact with nos RNA (Pinder and Smibert, 2013). Ccr4/Pop2/Not deadenylase to a target transcripts induces deadenylation and subsequent degradation.

1.5.1.2 Smg-mediated mRNA degradation

Hsp83 mRNA is rapidly degraded in the bulk of the early embryo while Hsp83 mRNA at the posterior is protected from degradation. As a result of this degradation/protection mechanism Hsp83 mRNA is localized to the posterior of the embryo (Bashirullah et al., 1999; Ding et al., 1993). Smg has been shown to be involved in the degradation of Hsp83 transcripts in the bulk of the embryo. Smg binds to SREs within the Hsp83 mRNA’s ORF to mediate this destabilization (Semotok et al., 2005; Semotok et al., 2008). Hsp83 mRNA remains abundant and distributed throughout the embryo and fails to localize in smg mutant embryos, indicating that Smg plays a role in the degradation and localization of Hsp83 mRNA. Mutations to either the SREs within the Hsp83 mRNA or the Smg RNA-binding domain also prevent Hsp83 degradation. Smg protein interacts with the Ccr4/Pop2/Not deadenylase, and induces the deadenylation of Hsp83 transcripts (Fig 1-4C). Hsp83 transcripts in smg mutant embryos persist as polyadenylated mRNAs. Thus, Smg induces the degradation of Hsp83 mRNA through recruitment of the Ccr4/Pop2/Not deadenylase. Smg does not repress the translation of Hsp83 and the complex of Smg and the Ccr4/Pop2/Not deadenylase appears to be distinct from that of Smg with Cup (Semotok et al., 2005). Therefore Smg employs three distinct mechanisms for post- transcriptional regulation through its ability to recruit Cup, Ago, or the Ccr4/Pop2/Not deadenylase to target mRNAs.

Unlocalized nos mRNA is also rapidly degraded from the bulk cytoplasm (Bashirullah et al., 1999) and Smg plays a modest role in mediating this degradation (Semotok et al., 2005; Semotok and Lipshitz, 2007; Zaessinger et al., 2006). Piwi-associated RNAs (piRNAs) have also been implicated in the degradation of nos mRNA. Embryos that are defective for piRNA-mediated regulation showed increased levels of nos mRNA as well as head defects, indicative of ectopic Nos protein. Sites complimentary to piRNAs have been identified within the 3’UTR of nos and deletion of these regions or injection of 2’O-Me anti-piRNAs also results in nos mRNA decay defects as well as head defects. Interactions between Smg and Aubergine or Ago3, Argonaute family members associated with piRNA-mediated repression, were also identified suggesting that piRNAs and their associated proteins coordinate with Smg to mediate degradation of nos mRNA (Rouget et al., 2010).

1.5.1.3 Smg is a major regulator of mRNA stability

Maternally loaded mRNAs and proteins direct early Drosophila development and are loaded into the embryo during oogenesis. Upon egg activation, a subset of these maternal mRNAs are degraded (Tadros and Lipshitz, 2009). In addition, two-thirds of these destabilized maternal transcripts were stabilized in smg mutant activated eggs, indicating that Smg is a major regulator of transcript stability in Drosophila (Tadros et al., 2007). This class of transcripts that are dependent on Smg for degradation was also found to be enriched in GO term categories involved in the cell cycle, which may provide insight into the cell cycle defects seen in smg mutant embryos (Benoit et al., 2009; Dahanukar et al., 1999).

1.5.2 Mammalian Smg

Two Smg homologs are found in mammals. Human Smg1 is found on chromosome 14 and contains all three conserved regions with Drosophila Smg: the SSR1, SSR2 and SAM domains (Aviv et al., 2003; Baez and Boccaccio, 2005). When transfected into BHK cells, hSmg1 repressed the translation of a reporter that contains SREs without affecting mRNA stability (Baez and Boccaccio, 2005). The mechanism of repression was not addressed, although it is expected that hSmg1 will bind to RNA through its SAM domain.

When expressed in fibroblasts, hSmg1 was found in cytoplasmic granules reminiscent of stress granules. These granules contained polyadenylated RNA as well as TIAR and TIA-1, proteins commonly found in these foci. Stress granules are cytoplasmic aggregates which contain translationally repressed RNAs as well as various RNA binding proteins. They are reversible and formed due environmental cues that can cause global translation inhibition (Stoecklin and Kedersha, 2013). Similar to stress granules, the presence of cytoplasmic Smg foci was also influenced by the translational status of the cells (Baez and Boccaccio, 2005). Whether these foci are important for known mechanisms of Smg-mediated repression or represent a novel function remains to be determined. Interestingly, Drosophila Smg has also been observed in foci in the bulk of the embryo, however the composition of these foci remains unknown (Zaessinger et al., 2006).

1.5.3 The S. cerevisiae homolog Vts1p

Vts1p was initially identified as a suppressor of growth defects and vacuolar transport defects in a temperature-sensitive allele of the Q-SNARE VTI1 (Dilcher et al., 2001). Vts1p was identified as a Smg family member since it contains an SSR1 domain as well as Smg-like SAM domain. Vts1p also binds to SRE stem loops with the same specificity as Smg (Aviv et al., 2003) and finally, binding of Vts1p to a reporter RNA has been shown to induce SRE-dependent transcript degradation (Aviv et al., 2003) suggesting that Vts1p regulates target transcripts by inducing their decay.

To better understand Vts1p function, identification of Vts1p targets has been undertaken by a number of groups. In two studies, Vts1p was immunoprecipitated and the mRNAs that co- purified with Vts1p were reverse transcribed and hybridized to microarrays for identification. These RNAs were nuclear encoded and enriched in SREs compared to the whole genome (Aviv et al., 2006b; Hogan et al., 2008). Select targets were confirmed by their ability to co-purify with Vts1p but not with a Vts1p RNA binding mutant. In addition, steady state levels of some targets were also measured and showed increased expression in a vts1 ∆ strain (Aviv et al., 2006b). Some of the genes have been identified in cellular functions such as sporulation and germination (Aviv et al., 2006b), which has been shown to be enhanced in a vts1 ∆ strain (Deutschbauer et al., 2002). Another approach employed microarray analysis to identify genes with increased expression in a vts1 ∆ strain as a means to identify targets. As validation, steady state levels were measured by Northern blot for four genes identified by the microarray. These genes showed increased steady state expression in a vts1 ∆ strain as compared to wild type, suggesting they could also be regulated by Vts1p (Oberstrass et al., 2006).

In addition to its function in mRNA degradation and potential roles in vesicle transport and sporulation described above, Vts1p has also been implicated in other cellular functions. Vts1p was identified as a multi-copy suppressor of dna2-K1080E , a mutant allele of DNA2 involved in Okazaki fragment processing. Post-transcriptional regulation is not likely involved in this function as an overexpressed Vts1p mutant that was defective for RNA binding was still able to suppress the phenotype (Lee et al., 2010). Vts1p is also able to interact with Dna2p in vitro and stimulate its endonuclease activity (Lee et al., 2010). Finally, overexpression of Vts1p was also found to promote termination read-through in a sensitized background, similar to nonsense

28 suppression in a [ NSI+ ] strain. This was not directly due to Vts1p’s ability to form prion aggregates since Vtsp1 overexpression or deletion did not induce or eliminate [ NSI +] respectively (Nizhnikov et al., 2012). The mechanism by which Vts1p promotes both these activities remains to be determined.

1.6 Thesis Rationale

Vts1p has been shown to specifically bind and regulate mRNAs in vivo (Aviv et al., 2003), therefore the goal of my project was to understand the molecular mechanism of Vts1p-mediated repression. Given the in vitro data suggesting that Vts1p may dimerize (Tang et al., 2007), the first objective of my thesis was to determine the role that dimerization plays in Vts1p-mediated degradation. I have shown that Vts1p dimerizes in vivo and that dimerization is required for full repression of a reporter construct that recapitulates Vts1p-mediated repression. Degradation kinetics of the reporter mRNA show that decay of the reporter is also compromised with Vts1p mutants that disrupt dimerization, therefore the stability defects observed contribute, at least in part, to the impaired repression by Vts1p dimerization mutants. Finally, my data supports the idea that dimerization increases the efficiency of Vts1p binding to target mRNAs with two or more SREs.

My second objective was to understand how Vts1p degrades target transcripts. I have shown that Vts1p interacts with the Ccr4p/Pop2p/Not deadenylase, which supports a model where, similar to Smg, Vts1p recruits the deadenylase to a target transcript to induce deadenylation and subsequent decay. I have also shown that Vts1p contains two regions, the N- and C-termini, that are sufficient for repression when tethered to a reporter. Both these regions can induce transcript decay, however the N-terminal region also mediates translational repression. Finally, I have also identified that translational repression is mediated through a conserved motif within the Vts1p N- terminus.

Chapter 2

Materials and Methods

2 Materials and Methods 2.1 Yeast Strains

All strains were derived from BY4741 (Brachmann et al., 1998). All deletion strains are described in Winzeler et al, (1999). The ccr4 ∆ vts1 ∆ mutant strain (MATa ccr4 ∆::NATMX vts1 ∆::KANMX leu2 ∆0 his3 ∆1 ura3 ∆0 met15 ∆0) was created by PCR based gene disruption (Baudin et al., 1993). Plasmid-based GFP reporters were created that consist of the GFP ORF under the control of a galactose inducible promoter and three SREs in the 3’UTR (GFP- 3XSRE+/GFP-3XSRE-). Additionally, GFP reporters containing either the nos 3’UTR (GFP nos 1-186+ and GFP nos 1-186-) or three TAR hairpins within the 3’UTR (GFP-3XTAR+ and GFP- 3XTAR-) were also integrated into the TRP1 locus ( trp1 ∆::Gal GFP reporter-URA3) . The presence of the reporter was detected through selection of the URA3 gene. All strains were transformed by standard techniques and plasmids were maintained by growth in selective media.

