The DEAD-Box Protein Dhh1p Couples mRNA Decay and Translation by Monitoring Codon Optimality by Aditya Radhakrishnan A dissertation submitted to The Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy. Baltimore, Maryland December, 2016 c Aditya Radhakrishnan 2016 All rights reserved Abstract Recent experimental findings have substantially advanced the notion that the codon- dependent rate of translation elongation is a major determinant of mRNA stability. While the role of the ribosome in identification and decay of aberrant mRNAs has been well established, how the process of translation elongation and mRNA decay communicate is less well understood. Here, we report that the yeast DEAD-box protein Dhh1, long implicated in regulation of translation and activation of mRNA decay, acts as a sensor of codon optimality that targets an mRNA for decay. First, we find that Dhh1 specifically associates with and degrades mRNAs of low codon optimality. We show that messages with greater numbers of slowly translating ri- bosomes are preferential targets for Dhh1 mediated decay. Moreover, we note that these mRNAs are degraded by Dhh1-specific mechanism separate from the standard cellular ribosome quality control apparatus. We find that overexpression of Dhh1 leads to accumulation of ribosomes specifically on mRNAs of poor optimality. We supplement this with high-throughput sequencing analysis to show that Dhh1 over- expression leads to ribosomal stalling on specific non-optimal codons. Taken together ii Abstract iii with the finding that Dhh1 is found to associate with ribosomes in vivo, these data suggest that Dhh1 acts a sensor for translation elongation, efficiently coupling codon optimality with mRNA decay. Rachel Green, Ph. D. (Sponsor and Reader) Professor Department of Molecular Biology and Genetics Johns Hopkins University School of Medicine Je↵ry Corden, Ph. D. (Reader) Professor Department of Molecular Biology and Genetics Johns Hopkins University School of Medicine Acknowledgments To Rachel, my sincerest thanks. The opportunity to work in such an intellectually engaging environment has been nothing short of a blessing. To have a mentor who is both understanding and supportive of my idiosyncrasies, even more so. The training I’ve received and experiences I’ve shared will forever stay with me. Particularly pertaining to presentation slides. To the varied, wonderful people I’ve had the privilege of calling my friends through my time in graduate school. To my academic family in Biophysics, and my adoptive family in BCMB. To the Green lab, both new and old, my thanks for the best set of work colleagues a guy could ask for. To Kristin, my long su↵ering baymate, and Anthony (V), my long su↵ering new baymate. To Chris, who ignited my passion for climbing stu↵, and to Boris who nurtured it. To Nick, who taught me how to make profiling samples, and to Karen, who taught me how to make profiling samples. To Julie and Beth, who ensured the only thing keeping me from getting my work done was me. To Colin and Kazuki, who brightened my days with unexpected snark. To Alan, with whom I could geek out about music theory. To Dan, who seemed to think I actually know what I’m doing at a computer. To Fuad and Jamie and Laura and iv Acknowledgments v Karole, who all have helped me maintain a sense of fun and perspective through the latter stages of my PhD. And to Anthony, who has been through this journey at the same time as me, and who is someone I’m truly lucky to call my friend. To all of you, thank you. The meaningless chats, beer, and jaunts to the Daily Grind have contributed more to the success of the thesis than you realize. To my family. To my new parents, and by new brother and sister-in-law, who endeavor to always make me loved. To all my extended family, ever ready to support and help out. To my parents, whose unhealthy obsession with my well-being and happiness has lead to me completing this PhD — as well as pretty much any other notable accomplishment or success I’ve encountered. And, finally, to my wife, as I’ve saved the best for last. I love you all. Contents Abstract ii Acknowledgments iv List of Tables ix List of Figures x 1 Introduction 1 1.1 Mechanisms of RNA Decay . 2 1.1.1 Deadenylation and exosome-mediated mRNA decay . 3 1.1.2 Decapping-mediated mRNA decay . 4 1.1.3 Decay of aberrant mRNAs . 5 1.2 General connections between translation and mRNA decay . 8 1.2.1 Codon selection informs mRNA stability . 9 1.3 Figures................................... 11 2 Dhh1 represses translation by modulating ribosome occupancy 15 vi Contents vii 2.1 Dhh1 is at the nexus of mRNA decay and translation repression . 16 2.2 Tethering of me31b and orthologs efficiently represses translation . 18 2.3 Both RecA domains of Dhh1 are necessary for translation repression . 19 2.4 Dhh1-tethered mRNA predominantly sequesters ribosomes . 