Mechanism of Repression by the Sequence-Specific mRNA-Binding Protein Vts1p by Melissa Alice Bieman A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Biochemistry University of Toronto © Copyright by Melissa A. Bieman 2014 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. ii 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. iii 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. iv 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 v 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 vi 3.3 Discussion ......................................................................................................................... 46 Chapter 4 The N-terminus of Vts1p mediates translational repression ........................................ 49 4.1