2.2 Plasmids

Plasmids expressing Vts1p-VSV-Flag and Vts1p-HA were generated by L. Rendl (Rendl et al., 2008) and express 574 bases and 396 bases upstream and downstream of the VTS1 ORF within a centromeric yeast expression vectors (Sikorski and Hieter, 1989). The dimerization mutants and truncation mutants are all created within this context. Amino acids 97-237 of the lambda phage c1 repressor dimerization domain were fused to the 5’ end of the VTS1 ORF. The Vts1-TAT constructs were created with an oligo duplex that contained the VSV tag as well as amino acids 65-81 which express the BIV Tat RNA-binding domain (Chen and Frankel, 1994; Puglisi et al., 1995).

2.3 Immunoprecipitations

Yeast cells were disrupted in lysis buffer (150 mM KCl, 30 mM Hepes pH 7.4, 0.4 mM AEBSF, 2 mM benzamidine, 1.5 µg/mL pepstatin A, 20 µg/mL leupeptin, 30 µg/mL chymostatin and 0.5 mM DTT) and lysed by vortexing in the presence of glass beads for ten rounds of 15 second pulses followed by 45 seconds on ice. Extracts were clarified at 13,500 rpm for 10 min and Tween-20 was added to a concentration of 0.1%. Protein concentration was assayed with Bio-

Rad Protein Assay Reagent and all extracts for a given experiment were adjusted to the same protein concentration using lysis buffer. A typical experiment employed 5 µL of anti-HA agarose beads (Santa Cruz) or 15 µL anti-Flag M2 affinity gel (Sigma) which were added to 1200 µg of yeast extract at a total protein concentration of 6 mg/mL. Immunoprecipitations were mixed end-over-end for 3 hours at 4 o C. The beads were washed four times with lysis buffer. Proteins were eluted from anti-HA beads by boiling in 2X SDS-PAGE loading buffer. Proteins bound to anti-Flag beads were washed end-over end twice in elution buffer (100mM KCl, 100mM Hepes pH 7.4) for 10 min at room temperature and then eluted for 10 minutes at room temperature in 50 µl of elution buffer supplemented with 200 µg/mL FLAG peptide. Where indicated, 0.35 g/L RNase A was added to the samples during the 3 hour immunoprecipitation. Western blots were used to detect VSV-FLAG and HA-tagged proteins using VSV and HA antibodies, respectively.

2.4 Flow Cytometry

Yeast cells were grown in media containing 2% (w/v) raffinose to an OD600 nm of 0.3-0.6. Galactose to a final concentration of 2% was then added to induce reporter expression. After 4 hours, cells were pelleted via centrifugation and resuspended in 50 mM sodium citrate pH 7.7 and GFP intensity was measured on a Becton Dickinson FACS Calibur flow cytometer and analyzed by FlowJo software.

2.5 Transcriptional Pulse Chase

Yeast cells were grown in media containing 2% (w/v) raffinose to an OD600 nm of 1-1.5. Cultures were cooled to 20 oC for one hour and galactose to a final concentration of 2% was added to the culture to induce reporter expression. 16 minutes post-galactose induction, transcription was inhibited by the addition of glucose to final concentration of 4%. Aliquots of cells were taken at each time point and added to 2.7 volumes of ice cold glucose media. Cells were resuspended in LET buffer (25 mM Tris pH 8.0, 100 mM LiCl, 20 mM EDTA) and RNA was harvested through glass bead lysis followed by extensive phenol:chloroform extraction and ethanol precipitation. RNA was resolved on agarose-formaldehyde gels and transferred to nitrocellulose. ACT1 or SCR1 RNA was used for normalization of the reporter mRNA levels. Northern blots were exposed to PhosphorImager screens and analyzed with ImageJ software.

2.6 SRE searching

138 putative Vts1p targets were identified by microarray following immunoprecipitation of Vts1p (Aviv et al., 2006b; Hogan et al., 2008). UTR sequences for these targets were extracted from high-throughput RNA sequencing data (Nagalakshmi et al., 2008; Yassour et al., 2009). Sequences that contained the target ORF and the longest identified 5’ and 3’ UTR, not necessarily from the same data set, were then subjected to a simple algorithm that searched for SREs that contained a four base pair stem of AU, CG, and GU base pairs with the loop sequence of CNGGN 0-4 within these sequences.

Chapter 3

The SSR1 Domain is a Dimerization Domain Involved in Vts1p-mediated Repression

Contribution of Work: I performed all the work presented in this Chapter.

3 The SSR1 domain is a dimerization domain involved in Vts1p-mediated repression

3.1 Introduction

The Smg family of proteins are conserved from yeast to human and regulate gene expression post-transcriptionally through their ability to bind to mRNA (Aviv et al., 2003; Baez and Boccaccio, 2005; Dahanukar et al., 1999; Nelson et al., 2004; Smibert et al., 1999; Smibert et al., 1996). Family members interact with mRNAs with the same specificity through a conserved SAM domain that binds to stem loop structures known as SREs (Aviv et al., 2003; Dahanukar et al., 1999; Green et al., 2003; Smibert et al., 1999; Smibert et al., 1996). Smg family members can also contain other conserved elements: SSR1 and SSR2 that have not been well characterized (Aviv et al., 2003; Smibert et al., 1999). Drosophila Smg represses the translation and also induces target transcript degradation through multiple mechanisms. This is achieved through the recruitment of different trans-acting factors to the target mRNA. Smg recruits the eIF4E-binding protein Cup as well as the miRISC component Ago1 to nos mRNAs to mediate translational repression (Nelson et al., 2004; Pinder and Smibert, 2013) . Additionally, Smg recruits the Ccr4/Pop2/Not deadenylase to Hsp83 mRNA, which induces its deadenylation and subsequent decay. The S. cerevisiae homolog Vts1p also interacts with mRNA through its SAM domain with the same specificity as Smg (Aviv et al., 2003). Recruitment of Vts1p to mRNAs results in repressed expression of a reporter gene and also decreased target mRNA stability. In addition to its SAM domain, Vts1p also contains the conserved SSR1 domain.

The SSR1 domains of Vts1p and Smg have been shown to be homologous to a family of dimerization domain called D domains (Fig 3-1) (Tang et al., 2007). These domains are highly conserved amongst WD40 repeat F box proteins that are a component of the SCF E3 ubiquitin ligase machinery. Multiple F box proteins have been shown to form homotypic dimers (Patton et al., 1998; Suzuki et al., 2000; Tang et al., 2007; Welcker and Clurman, 2007; Wolf et al., 1999). These domains contain a high degree of hydrophobicity at the dimerization interface however

Figure 3-1 Protein alignment of D domains Structure-based sequence alignment of D domains in F box and non-F box containing proteins. Conserved hydrophobic residues are highlighted in yellow. Red boxes show mutations shown in Tang et al, (2007) to disrupt dimerization. Smaug and Vts1p are found at the bottom of the alignment. Adapted from Tang et al, (2007).

very few of these residues are actually conserved, which has been postulated to mediate specificity of D domain homodimer formation (Tang et al., 2007). Dimerization of the F box proteins is required for substrate binding and degradation of various targets, although in humans, dimerization may only be required for the recognition of sub-optimal degrons (Dixon et al., 2003; Welcker and Clurman, 2007).

Consistent with the homology between SSR1 and D domains, this suggests the Vts1p SSR1 domain dimerizes in vitro (Tang et al., 2007). Through in vivo biochemical analysis, I have confirmed that Vts1p dimerizes in vivo through its SSR1 domain. In addition, mutations to the SSR1 domain that disrupt dimerization result in impaired repression of a reporter construct. This defect is likely due to changes in mRNA stability since dimerization mutants show slower decay of target mRNAs. Finally, the dimerization of Vts1p likely stabilizes the interaction between Vts1p and target transcripts bearing two or more SREs.

3.2 Results

3.2.1 Vts1 dimerizes in vivo though it’s SSR1 domain

To assess the role of dimerization in Vts1p function I first set out to ask if Vts1p forms dimers in vivo. Two differentially tagged versions of Vts1p, one carrying an HA tag and another carrying a VSV tag, were co-expressed in yeast under control of native VTS1 gene regulatory elements and extracts prepared from these cells were used in immunoprecipitation experiments. Consistent with Vts1p interacting with itself, the VSV-tagged protein could be detected in material immunoprecipitated with an anti-HA antibody (Fig 3-2) in a manner that was dependent upon the presence of HA-tagged Vts1p. Since Vts1p is an RNA-binding protein, it is possible that this co- immunoprecipitation is mediated by an mRNA, for example an mRNA carrying two or more SREs. The addition of RNase A however did not affect the co-immunoprecipitation of VSV- tagged Vts1p with HA tagged Vts1p, indicating that the interaction of Vts1p with itself is RNA independent and suggesting that the interaction is mediated through protein/protein interactions (Fig 3-2).

To confirm that Vts1p’s interaction with itself involves the SSR1 domain, mutations were created within the SSR1 domain that are predicted to disrupt dimerization in vitro. These

Figure 3-2 Vts1p dimerizes in vivo VSV and HA tagged Vts1p constructs were expressed alone or co-expressed in a vts1 ∆ strain as indicated. HA-tagged Vts1p was immunoprecipitated with HA-agarose and Vts1p-VSV was detected in the resulting immunoprecipitates by western blot. 0.35 g/L RNase A was added to the co-immunoprecipitation where indicated.

mutations changed one of two pairs of conserved, surface exposed hydrophobic residues to glutamates, and have been shown to affect dimerization in other D domains (Tang et al., 2007). While VSV-tagged version of Vts1p carrying these mutations, Vts1p V210 M211/EE or Vts1p I214 L215/EE , are expressed at levels similar to wild-type Vts1p, neither was co-immunoprecipitated with HA-tagged Vts1p (Fig 3-3). Taken together these data indicate that Vts1p dimerizes in vivo through its SSR1 domain.