20 2.5 Dhh1-mediated ribosome occupancy is not a termination defect . 23 2.6 Materials and methods . 26 2.7 Figures................................... 34 3 Dhh1 stimulates decay of mRNAs with low codon optimality 42 3.1 Codon optimality underlies efficient translation . 43 3.2 Metrics for codon optimality . 44 3.2.1 CAI and tAI: Supply and demand at the codon level . 46 3.3 Dhh1p stimulates the degradation of mRNAs with low codon optimality 49 3.4 Dhh1p binds preferentially to mRNAs of low codon optimality . 52 3.5 Materials and methods . 54 3.6 Figures................................... 58 4 Dhh1 monitors codon optimality through ribosome elongation 68 4.1 Decay is stimulated by increasing numbers of slow-moving ribosomes 69 4.2 Dhh1p physically binds to the eukaryotic ribosome . 71 4.3 Ribosome occupancy is enhanced upon Dhh1 binding . 72 4.4 Discussion . 73 Contents viii 4.5 Materials and methods . 77 4.6 Figures................................... 81 Bibliography 90 Vita 109 List of Tables 2.1 Characterized functions of Dhh1 and orthologs . 17 2.2 Reporter ribosome profiling samples and GEO sample numbers . 24 3.1 Additional ribosome profiling samples and GEO sample numbers . 43 4.1 Publically available yeast strains generated for this study . 80 ix List of Figures 1.1 There is heterogeneity in the stability of mRNA transcripts . 11 1.2 Canonical mRNA decay in S. cerevisiae ................. 12 1.3 Decay of aberrant mRNAs in S. cerevisiae ............... 13 1.4 Codon optimality vs. mRNA stability in mice and yeast . 14 2.1 Characteristic sequence motifs in Dhh1 . 34 2.2 The crystal structure of Dhh1 . 35 2.3 me31b and Dhh1 repress translation in D. melanogaster ........ 36 2.4 Dhh1 requires both RecA-like domains to repress translation . 37 2.5 Catalytic activity in Dhh1 is required to sediment reporter mRNAs with polyribosomes . 38 2.6 Tethering Dhh1 increases ribosome density on reporter mRNAs . 39 2.7 Nucleotide resolution into ribosome occupancy by ribosome profiling . 40 2.8 Dhh1 does not a↵ect translation termination . 41 3.1 Experimentally observed vs. computationally predicted half-lives. 58 3.2 Codon contributions to mRNA half-life . 59 3.3 Codon e↵ects on mRNA stability is a tunable phenomenon . 60 3.4 Codon composition of HIS3 reporter mRNAs . 61 3.5 Dhh1 selectively stimulates decay of mRNAs with low codon optimality 62 3.6 Dhh1-mediated decay of mRNAs depends on the level of codon optimality 63 3.7 Loss of Dhh1 stabilizes low optimality mRNAs genome wide . 64 3.8 Dhh1-mediated decay is not due to mRNA secondary structure . 65 3.9 Dhh1 preferentially binds with low optimality mRNAs . 66 3.10 Dhh1 associates with low optimality mRNAs genome-wide . 67 4.1 Dhh1 senses polarity of non-optimal codons within mRNAs . 81 4.2 Dhh1-mediated degradation is dependent on inefficient translation . 82 4.3 Dhh1-mediated degradation is dependent on ribosome pausing up- stream of non-optimal stretches . 83 4.4 Canonical RQC proteins do not sense polarity of non-optimal codons 84 4.5 Pull-down of Dhh1 suggests association with the ribosome . 85 x List of Figures xi 4.6 Catalytically active Dhh1 modulates ribosome occupancy on mRNAs with low codon optimality . 86 4.7 A-site occupancy by non-optimal codons is increased on Dhh1 overex- pression .................................. 87 4.8 Dhh1 preferentially sequesters ribosomes on messages with low codon optimality . 88 4.9 Dhh1 is a general sensor of ribosome speed during elongation . 89 Chapter 1 Introduction Note: Parts of this chapter were published in: Radhakrishnan, A. & Green, R. (2016). Connections Underlying Translation and mRNA Stability. J. Mol. Biol. 428 (18), 3558-3564. A coding mRNA lives its life in three distinct phases: birth by transcription, production of protein through translation, and finally death through decay. As the central nexus through which information flows in gene expression, mRNAs play a key role in regulation of gene expression.1 Given that protein synthesis depends on the availability of mRNA,2–4 understanding how cells regulate the availability of mRNA is of paramount importance to understanding gene expression. Specifically, the steady- state level of coding mRNAs is governed by transcription (which, for the purposes of this discussion, we take to include all subsequent processing steps required for efficient export and proper translation) and decay. Eukaryotic transcription is a highly complex event, requiring the concerted ef- 1 1.1. Mechanisms of RNA Decay 2 fort of numerous proteins, subject to multiple levels of spatial and temporal reg- ulation.5 Further sequence-mediated processing events (e. g. 7-methylguanosine capping, intron removal through splicing, 3’ terminal cleavage and polyadenylation) act to regulate the number of mRNA transcripts that are exported from the nucleus to the cytoplasm.