3.2.2 Dimerization mutants show defects in Vts1p-mediated repression and transcript decay

To determine the contribution that dimerization plays in Vts1-mediated repression, an in vivo reporter assay was employed to monitor repression by Vts1p. As described below I considered the possibility that dimerization might be particularly important in the regulation of mRNAs carrying two SREs. Thus I generated a reporter that consists of a GFP ORF under the control of a galactose-inducible promoter and contains a 186 nucleotide fragment of the Drosophila nos mRNA’s 3’ UTR that carries two SREs (Crucs et al., 2000) (GFP nos 1-186 +) (Fig 3-4A). The 5’SRE has been designated SRE1 and the 3’ site SRE2 and they are separated by ~100 bases (Smibert et al., 1996). As a control, a similar GFP reporter was constructed, GFP nos 1-186 -, where both SREs were mutated such that they abolish binding of Drosophila Smaug to the nos 3’UTR (Smibert et al., 1996) and should therefore abolished Vts1p binding. Flow cytometry was employed to measure GFP expression in vts1 ∆ cells rescued with various versions of Vts1p expressed under the control of native VTS1 gene regulatory elements. To assess the function of these various versions of Vts1p a fold repression value was calculated as the ratio of the mean fluorescence of the GFP nos 1-186-/GFP nos 1-186+ reporter. As expected when cells carry no Vts1p the GFP nos 1-186+ and GFP nos 1-186- construct are expressed a similar levels and hence the level of repression level is ~1.0. In contrast, in cells expressing wild-type Vts1p the GFP nos 1-186+ construct is repressed ~3.5 fold compared to the GFP nos 1-186- construct. Both dimerization mutants show repression of ~2.5 fold (Figure 3-4B). These defects were not due to a decrease in protein expression as observed by western blot (Fig 3-4C) indicating that dimerization of Vts1p is required for full Vts1p function.

To confirm that the results above were due to a defect in dimerization, an exogenous dimerization domain from the lambda phage c1 repressor (Bell et al., 2000) was added to the N-

Figure 3-3 Mutations to SSR1 domain disrupt dimerization Vts1p-HA and the dimerization mutants Vts1p V210E/M211E -VSV or Vts1p I214E/L215E -VSV were expressed alone or co-expressed in a vts1 ∆ strain as indicated. Vts1p-HA was immunoprecipitated with anti-HA agarose and VSV-tagged wild type Vts1p or the Vts1p dimerization mutants were detected in the resulting immunoprecipitates by western blot.

Figure 3-4 Dimerization mutants show defect in Vts1p-mediated repression (A) Schematic of GFP reporters used to monitor repression by Vts1p. The reporters are under the control of a galactose inducible reporter and contain nucleotides 1-186 of the nos 3’UTR that contains two SREs ( nos 1-186+ ). The nos 1-186- reporter contains mutations within the SRE that abolish Vts1p binding. (B) Vts1p V210 M211/EE and Vts1p I214 L215/EE were tested for defects in Vts1p-mediated repression where Vts1 constructs were expressed in a vts1 ∆ strain. Flow cytometry measures GFP intensity of the nos 1-186+ and nos 1-186- reporters and fold repression is calculated as the ratio of the mean fluorescence of SRE-/SRE+. The dimerization mutants were N-terminally tagged with the dimerization domain of lambda phage c1 repressor ( λ) to rescue the defects seen. Error bars show standard error significant difference was calculated using the Student’s t-test. (C) Western blot of Vts1p dimerization mutants showing that all constructs are expressed at similar levels.

terminus of the dimerization mutants. For both Vts1p dimerization mutants, the lambda c1 domain rescued repression back to wild type levels (Figure 3-4B). These data provide strong support for a model Vts1p dimerization is necessary for full Vts1p function.

To further explore the role that dimerization plays in Vts1p function I tested the role of dimerization in Vts1p-mediated transcript degradation. Transcriptional pulse chase experiments were performed where transcription of the GFP nos 1-186+ reporter was induced for a short period of time with galactose, then shut off with an excess of glucose. Yeast cells were then collected at various time points after the transcriptional shut off and the amount of GFP mRNA was assayed via northern blot. The GFP nos 1-186+ reporter has a half-life of greater than 8 minutes in cells lacking Vts1p while its half-life the presence of Vts1p is ~4 minutes (Fig 3-5). The levels of GFP nos 1-186+ reporter mRNA were significantly increased at 2, 4, 6 and 8 minutes in cells expressing dimerization defective Vts1p compared to wild-type Vts1p. As such the half-life of GFP nos 1-186+ reporter in cells expressing dimerization defective Vts1p increased to ~6 minutes, consistent with a role for dimerization in Vts1p-mediated mRNA decay. Thus, the defect observed in GFP protein levels as assayed by flow cytometry are likely due, at least in part, to defects in transcript degradation.

3.2.3 Dimerization affects Vts1p function through a combination of mechanisms

Dimerization may affect Vts1p-mediated transcript repression of the GFP nos 1-186+ mRNA through two mechanisms outlined in Figure 3-6. The first model takes into account the fact that this reporter mRNA carries two SREs in its 3’UTR and proposes that the two RNA-binding domains, present in a single dimer of Vts1p, could each contact one of the SREs in the same RNA molecule. In this scenario it is likely that the Vts1p dimer would be more stably bound to the target mRNA than would monomeric Vts1p molecules. In an alternative mechanism each SRE in a target transcript would recruit one SRE dimer. If each Vts1p monomer within a dimer can recruit the factors that destabilize the target mRNA, than dimerization would serve to increase the number of such factors brought to the target, thereby increasing the efficiency of repression.

Figure 3-5 Transcriptional pulse chase of Vts1p dimerization mutants (A) Transcription of the nos 1-186+ reporter was induced with galactose for 16 min and shut off with an excess of glucose in vts1 ∆ strains expressing wild type or dimerization mutants of Vts1p. Aliquots of cells were collected at the indicated time points and RNA was extracted to measure GFP transcript levels. ACT1 mRNA was used as a loading control. Error bars show standard error and red asterisks denote significant differences as assessed using the Student’s t-test. (B) Representative northern blots for each time course.

Figure 3-6 Models of dimerization-mediated repression of Vts1p (A) RNA binding model where dimerization serves to enhance RNA binding through stabilization of the Vts1p/mRNA interaction. (B) Recruitment model where dimerization allows recruitment of multiple repressive factors to a target transcript to mediate efficient degradation.

To test these two models I first generated versions of the GFP nos 1-186+ reporter where either SRE1 or SRE2 were mutated to block Vts1p binding. I then compared the repression of the reporters GFP nos 1-186+ , GFP nos 1-186,SRE1- and GFP nos 1-186,SRE2- by wild-type Vts1p and the Vts1p proteins that are defective for dimerization. By flow cytometry, GFP nos 1-186,SRE1- and GFP nos 1-186,SRE2- were all repressed ~2 fold by both wild-type Vts1p and dimerization defective Vts1p. If dimerization was affecting number of molecules of repressor recruited to a target mRNA then we would expect that a loss of dimerization would affect repression of reporters bearing one SRE. Therefore it is unlikely that dimerization recruits multiple repressors to a target transcript to mediate efficient mRNA degradation. When comparing reporters that contain two SREs, GFP nos 1-186+ was repressed ~4 fold (compared to GFP nos 1-186-), by wild-type Vts1p and ~3 fold by Vts1p mutants that are defective for dimerization (Fig 3-7). Since the loss of dimerization specifically affects repression of an mRNA carrying two SREs, these data are consistent with dimerization playing a role in enhancing the stability of Vts1p’s interaction with mRNAs carrying two SREs.

3.2.4 A large fraction of Vts1p target mRNAs possess two or more SREs.

These data suggest that dimerization could be relevant for the regulation of mRNAs bearing two or more SREs. Thus, to determine importance of dimerization for Vts1p’s regulation of endogenous target mRNAs I assessed the number of potential SREs present in Vts1p-bound mRNAs that were identified as co-purifying with immunoprecipitated Vts1p using microarrays (Aviv et al., 2006b; Hogan et al., 2008). Aviv et al. identified 79 Vts1p bound mRNAs while Hogan et al. identified 95 and 36 targets are shared between the two lists. The union of these two data sets results in list of 138 mRNAs and I searched these mRNAs for stem-loops with a minimum stem length of 4 base pairs and a loop sequence matching the CNGGN 0-4 consensus and found that ~59% of Vts1p targets have more than one potential SRE (Table A-1). This suggests that dimerization could be important for regulation of many Vts1p targets.

Figure 3-7 Dimerization of Vts1p stabilizes its interaction with mRNA Flow cytometry of nos 1-186 reporters that contain either 2 SREs or have 1 SRE mutated (SRE1- and SRE2-). Vts1p, Vts1p V210 M211/EE , or Vts1p I214 L215/EE was expressed in vts1 ∆ cells. Error bars show standard error and significance differences were assessed using the Student’s t-test.

3.3 Discussion

Here I have shown that: 1) Vts1p dimerizes in vivo through its SSR1 domain, 2) mutations that disrupt dimerization result in impaired repression and slower decay of target mRNAs, and 3) dimerization functions through enhancing Vts1p’s ability to interact with mRNAs carrying two and presumably more SREs.

Interestingly, many of the SREs found within Vts1p mRNAs are contained within open reading frames and previous work in both Drosophila and yeast indicate that SREs within ORFs are indeed functional (Aviv et al 2006 and Semotok et al., 2008). This raises the question as to how Vts1p and Smg are able to stably interact with mRNAs with SREs in the ORF in the face of ribosomes potentially transiting the transcript. Consider the case of a ribosome translating an mRNA with two SREs, each bound by one half of a Vts1p or Smg dimer. As the ribosome disrupts binding of the dimer to one SRE it will nonetheless have a chance to remain bound to the mRNA via the dimer’s interaction with the other SRE. For this mechanism to be effective it would require that the SREs be at least 30 nucleotides apart from one another (i.e. the footprint of a translating ribosome on an mRNA) so as to prevent the ribosome from disrupting both SREs at the same time. Taken together this model would suggest that dimerization of Vts1p and Smg would play a bigger role in regulating the expression of mRNAs that carry SREs exclusively in their ORFs.

Many other RNA-binding proteins also form dimers and higher order structures that regulate their activity. As described in Section 1.4.4, CPEB regulates translation of target transcripts by regulating their poly(A) tail length. Xenopus CPEB has also been shown to form dimers in vivo through its RRM and zinc finger, both of which are also the mRNA binding domains. Dimerization abrogates mRNA binding and also promotes the ubiquitin-mediated degradation of CPEB following phosphorylation at oocyte maturation (Lin et al., 2012). This degradation is required for progression through meiosis since mutants of CPEB that are no longer degraded, block progression to meiosis II (Mendez et al., 2002). Thus, dimerization is required for ubiquitin-mediated degradation of CPEB for proper progression through meiosis.

Dimerization can also promote mRNA binding of the Vg1RBP/Vera, IMP, CRD-BP, KOC, and ZBP-1 (VICKZ) family of proteins a conserved family of proteins that mediate localization of

47 their target mRNAs. They are composed of a number of RNA binding domains, two RRM domains and four KH domains, that are arranged in pairs with conserved linkers between the domains (Git and Standart, 2002). In Xenopus, all four KH domains of Vg1RBP/Vera are able to bind RNA, however the RRM domains show no RNA binding in vitro. Cross-linking of recombinant Vg1RBP/Vera show that a fragment consisting of KH domains 3 and 4 can also dimerize. Interestingly, RNA binding mutants within these domains or RNase A treatment of rabbit reticulocyte extracts expressing Vg1RBP/Vera constructs resulted in diminished interactions, suggesting that dimerization is RNA dependent (Git and Standart, 2002). Work with the human family member, IMP1, has shown similar results with no interaction seen in the absence of RNA. In this case, the first monomer forms an unstable complex with an mRNA and the addition of a second IMP1 monomer results in a stable complex on the mRNA. IMP1 also mediates protein-protein interactions through its third and fourth KH domain, however, the significance of the protein-protein interaction on complex formation has not been assessed (Nielsen et al., 2004). Although the VICKZ family members form multimers on the same RNA, this form of dimerization differs from that of Vts1p. I have shown that Vts1p co- immunoprecipitates in the presence of RNase A, indicating that RNA is not required for Vts1p dimerization and thereforeVts1p dimerizes before binding to target mRNAs.

The signal transducer and activator of RNA (STAR) protein family are involved in many post- transcriptional regulatory events including mRNA stabilization, translation, mRNA localization, and splicing. The family is defined by the STAR domain that consists of a KH RNA-binding domain flanked by two subdomain Qua1 and Qua2. The KH domain and Qua2 bind mRNAs while Qua1 is a homodimerization domain (Chen et al., 1997; Liu et al., 2001). Mutations that disrupt dimerization in Quaking are embryonic lethal in mice, highlighting the importance of dimerization for function (Chen and Richard, 1998). Dimerization mutants in Quaking and also the human SAM68 also show defects in alternative splicing indicating that dimerization is important for splicing regulation (Beuck et al., 2012; Meyer et al., 2010). STAR family members bind to a consensus site that consists bipartite sequence of a hexamer AC(C/U)UAA(C/U) and a half-site UAA(C/U) (Galarneau and Richard, 2005; Ryder et al., 2004). Both monomers are able to bind RNA, and it is though that each monomer interacts with the hexamer or the half site (Galarneau and Richard, 2009; Ryder et al., 2004). This differs from the model I propose for Vts1p in that STAR family members utilize their two RNA binding

48 domains to interact with discrete regions of the same binding site. Instead, Vts1p interacts with two SREs within the transcript that stabilizes Vts1p binding to the mRNA.

Finally, the bacterial protein carbon storage regulator (CsrA) is conserved across eubacteria and negatively regulates translation of genes involved in gluconeogenesis and glycogen biosynthesis during the transition to stationary phase (Romeo et al., 1993). The structure of CsrA reveals that it is a homodimer with two RNA binding sites on opposite sides of the molecule that are composed of β-strands from both monomers (Heeb et al., 2006; Rife et al., 2005; Schubert et al., 2007). CsrA binds to an unpaired GGA motif surrounded by semi-conserved residues that have been found in multiple copies in the 5’ leader sequence of CsrA targets (Dubey et al., 2005). Typically, one binding site covers the Shine-Dalgarno ribosome binding site, therefore CsrA binding blocks ribosome recruitment and represses translation (Baker et al., 2002). A CsrA dimer can interact with two binding sites on one transcript and mutations to either the CsrA RNA binding domain or the binding site in a reporter construct results in decreased repression. Interestingly, when one RNA binding domain is mutated in CsrA, the loss of the upstream binding site had no effect on repression (Mercante et al., 2009). This is very similar to the model I propose for Vts1p where Vts1p dimers mediate efficient repression of target transcripts that contain two or more SREs. Vts1p monomers however repress a target to similar levels regardless of whether one or two SREs are present. Mercante et al. suggest a model where the CsrA dimer first interacts with a high-affinity site which tethers CsrA to the transcript and can promote binding to a second lower affinity binding site that covers the Shine-Delgarno sequence. In contrast, there is no evidence that Vts1 interacts with SREs with different affinities and whether dimerization of Vts1p promotes cooperative binding remains to be investigated.

Chapter 4

The N-terminus of Vts1p Mediates Translational Repression

Contribution of Work: I performed the majority of work presented in this Chapter.

∆ A. Orlowicz created the Vts1p 170-523 -Tat and Vts1p SSR1 -Tat constructs and tested expression level by western blot (Figure 4-2)

A. Orlowicz mapped the Vts1p/deadenylase interaction and the Vts1p/Eap1p interaction to the C-terminus of Vts1p

4 The N-terminus of Vts1p mediates translational repression 4.1 Introduction

Sequence specific RNA binding proteins employ numerous mechanisms to regulate the expression of target transcripts. RNA-binding proteins that function in the cytoplasm typically recruit trans-acting factors to target transcripts that influence mRNA translation, stability and/or subcellular localization. For example, the Smg family of post-transcriptional repressors, bind to specific stem-loop structures known as SREs through their conserved SAM domains (Aviv et al., 2003; Baez and Boccaccio, 2005; Dahanukar et al., 1999; Green et al., 2003; Smibert et al., 1999; Smibert et al., 1996). This results in translational repression and/or mRNA degradation of the target transcript through a number of mechanisms. Drosophila Smg recruits the eIF4E- binding protein Cup and Ago1, a component of the miRISC complex, to mediate translational repression (Nelson et al., 2004; Pinder and Smibert, 2013). Smg has also been shown to induce the deadenylation of target mRNAs through the recruitment of the Ccr4/Pop2/Not deadenylase, thereby triggering transcript destabilization (Semotok et al., 2005).

The S. cerevisiae homolog Vts1p also binds to SREs thorough its SAM domain to mediate repression of target mRNA expression through its ability to induce transcript degradation (Aviv et al., 2003). Vts1p-mediated repression also requires Ccr4p, the catalytic subunit of the Ccr4p/Pop2p/Not deadenylase.

To explore the mechanisms that underlie Vts1p’s ability to repress the expression of target mRNAs I first asked if Vts1p functions through the recruitment of the Ccr4p/Pop2/Not deadenylase in a mechanism similar to Smg. I also performed a structure-function analysis of Vts1p to identify minimal regions that are sufficient for repression. These data combined with other work from the Smibert lab indicated that Vts1p recruits the deadenylase to target transcripts to induce deadenylation and subsequent degradation. Also, I have identified two regions of Vts1p, one contained within the protein’s N-terminus and another located in C- terminal sequences that are sufficient for repression and can mediate mRNA degradation of target mRNAs. In addition, I have identified a conserved 10-amino acid motif within the N terminal region that mediates translational repression of a target mRNA.

4.2 Results

4.2.1 Vts1 p interacts with Ccr4p/Pop2/Not deadenylase

Drosophila Smaug induces transcript decay through its ability to interact with, and recruit the Ccr4p/Pop2p/Not deadenylase complex to target mRNAs (Semotok et al., 2005). In addition, Vts1p-mediated repression is dependent upon Ccr4p (Aviv et al., 2003). Thus, to explore the molecular mechanisms that underlie Vts1p-mediated repression of target mRNA expression I set out to ask if Vts1p interacts with the Ccr4p/Pop2p/Not complex. I performed co- immunoprecipitation experiments using whole-cell lysates from cells expressing Vts1p tagged with a FLAG and VSV epitopes and Pop2p tagged with an HA epitope both expressed under the control of native VTS1 and POP2 gene regulatory elements, respectively. Lysates from cells expressing only one of the epitope-tagged proteins served as negative controls. Vts1p-Flag was immunoprecipitated using anti-FLAG resin, and the immunoprecipitates were analyzed by western blot. Pop2p-HA was present in the anti-FLAG immunoprecipitate when the Vts1p-Flag protein was present (Fig 4-1). As a control, Pop2p-HA was not immunoprecipitated from an extract lacking Vts1p-Flag. The co-immunoprecipitation of Vts1p with Pop2p was RNA independent, as it was observed in the presence of RNase A and when Vts1p harbored an amino acid change (A498Q) that blocks its ability to bind RNA binding (Vts1p RBD−) (Aviv et al. 2003). This suggests that Vts1p physically associates with a component of the Ccr4p/Pop2p/Not deadenylase complex and supports a model in which Vts1p recruits the deadenylase to a target transcript to initiate deadenylation-dependent mRNA degradation. Consistent with this model, additional work from the Smibert lab has shown that Vts1p stimulates mRNA degradation through deadenylation mediated by the Ccr4p/Pop2p/Not deadenylase complex (Rendl et al., 2008).

Figure 4-1 Vts1p-interacts with Pop2p Vts1p-VSV-FLAG and Pop2p-HA were expressed in yeast alone or in combination as indicated. Extracts were immunoprecipitated with anti-FLAG resin. Crude extract (input) and immunoprecipitates were analyzed by Western blot. RNase A or the Vts1p RNA binding mutant (Vts1p RBD-) were employed as indicated.

4.2.2 Establishing a tethering assay to study the mechanisms that underlie Vts1p-mediated repression.

To further explore the molecular mechanism by which Vts1p represses the expression of target mRNAs, an in vivo assay was created to identify minimal regions of the protein that could repress the expression of a target mRNA. To do this I employed the 16 amino acid long RNA- binding motif of the Bovine Immunodeficiency Virus (BIV) Tat protein which interacts specifically and with high affinity to TAR RNA stem/loop structures (Chen and Frankel, 1994; Puglisi et al., 1995). In frame fusion of various regions of Vts1p to the Tat RNA-binding motif allow me to test their ability to repress the expression of a GFP reporter mRNA carrying three TAR RNA stem-loops inserted into its 3’ UTR (GFP 3XTAR+). As a control a similar GFP reporter was generated where the TAR stem loops were mutated such that the TAT RNA-binding motif can no longer bind (GFP 3XTAR-) (Fig 4-2A). The TAR stem-loops recruit Tat fusion proteins to the reporter RNA, which tethers the protein of interest to the RNA. Full-length Vts1p tagged with the TAT RNA-binding motif repressed expression of GFP 3XTAR+ ~2.9 fold compared to the GFP 3XTAR- reporter as assayed via flow cytometry. In contrast, Protein A and Renilla luciferase protein tagged with the Tat RNA-binding motif had no effect on GFP 3XTAR+ expression (Fig 4-2C).

4.2.3 Vts1p carries two independent repression domains

To determine which regions of Vts1 are sufficient to repress target mRNA expression A. Orlowicz and I assayed the ability of a series of tethered constructs (Fig 4-2B) to repress GFP 3XTAR+ expression compared to GFP 3XTAR- expression via flow cytometry. We found that a C-terminal fragment of Vts1p (amino acids 170-523) was able to repress GFP 3XTAR+ expression by ~1.4 fold, while an N terminal fragment (amino acids 1-237) repressed expression ~2.9 fold (Fig 4-2C). The fact the Vts1p 1-237 was a more effective repressor than Vts1p 170-523 was not a result of higher expression levels of the former fragment as western blots suggest it is expressed at lower levels (Fig 4-2D).

Figure 4-2 Vts1p functions through two separate regions (A) A GFP reporter was generated that carries 3 TAR hairpins inserted into the reporter’s 3’ UTR. This reporter is under the control of a galactose inducible promoter (GFP 3XTAR+). GFP 3XTAR- reporters contain mutations within the hairpins that abolish Tat RBD binding. (B) A series of Vts1p-Tat fusion constructs were tested by in vivo tethering assay. Conserved domains are shown in gray and black boxes represent the Tat RNA binding domain. (C) Flow cytometry of Vts1p-Tat fusion proteins shown in (B). Tat fusion proteins were expressed in a vts1 ∆ strain. Fold Repression represents ratio of the mean fluorescence of the GFP 3XTAR-/GFP 3XTAR+. Protein A and renilla luciferase-Tat fusion proteins were tested as a negative control. Error bars show standard error and asterisks denote significant differences assessed using the Student’s t- test (D) Western blot showing all fusion proteins are expressed at similar levels. PSTAIR was employed as a loading control.

Both Vts1p 1-237 and Vts1p 170-523 contain the SSR1 domain and is possible that the repression mediated by the N and C-termini was solely dependent upon SSR1. To test this possibility a construct lacking the SSR1 domain was tethered to the TAR reporters. Consistent with my results detailed in Chapter 3, suggesting that dimerization is not essential for Vts1p function, this construct was able to repress GFP 3XTAR+ expression (Fig 4-2C). Taken together, these data suggest that there are two separate regions within Vts1p, the N-terminus and C-terminus, that can repress target transcripts, where the Vts1p 1-237 is the more potent of the two repressive domains.

Further support for these two regions of Vts1p functioning through different mechanisms comes from work showing that the regions required for the Vts1p’s interaction with the Cce4p/Pop2p/Not deadenylase reside downstream of amino acid 237 (Orlowicz and Smibert, unpublished data). Eap1p has also been shown to interact with Vts1p and promote decapping of a target mRNA (Rendl et al., 2012). The interaction between Eap1p and Vts1p has also been mapped to the SAM domain (Orlowicz and Smibert, unpublished results). Therefore, none of the existing mechanisms, or known binding partners of Vts1p-mediated repression function through the N-terminus, and this region is utilizes a novel mechanism to degrade target transcripts.

4.2.4 Vts1p 1-237 and Vts1p 170-523 mediate transcript degradation

To determine the mechanisms by which the N and C terminal portions of Vts1p represses target transcripts, transcriptional pulse chase experiments were employed to assess the stability of the GFP 3XTAR+ mRNA. In the presence of full length Vts1p tagged with TAT, GFP 3XTAR+ mRNA had a half-life of ~9 minutes while GFP 3XTAR- mRNA had a half-life of > 15 minutes (Fig 4-3A). Thus, tethered full-length Vts1p is able to induce transcript decay. Similarly, GFP ∆ 3XTAR- mRNA had a half-life cells expressing Vts1p 1-237 , Vts1p 170-523 , and Vtsp1p SSR1 of >15 minutes, >15 and ~11 minutes, respectively. In contrast, GFP 3XTAR+ mRNA in cells ∆ expressing Vts1p 1-237 , Vts1p 170-523 , and Vtsp1p SSR1 had a half-life of ~7 minutes, ~13 minutes ∆ and 7 minutes, respectively (Fig 4-3B, C, D). Since the Vts1p SSR1 construct can still mediate degradation, this suggests that regions outside of the SSR1 domain are involved in degradation

Figure 4-3 Transcriptional pulse chase of Vts1p-Tat fusion proteins Transcriptional Pulse Chase experiments were conducted on Vts1p-Tat (A), Vts1p 1-237 -Tat (B), ∆ Vts1p 170-523 -Tat (C), and Vts1p SSR1 -Tat (D) expressed in a vts1 ∆ strain. Graphs show degradation of GFP 3XTAR+ reporter in solid lines and GFP 3XTAR- in dotted line. ∆ Vts1p SSR1 -Tat also shows degradation of full length Vts1p since the degradation kinetics were different with this set of time courses. Representative Northern blots of transcript levels are shown below each graph. Error bars denote standard error.

of the transcripts suggesting that Vts1p has two separate regions that are able to induce transcript decay. Also consistent with the flow cytometry data, the Vts1p 1-237 carries a more potent degradation activity than does Vts1p 170-523 .

4.2.5 A conserved motif within the N-terminus is required for repression

In order to identify the molecular mechanism by which the N-terminus of Vts1p represses target transcripts, I sought to find conserved motifs within this region. An iterative PSI-blast was performed with Vts1p 1-186 , which lacks the SSR1 domain to prevent its high level of conservation from influencing the PSI-blasts results. This approach identified a ten-amino acid motif (AHPGAVLLSP) that showed perfect identity amongst the Vts1p proteins of other fungal species (Fig 4-4A).

A series of alanine mutations were created to test whether this motif is required for repression by Vts1p. Three mutants were constructed where the first half of the motif was mutated to AAAAAVLLSP (Vts1p 3A ), the second half was mutated to AHPGAAAAAA (Vts1p5A ), or the entire motif was mutated to alanines (Vts1p 8A ). The mutants were expressed in the context of Vts1p 1-237 tagged with TAT to test their ability to repress in the tethering assay. Each mutant repressed GFP 3XTAR+ expression ~1.5 fold compared to the GFP 3XTAR- in contrast to wild type Vts1p 1-237 that has a fold repression of ~3.25 (Fig 4-4B). These defects were not due to a decrease in protein expression as observed by western blot (Fig 4-4B). Thus this motif plays an important role in the function of the Vts1p N-terminal fragment.

I next tested the role of this motif in Vts1p-mediated repression in the context of full-length Vts1p targeted to a transcript via native Vts1p RNA-binding domain. In this case, a previously generated GFP reporter (Aviv et al., 2003) that contains three SREs in the 3’UTR (GFP 3XSRE+) was used to test repression by flow cytometry. The motif mutants again showed a severe defect in Vts1p-mediated repression while having no effect on the stability of Vts1p (Fig 4-4C), confirming that this N-terminal motif is important for Vts1p-mediated repression.

Figure 4-4 A 10 amino acid motif is important for Vts1p function (A) An iterative Psi-BLAST of Vts1p1-186 revealed a 10 amino acid motif highlighted in yellow conserved amongst fungal Vts1p homologs. (B) Flow cytometry of Vts1p1-237-Tat constructs containing the motif mutants were tested for repression with the in vivo tethering assay. Three mutants were constructed where the first half of the motif was mutated to AAAAAVLLSP (Vts1p 3A ), the second half was mutated to AHPGAAAAAA (Vts1p5A ), or the entire motif was mutated to alanines (Vts1p 8A ). (C) Repression of GFP-3XSRE+/- reporters by full length Vts1p containing the motif mutations. Error bars denote standard error and asterisks show significant differences to the Vts1p sample using the Student’s t-test.

4.2.6 The N-terminal motif functions in Vts1p-mediated translational repression

To further explore the molecular mechanism by which the N-terminal motif represses a target transcript transcriptional pulse chase experiments were performed to measure the stability of a reporter transcript in the presence of full-length Vts1p or the full-length Vts1p 8A mutant. No significant effect on the half-life of GFP 3XSRE+ mRNA was observed when the motif was mutated (Fig 4-5A, B), indicating that the motif is not involved in transcript degradation. This suggests that the motif may function in translational repression of its target. This is the first evidence that Vts1p can directly repress the translation of target mRNAs.

To confirm that the motif is mediating translational repression of the reporter transcript, steady state levels of GFP protein and mRNA were measured in cells that expressed Vts1p or Vts1p 8A . Protein and mRNA levels were unchanged with the GFP 3XSRE- reporter confirming that Vts1p has no effect on expression of a non-target mRNA (Fig 4-5C). When the GFP 3XSRE+ reporter was expressed in these cells, expression of Vts1p 8A resulted in a slight increase in the amount of GFP transcript (1.3 fold), however there was a larger increase in the amount of GFP protein when Vts1p 8A was expressed (2.3 fold) (Fig 4-5C). Since the change in RNA levels does not account for the changes seen in GFP protein this strongly suggest that Vts1p mediates translational repression through its N-terminal motif.

4.3 Discussion

Taken together my data combined with other data from the Smibert lab indicates the Vts1p employs several mechanisms to repress the expression of target mRNAs. I have shown that Vts1p interacts with the Ccr4p/Pop2p/Not deadenylase. This result combined with the fact that Vts1p stimulates the deadenylation of Vts1p target mRNAs in a Ccr4p dependent manner (Rendl et al., 2008) supports a model whereby Vts1p recruits the Ccr4p/Pop2p/Not complex to trigger transcript deadenylation. Additional work indicates that following deadenylation transcripts are decapped by Dcp1/Dcp2 and then degraded in the 5’-3’ direction by Xrn1p (Rendl et al., 2008).

The eIF4E-binding protein, Eap1p, also functions in Vts1p-mediated transcript decay (Rendl et al., 2012). The interaction between Eap1p and eIF4E is required for its role in this process which

Figure 4-5 The N-terminal motif of Vts1p mediates translational repression (A) Transcriptional pulse chase experiments of full length Vts1p and Vts1p 8A with GFP- 3XSRE+/- reporters in a vts1 ∆ strain. Error bars denote standard deviation. (B) Representative Northern blots for each time course. (C) Steady state levels of GFP-3XSRE+ and GFP-3XSRE- mRNA and protein were measured 4 hours after transcriptional induction by Northern blot and flow cytometry respectively. All values shown are normalized to wild type Vts1p GFP 3XSRE+. Error bars denote standard error and asterisks show significant difference assessed using the Student’s t-test as compared to the Vts1p GFP 3XSRE+ sample.

involves Eap1p’s ability to stimulate decapping. Eap1p co-immunoprecipitates with Vts1p suggesting a model whereby Vts1p recruits the Ccr4p/Pop2p/Not complex and Eap1p to induce rapid transcript decay, stimulating removal of both the poly(A) tail and cap. The ability of Vts1p to interact with both the deadenylase and Eap1p maps downstream of residue 236 suggesting that the ability of Vtsp1 170-523 to repress target gene expression and induce mRNA decay involves its interaction these factors.

My work has also demonstrated that amino acids 1-237 of Vts1p are also able to repress the expression of target mRNAs through a mechanism that is independent of that employed Vtsp1 170- 523 . Vts1p 1-237 is able to both induce transcript decay and through an independent mechanism repress translation and I have identified a 10-amino acid motif which is required for translational repression by both Vts1p 1-237 and full length Vts1p. This represents the first evidence of translational repression mediated by Vts1p.

Similar to Vts1p, the Drosophila homolog Smg also recruits the Ccr4/Pop2/Not deadenylase and an eIF4E-binding protein to target mRNAs to regulate their expression (Nelson et al., 2004; Semotok et al., 2005). Thus these mechanisms likely represent conserved modes of function employed by Smg family members to regulate target mRNAs. Smg also recruits Ago1 to mediate translational repression of transcripts (Pinder and Smibert, 2013) while this mechanism is not an option in S. cerevisiae as its genome does not encode any Ago proteins.

Smg can differentially regulate the expression of its target mRNAs. For example, Smg represses nos translation while it plays only a modest role in nos mRNA decay (Baez and Boccaccio, 2005; Dahanukar et al., 1999; Nelson et al., 2004; Pinder and Smibert, 2013; Semotok et al., 2005; Smibert et al., 1999; Smibert et al., 1996; Tadros et al., 2007). In contrast, Smg is the major regulator of Hsp83 mRNA stability while it does not affect Hsp83 translation (Semotok et al., 2005; Semotok et al., 2008). While the molecular mechanisms that underlie this differential regulation are not understood it is possible that different cis-elements within target Smg mRNAs influence Smg function. Whether Vts1p differentially regulates the expression of its target mRNAs remains untested.

Chapter 5

Conclusions and Future Directions

5 Conclusions and Future Directions 5.1 Conclusions

In this thesis, I have described the role of the SSR1 domain in Vts1p and show that it is a dimerization domain in vivo. I have also created dimerization mutants which abolish Vts1p dimerization and found that dimerization is required for full Vts1p-mediated repression and robust destabilization of a reporter containing two SREs. Finally, my data suggests that dimerization of Vts1p promotes Vts1p function by enhancing binding of Vts1p to a target transcript carrying two or more SREs. Analysis of Vts1p mRNA targets that were identified by immunoprecipitation-microarray experiments suggest that dimerization may be important for proper repression for the majority of target transcripts since a majority contain multiple SREs.

I have also shown that Vts1p interacts with the Ccr4p/Pop2p/Not deadenylase in an RNA independent fashion. These data are consistent with the model that, similar to Smg, Vts1p recruits the Ccr4p/Pop2p/Not deadenylase to target transcripts to induce deadenylation and subsequent degradation.

Finally, I have created a tethering system that employs the interaction between the BIV Tat RBD and TAR mRNA hairpins to identify regions within Vts1p that are sufficient for repression. I have shown that two separate regions of Vts1p, the N-terminus and the C-terminus, can repress when tethered to a reporter suggesting that Vts1p employs multiple mechanisms to regulate the expression of target mRNAs. Indeed, the C-terminal region of the protein interacts with both the Ccr4p/Pop2/Not deadenylase and the eIF4E-binding protein Eap1p suggesting that the C- terminus of Vts1p regulates gene expression by recruiting these factors to target mRNAs. I have also shown that the N-terminus contains a conserved motif that functions in Vts1p-mediated translational repression while it plays no role in transcript decay. This is the first evidence that Vts1p mediates the translational repression of a target transcript.

5.2 Future Directions

5.2.1 Characterization of Vts1p dimerization to mRNA targets

Our data suggests that dimerization of Vts1p stabilizes its interaction with mRNAs carrying two or more SREs. To directly test this, in vitro electrophoretic mobility shift assays would be employed to assess the affinity of wild-type Vts1p and the Vts1p dimerization mutants for nos 1- 186+ RNA. Full-length Vts1p and the dimerization mutants would be expressed and purified from E. coli as His-tagged fusions and I note that this approach has been successfully used to purify the full-length protein (Lee et al., 2010). Purified proteins would then be incubated with radiolabelled nos 1-186+ RNA and run on native gels. If dimerization is required to stabilize the binding of Vts1p to RNA, more dimerization mutant protein would be required to shift the same amount of RNA compared to wild-type Vts1p. If I were unable to express the dimerization mutant proteins in E. coli I would over-express wild-type and mutant proteins in vts1 ∆ yeast cells using the GPD promoter and perform gel shifts using total protein extracts. In order to compare the ability of Vts1p and the dimerization mutants to interact with RNA, all of these constructs need to be expressed at comparable levels, which would be assessed using western blots. If there are differences in the expression of the wild-type Vts1p and the dimerization mutants I would use tagged versions of these proteins from yeast in gel shift assays.

Additionally, I would test this model with bona fide Vts1p targets by comparing the ability of wild-type Vts1p and dimerization mutant Vts1p to regulate targets with two SREs. From the immunoprecipitation-microarray data (Aviv et al., 2006b; Hogan et al., 2008), I have identified twenty-four potential mRNA targets that contain two SREs (Table A-1). These SREs are non- overlapping, have a loop sequence of CNGGN 0-3, and are spaced 60-1859 base pairs apart. To determine whether these transcripts would be suitable for analysis, a subset would be tested to determine whether they are bona fide targets of Vts1p. I would first confirm that these mRNA are enriched in wild-type Vts1p immunoprecipitates and not with immunoprecipitates of a mutant version of Vts1p that does not bind to RNA using RT-qPCR.

Once Vts1p-bound transcripts have been confirmed I would assess if their expression is regulated by Vts1p and confirm that putative SREs are functional. To do this reporter constructs would be created consisting of the transcript ORF fused to the GFP ORF and under the control of

66 transcript’s 5’ and 3’ regulatory elements. Flow cytometry would then allow me to compare reporter expression in wild-type and vts1 ∆ cells to identify those mRNAs that are regulated by Vts1p. For these transcripts, putative SREs would be mutated to block Vts1p binding to generate reporters that carry one or zero SREs. Flow cytometry will then be employed to monitor repression of these reporters when Vts1p or the Vts1p dimerization mutants are expressed in vts1 ∆ cells. If my model is correct reporters carrying two SREs will require dimerization for full repression while mutant versions of the reporters which carry only one functional SRE will not.

Comparison of the results of these experiments with data presented in Chapter 3 could suggest that dimerization is particularly important for mRNAs where SREs are present in the target’s ORF. To test this directly I would construct a reporter based on the GFP nos 1-186 + reporter where the stop for the GFP ORF was mutated and another created immediately downstream of the nos 3’UTR sequences such that translation would proceed through the nos sequences. This would also require mutation of all in frame stop codons within the nos sequences. I would then compare the level of repression of this modified reporter mediated by wild-type Vts1p and dimerization mutant Vts1p to a similar reporter where translation terminates upstream of the nos sequences.

5.2.2 Mapping the Vts1p/deadenylase interaction

I have shown in Section 4.2.1 that Vts1p interacts with the Ccr4p/Pop2p/Not deadenylase, which is consistent with the model that Vts1p recruits the deadenylase to a target transcript to induce poly(A) tail shortening. To further test this model, it will be necessary to map the region of Vts1p that interacts with the deadenylase. The deadenylase interacts with sequences between residues 237-523. I propose to create a series of progressively smaller N- and C-terminal truncation within this region to identify the minimal region sufficient for deadenylase binding. The ability of this minimal binding fragment to induce transcript deadenylation and transcript decay would be tested by fusing it to the Tat RNA-binding motif. Reporter mRNA stability would be measured in transcriptional pulse chase experiments and deadenylation activity will be measured through deadenylation assays as described in Rendl et al, 2008. Briefly, each RNA sample from a transcriptional-pulse chase experiment would be treated with an oligonucleotide that hybridizes ~300 base pairs upstream of the polyadenylation site thus forming a DNA/RNA hybrid that can be cleaved by RNase H. This creates a short RNA fragment which carries the

67 poly(A) tail and can be resolved on a denaturing urea/polyacrylamide gels and the range of poly(A) tail lengths can be measured after northern blot analysis.

Once a minimal region that is sufficient for deadenylation of the reporter is identified, further point mutations will be made within this region with the goal of creating a subtle mutation within Vts1p that abolishes the deadenylase interaction. These deadenylation interaction mutants would be tested for their ability to co-purify with Pop2p in co-immunoprecipitation experiments. Once a deadenylase interaction mutant has been identified, the importance of this interaction can be assayed by measuring the stability and deadenylation of the GFP-3XTAR+ reporter as described above.

5.2.3 Characterization of mRNA degradation by Vts1p1-237

As shown in Figure 4-4, Vts1p 1-237 can induce the degradation of a reporter construct. However, the conserved motif identified within the N-terminus, which is required for repression, is not required for this degradation (Fig 4-7). This indicates that another region of Vts1p 1-237 is capable of inducing degradation of an mRNA transcript. The experiments outlined here, will identify this region and the factors that it interacts with to mediate transcript decay.

An iterative PSI-blast performed on amino acids 1-186 of Vts1p that identified the motif responsible for translational repression also identified three other motifs (Fig 5-1). These motifs are not as well conserved amongst the fungal homologs, but would be good candidates for testing whether mutations to these motifs affect transcript degradation by the N-terminus. In addition, there is a Q rich region within amino acids 1-186. While it is not conserved, it would still be worth testing if it plays a role in transcript decay given the importance of such regions in P body function. These motifs would be individually mutated to alanines in the context of the Vts1p 1-237 TAT-tagged construct and their ability to degrade the GFP-3XTAR+ reporter would be assayed with transcriptional pulse chase experiments similar to those described in Chapters 3 and 4.

Another possibility is that the SSR1 domain is mediating this effect. To test this, a Vts1p 1-186 -Tat construct that lacks the SSR1 domain and a similar construct carrying the lambda c1 repressor dimerization domain will be assayed for their ability to induce the degradation of the GFP 3XTAR+ mRNA. In addition, I would test the ability of Vts1p 1-237 -Tat constructs that are

Figure 5-1 Protein alignment of Vts1p 1-186 An iterative Psi-BLAST of Vts1p 1-186 revealed the conserved 10 amino acid motif highlighted in yellow described in Chapter 4. Three other motifs were identified that are also show conservation and are highlighted in blue and the non-conserved poly Q region is highlighted in green.

defective for dimerization along with the similar mutant constructs carrying the lambda c1 dimerization domain to induce GFP 3XTAR+ mRNA degradation. The results of these experiments could suggest a number of roles for the dimerization domain in transcript decay, including: 1) no role, 2) an indirect role, such as in mRNA binding, that can be rescued by the lambda N dimerization domain, 3) a direct role in transcript decay that is unrelated to dimerization (i.e. transcript decay requires SSR1 but does not require dimerization and 4) a direct role that is related to dimerization (i.e. dimerization mutants show defects in transcript decay that cannot be rescued by the lambda N dimerization domain). If the results suggest options 3 or 4, the structure of the Vts1p SSR1 domain could be modeled from existing crystal structure data from other D domain proteins. The D domain crystal structures from yeast Cdc4p and human β- TrCP1 have been solved and form very similar folds (Tang et al., 2007) and given the similarity of the SSR1 domain sequence to these other proteins, it will likely fold in a similar fashion as well. This model could then be employed to make mutations to expected surface-exposed residues on the SSR1 domain and assess their role in mRNA degradation by measuring the stability of reporter constructs.

Once the regions of Vts1p 1-237 that are involved in transcript decay are identified I would then investigate the molecular mechanisms involved. To do this I would employ immunoprecipitation experiments and mass spectrometry to identify proteins that interact with wild-type Vts1p 1-237 and not to mutant versions of this protein that are defective in mRNA decay.

5.2.4 Translational repression by the N-terminus

I have shown in Section 4.2.8 that the N-terminal motif of Vts1p mediates translational repression of a target transcript. The subsequent experiments will further characterize this mechanism. To test if Vts1p mediates translational repression at the level of initiation, polysome gradients will be employed to determine the amount of reporter mRNA associated with polysome fractions. If the motif is mediating translational repression at the level of initiation, the polysome gradients in cells expressing the Vts1p 8A mutant would show more of the GFP- 3XSRE+ reporter associated with polysomes as compared to wild type cells, which would suggest that N-terminal motif of Vts1p is repressing the translation of target transcripts.

One model for how Vts1p may be repressing translation is that the motif may serve as a binding site for another protein to inhibit translation. Preliminary evidence from immunoprecipitation- LC/MS/MS experiment that compared proteins that co-purified with wild type Vts1p and the Vts1p 8A mutant suggests that Pab1p may interact with the motif. Given Pab1p’s role in enhancing translation initiation, this suggests that Vts1p may inhibit Pab1p activity. To investigate this further, I will first confirm that Vts1p does interact with Pab1p by co- immunoprecipitation experiments. If this interaction is observed, I will repeat the experiments with the Vts1p 8A mutant, to confirm that Pab1p no longer interacts this mutant.

Vts1p’s interaction with Pab1p could result in the displacement of Pab1p from the poly(A) tail thereby repressing translation. To determine whether Pab1p has reduced binding to a poly(A) tail of a Vts1p target transcript, I propose to immunoprecipitate Pab1p and measure the amount of associated GFP-3XTAR+/- reporter transcripts by quantitative real-time PCR (qRT-PCR) in vts1 ∆ cells expressing Vts1p 1-237 -Tat or the 8A mutant version of this protein. If the N terminal motif displaces Pab1p from the mRNA, I would expect a lower amount of GFP-3XTAR+ RNA to co-purify with Pab1p as compared to a GFP-3XTAR- control in a manner that is dependent upon the wild-type motif. This is similar to experiments that have shown that the GW182 interaction with PABP in Drosophila S2 cells results in its release from a transcript that is repressed by miRNAs (Zekri et al., 2013).

Since Vts1p 1-237 also induces transcript decay it is possible that Vts1p induces deadenylation which would also result in Pab1p displacement. To control for this the immunoprecipitation- qRT-PCR experiments could be repeated with a reporter construct that cannot be deadenylated. This reporter will contain an internal poly(A) tail to which Pab1p can bind followed by a hammerhead ribozyme 3’ end that will inhibit deadenylation (Duvel et al., 2002). If Pab1p displacement still occurs with the internal poly(A) construct, this indicates that the motif is not functioning through deadenylation.

5.2.5 Relative contributions of Vts1p-mediated mechanisms to repression

Vts1p utilizes at least three mechanisms to repress target transcripts: recruitment of the Ccr4p/Pop2//Not deadenylase to induce deadenylation (Rendl et al., 2008), recruitment of Eap1p

71 to promote decapping (Rendl et al., 2012), and Vts1p mediates translational repression through the N-terminal motif as described in Chapter 4. To fully understand the role of these mechanisms in Vts1p-mediated repression, it will be necessary to determine the contribution of these mechanisms to repression of a target transcript.

To determine the importance of deadenylation, decapping and translational repression to Vts1p- mediated repression, mutants that specifically disrupt each activity will be assayed for repression individually and also in combination. The creation of the Vts1p deadenylation mutant is described in Section 5.2.2, a Vts1p point mutant has also been made which disrupts Vts1p’s interaction with Eap1 (Vari and Smibert, unpublished results), and finally, the Vts1p 8A mutant can be employed to disrupt translational repression. These mutants can be combined in various configurations and repression of the GFP 3XSRE+ reporter can be determined compared to the GFP 3XSRE- reporter by flow cytometry. Additionally, repression of other reporters such as the GFP nos 1-186+ and existing reporters of bona fide targets such as GFP-NNF1 (Aviv et al., 2006b), GFP-YIR016W (Rendl et al., 2008) that both contain one SRE in their ORF as well as GFP reporters for targets containing two SREs that were described in Section 5.2.1 could also be employed. These reporters would begin to ask the question whether specific mechanisms are required to repress specific transcripts.

The contribution of each mechanism towards Vts1p-mediated repression can also be investigated on a genome wide scale. DNA microarrays can be employed to identify transcripts that increase in expression when Vts1p in mutated. Transcripts levels for each mutant alone or in combination would be compared to a wild type strain that would display full repression and also a vts1 ∆ strain where target transcripts would be completely derepressed. These microarrays would allow identification of genes that require a specific mechanism or combination of mechanisms for repression. Defining features of these various classes of transcripts could then be identified afterward.

Finally to look at translational effects of Vts1p at a genome wide scale SILAC followed by mass spectrometry will be employed. Briefly, yeast cells would be metabolically labeled with different isotopes of lysine and arginine. In this case, wild type cells would be labeled with normal amino acids (light), the mutant Vts1p proteins would be labeled with a medium-heavy amino acid, and a vts1 ∆ strain would be labeled with heavy amino acids. Cells would then be

72 mixed together in equal proportion so that processing is identical for all samples and protein extracts are made. Proteins would be trypsinized and subjects to LC/MS/MS. Identical peptides from each sample can be distinguished because of their mass differences. The ratio of signal intensities for peptide pairs reflects the abundance ratios for the corresponding proteins. Similar to the microarray data, targets in wild-type cells will be fully repressed, while targets in the vts1 ∆ cells derepressed. The cells expressing Vts1p mutants would represent the amount of repression for a specific mechanism or combination of mechanisms, a combination of mRNA decay and translational repression. Translational repression may be identified for targets that don’t display changes at the transcript level by microarray or if the changes at the transcript level don’t correspond to the changes at the protein level.

Alternatively, ribosome profiling could be employed to measure translation genome-wide in cells expressing the mutant Vts1p proteins. Briefly, cells are lysed in the presence of translational inhibitors so that ribosomes remain associated with the mRNA transcripts being translated. A ribosome bound to an mRNA protects a ~30 nucleotide fragment from nuclease digestion and these fragments can be identified through deep-sequencing (Ingolia et al., 2012). The number of sequencing reads from a specific transcript reflects the ribosome occupancy and therefore the translation of that mRNA. One advantage to this approach as compared to SILAC is that ribosome profiling only measures protein synthesis and not protein degradation. Also, ribosome profiling may also reveal some mechanistic details about translational repression by Vts1p. For example, if translational repression by Vts1p results in stalled ribosomes within the ORF, this would be reflected by a large number of sequencing reads at a specific location within the transcript. This approach would be an alternative to purifying polysomes through sedimentation and identify the associated transcripts by microarray, which may not be sensitive enough to detect subtle changes in translation we may see with the Vts1p mutants.

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Appendices

Table A-1 List of Vts1p targets and their SREs from immuniprecipitation/microarray data

a Position represents 5' end of the stem-loop Number of Gene SREs Position a, loop size Location Minimum SRE distance YBR153W 0 YCL027W 0 YDL022W 0 YDR462W 0 YER031C 0 YER081W 0 YER167W 0 YGR057C 0 YGR256W 0 YHR030C 0 YIL074C 0 YIL124W 0 YKR034W 0 YKR045C 0 YML001W 0 YMR152W 0 YNL145W 0 YNL180C 0 YOR101W 0 YBL032W 1 426,7 ORF YBR126C 1 268,4 ORF YBR265W 1 89,4 ORF YDL054C 1 1376,7 ORF YDL133W 1 1269,4 ORF YDL192W 1 589,7 ORF YDL198C 1 1194,4 ORF YDR275W 1 1073,7 3' UTR YDR513W 1 564,6 ORF YEL060C 1 1039,4 ORF YFL045C 1 658,5 ORF YFR017C 1 532,4 ORF YGL187C 1 252,4 ORF YGR068C 1 1181,6 ORF YHR096C 1 669,6 ORF YIR016W 1 266,4 ORF YJL122W 1 191,5 ORF

YJR016C 1 1125,4 ORF YJR112W 1 154,5 ORF YKL185W 1 470,7 ORF YKR058W 1 612,5 ORF YLR019W 1 1735,6 3' UTR YLR220W 1 769,8 ORF YML038C 1 653,7 ORF YMR199W 1 757,8 ORF YNL037C 1 734,7 ORF YNL116W 1 198,4 ORF YOL105C 1 1977,5 ORF YOR047C 1 765,6 ORF YOR125C 1 411,6 ORF YOR359W 1 443,6 ORF YPL031C 1 494,4 ORF YPL061W 1 832,4 ORF YPL168W 1 541,6 ORF YPR056W 1 507,5 ORF YPR084W 1 228,4 ORF YAR015W 2 195,6; 301,8 ORF 106 YBL006C 2 274,5; 392,6 ORF 118 YBR057C 2 73,5; 512,6 ORF 439 YBR129C 2 562,4; 622,5 ORF 60 YCL018W 2 247,7; 1000,4 ORF 753 YCR012W 2 1176,4; 1255,5 ORF 79 YDL209C 2 671,7; 797,4 ORF 126 YDL211C 2 1071,7; 1072,5 ORF 1 YDR342C 2 401,7; 801,4 ORF 400 YEL011W 2 1268,8; 1716,8 ORF 448 YFR014C 2 414,6; 879,8 ORF 465 YFR015C 2 123,5; 1982,7 ORF 1859 YGL003C 2 1028,4; 1424,4 ORF 396 YGL127C 2 233,6; 347,4 ORF 114 YGR046W 2 607,4; 880,4 ORF 273 YGR065C 2 730,5; 981,4 ORF 251 YHR183W 2 598,6; 604,5 ORF 6 YIR017C 2 492,5; 902,7 ORF 410 YKR051W 2 146,7; 1203,8 ORF 1057 YLL024C 2 588,7; 1320,4 ORF 732 YLR177W 2 1798,4; 1657,7 ORF 141 YLR355C 2 476,7; 701,7 ORF 225 YMR105C 2 798,8; 1591,5 ORF 793 YMR235C 2 493,4; 254,6; ORF 239 YMR268C 2 1036,4; 1171,7 ORF 135

YMR272C 2 272,5; 1198,7 ORF 926 YNL052W 2 430,4; 230,7 ORF 200 YOL119C 2 340,8; 629,6 ORF 289 YOR044W 2 196,8; 777,6 ORF 581 YOR324C 2 59,4; 588,6 5' UTR, ORF 529 YPL111W 2 201,4; 920,5 ORF 719 YPL133C 2 955,6; 1212,7 ORF 257 YPR083W 2 84,5; 1721,5 ORF 1637 YAL005C 3 584,7; 1316,4; 1955,4 ORF 639 YBR157C 3 740,7; 993,4; 1117,5; 2 in ORF, 1 spans stop codon 253 YCR065W 3 624,5; 1214,4; 1635,7 ORF 421 YCR075C 3 233,4; 329,6; 777,5 ORF 96 YDL055C 3 277,4; 794,6; 1076,7 ORF 282 YDL245C 3 298,8; 526,8; 1258,7 ORF 228 YEL007W 3 674,6; 1250,7; 1349,4 ORF 576 YEL064C 3 311,6; 401,4; 477,7 ORF 76 YER004W 3 193,4; 390,4; 827,4 ORF 197 YER018C 3 136,5; 416,5; 668,8 ORF 252 YER019W 3 311,8; 1055,4; 1455,5 ORF 400 YER103W 3 1264,4; 1354,7; 1888,4 ORF 90 YER145C 3 288,8; 340,7; 646,4 ORF 52 YGL038C 3 191,6; 655,6; 1285,4 1 in 5' UTR, 2 in ORF 464 YGL225W 3 300,6; 610,6; 892,6 ORF 282 YGR079W 3 271,8; 750,8; 1157,8 ORF 407 YHL028W 3 264,4; 1592,4; 1845,5 ORF 253 YHR092C 3 669,7; 1396,5; 1561,5 ORF 165 YJL084C 3 878,6; 907,5; 5763,8 ORF 29 YKL035W 3 241,5; 1294,7; 1559,4 ORF 265 YKL052C 3 207,7; 234,8; 246,4 ORF 27 YLR081W 3 254,8; 262,7; 1631,7 ORF 8 YML126C 3 231,4; 595,4; 1198,7 ORF 364 YNL053W 3 950,5; 1130,5; 1839,4 2 in ORF, 1 in 3' UTR 180 YOL020W 3 465,6; 1388,5; 1433,6 ORF 923 YOR038C 3 545,6; 1906,4; 2086,5 ORF 180 YOR043W 3 697,6; 916,8; 1070,4 ORF 219 YOR066W 3 505,4; 599,4; 1430,8 1 in 5' UTR*, 2 in ORF 94 YOR157C 3 163,5; 382,4; 727,6 1 spans start codon, 2 in ORF 219 YOR337W 3 199,8; 317,7; 1985,7 ORF 118 YBL058W 4 380,5; 476,7; 934,8; 947,6 ORF 13 YDL240W 4 715,5; 862,4; 1430,5; 2499,4 ORF 147 YER073W 4 905,7; 1124,7; 1643,5; 1742,8 3 in ORF, 1 in 3' UTR 99 YHR018C 4 135,7; 702,7; 1272,5; 1523,7 3 in ORF, 1 in 3' UTR 251 YHR094C 4 118,6; 580,7; 866,4; 1472,5 ORF 286 YJL157C 4 637,4; 817,4; 892,4; 1449,8 ORF 75

YKR093W 4 387,4; 1025,6; 1616,4; 1871,4 ORF 255 YLR310C 4 376,4; 431,4; 1324,5; 2847,4 ORF 55 YMR001C 4 682,7; 1047,6; 1633,5; 2058,7 ORF 365 YMR011W 4 353,4, 539,7, 1425,6, 1491,4 ORF 186 YNL208W 4 298,8; 467,5, 476,5, 548,5 ORF 9 YBL075C 5 605,6; 1860,4; 1961,4; 1994,6; 2138,7 4 in ORF, 1 in 3' UTR 101 YCL036W 5 82,8; 273,5; 1107,5; 1179,7; 1442,5 ORF 16 YER147C 5 640,5; 1229,4; 1252,5; 1425,5; 1541,5 ORF 23 YHR050W 5 517,6; 684,8; 1057,8; 1261,8; 1387,6 ORF 167 YIL123W 5 683,4; 1509,5; 1959,5; 2012,7; 2021,4 1 in 5' UTR, 4 in ORF 9 YIL155C 5 274,6; 470,5; 790,8; 1012,6; 1618,4 ORF 196 YLR304C 5 302,7; 617,4; 775,4; 1630,4; 1924,5 ORF 158 76,8; 379,4; 672,6; 981,4; 1325,4; YBR068C 6 1899,6 5 in ORF, 1 in 3' UTR 303 227,4; 477,5; 1331,8; 1410,7; 1455,8; YCR057C 7 1542,6; 2362,6 ORF 45

* Yassour et al. have identified the coordinates for the 5’ UTR of YOR066W as nucleotides 448863-449436 of chromosome 15, while Nagalakshmi et al. has identified the 5’UTR as nucleotides 449423-449436. There is a 560 nucleotide difference between these datasets and as such, the SRE identified in the 5’ UTR is only present in the longest 5’ UTR.