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DORAZIO-DISSERTATION-2020.Pdf (6.986Mb) CHARACTERIZING MOLECULAR PATHWAYS THAT RESCUE STALLED RIBOSOMES AND DECAY PROBLEMATIC MESSENGER RNAS by Karole Nicole D’Orazio A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland January 2020 Abstract Translation through problematic sequences in mRNAs leads to ribosome collisions that trigger a collection of quality control events including ribosome rescue, degradation of the stalled nascent polypeptide via the Ribosome- mediated Quality control Complex (RQC), and targeting of the mRNA for decay (No Go Decay or NGD). Using reverse genetic screens in yeast, we identify Cue2 as the key endonuclease that is recruited to stalled ribosomes to promote NGD. Following Cue2-mediated cleavage, ribosomes upstream of the cleavage site translate to the end of the truncated mRNA and are rescued by the Dom34:Hbs1 complex. We also show that the putative helicase Slh1 reduces ribosome occupancy on intact problematic mRNAs and thereby reduces endonucleolytic cleavage by Cue2. The synergistic activities of Cue2 and Slh1 define two parallel pathways that allow cells to recognize and respond to ribosomes trapped on problematic mRNAs. From the same study in yeast, we also identify a set of novel candidate genes that act on NGD-substrates. In preliminary experiments we note that these factors are involved in many different processes involving translation elongation, deubiquitination processes, and mRNA decay and future work will further characterize these genes. ii Rachel Green (Sponsor and reader), Professor, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine Brendan Cormack (Reader), Professor, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine iii Preface Acknowledgments When I think about the people that gave me the chance to become a scientist, before I knew what being a scientist meant, I think of Dr. Ed Luk. He was a new professor at SUNY Stony Brook when I joined his lab and his patience, knowledge of biochemistry, mentorship skills, and incredible positivity and determination set the stones for my path to Johns Hopkins University. He helped me get into the graduate school of my dreams and has given me motivation all along the way. It has been an incredible honor to train under the advisory of Dr. Rachel Green in the Molecular Biology and Genetics Department at Johns Hopkins University. Rachel’s passion for science and the enjoyment that she gets out of our work is a driving force for everyone in her lab. I want to thank her for sharing her brilliance with me and for sending me all over the world to carry out my experiments. She forced me to push the boundaries of my comfort zone, setting no limits on the type of science I could do or type of scientist I could be. She taught me to be fearless in both science and in life and I will be forever grateful for that. Rachel also promoted my work and me in a way that I never thought anyone would, giving me the confidence I needed to pursue a career in science and I will never forget that. Thank you to my incredible lab members, past and present, who have taught me so much about how to be a scientist. Thank you specifically to Boris Zinshteyn for teaching me to question everything, Laura Lessen for teaching me iv mental strength, Daniel Goldman for teaching me patience, and Colin Wu for teaching me determination. I am incredibly lucky to have such a dedicated and supportive thesis committee. Thank you to Dr. Scott Bailey for clearly having my back through every thesis committee meeting, Dr. Brendan Cormack for spending countless hours helping me with genetics and analyzing pathways, Dr. Carol Greider for treating me like her own lab member and Dr. Geraldine Seydoux for giving me a boost whenever I needed help with science or life decisions. They have all gone above and beyond to help me throughout my time here. Thank you to Dr. Grant Brown as well for teaching me how to be a yeast geneticist and for trusting me to come to his lab and perform endless genetic screens. My friends were there to go through the uncertainties and disappointments that come along with graduate school and to celebrate the successes that I never thought I would accomplish. Thank you to my friends in Baltimore: the ones who have been there since day one - Chris Cho, Kayarash Karimian, Joyce Lee, and Chirag Vasavda; the ladies who lunch - Meiling May and Miriam Akeju; and the others I can always count on - Allison Daitch, Byron Ho, Maxime Cheve, and Ryan McQuillen. Thank you to my friends on Long Island who bring so much happiness and balance to my life – Melissa Milhim, Rachael Patane, Nicole Tollinchi, and Jessie Vitrano. Thank you to the Clarke and Zuniga families for supporting me like family. v Thank you to my person, Jessica Zuniga. You help me through everything in life and I have no idea what I would do without you. My ride or die, number one AP fan. Most of all thank you to my parents Dominic and Carol D’Orazio. You provide me with never-ending support and the comfort that, wherever life takes me, I will always have my family by my side. Thank you to my extended family for providing me so much guidance, especially my cousin John for being my traveling partner and my Grandma Ann for being the strongest woman I know. Thank you to my brothers and sisters and all of their children – Ann, Ryan, Justin, Noah, Lucas and Avery Dumond; Max, Kristen, Max Jr, Anthony and Alivia D’Orazio; Dominic, Michele, Nikki, and Isabel D’Orazio; Liz, Troy, Troy Jr., Dylan, and Evan Silva; Vito, Angela, Dominic and Angelena D’Orazio; Andrea D’Orazio, Triny Alarcon, Liliana Alarcon, and Leo Alarcon; and Brianna D’Orazio – for the love and encouragement you have given me and for being the most fun group of people to go through life with. vi Dedication I would like to dedicate this dissertation to Rosanna D’Orazio and our parents Dominic and Carol D’Orazio, Colleen Clarke and her parents Kevin and Mary Clarke, and Leo Alarcon and his parents Trinidad Alarcon and Andrea D’Orazio. Thank you for motivating me to pursue a career research with the hopes of giving sick children and their families an easier road to travel. vii Table of Contents Abstract…………………………………………………………………………………...ii Acknowledgments…………………………………………………………………..…..iv List of Tables.……………………………………………………………………………xi List of Figures…………………………………………………………………………..xii Chapter I: Introduction……………………………………….………… ..........……….1 1.1 Translation dependent control of cellular mRNA levels…….…………..2 1.2 Ribosome signaling decay of problematic mRNAs……………………..5 1.2.1 No Go Decay…………………….………………………………5 1.2.2 Non-Stop Decay…………………….…………………………..7 1.3 Thesis contributions…………………….…………………….…………….9 1.3.1 The endonuclease Cue2 cleaves mRNAs at stalled ribosomes during No Go Decay……………………………….9 1.3.2 Deletion screens for factors involved in mRNA decay and translation suppression in response to ribosome stalling…..9 Chapter II: The endonuclease Cue2 cleaves mRNAs at stalled ribosomes during No Go Decay…………………….…………………….…………………….………..10 2.1 Introduction…………………….…………………….………………….…12 2.2 Results…………………….…………………….…………………….……16 2.2.1 Screening for factors involved in NGD……….……………….16 2.2.2 CUE2 domain structure and homology modeling……….…..18 2.2.3 Characterizing roles of CUE2 in NGD in vivo……….……….20 viii 2.2.4 Contribution of Cue2 to NGD is increased in specific genetic backgrounds…..………………….……….……………………………21 2.2.5 Ribosome profiling provides high-resolution view of Cue2 cleavage sites…………………..……………………………………...23 2.2.6 In vitro reconstitution of Cue2 cleavage on isolated colliding ribosomes……….…………….……….……………………………….27 2.2.7 Ribosome profiling provides evidence that Slh1 inhibits ribosome accumulation on problematic mRNAs……….……..……29 2.2.8 Genome-wide exploration of endogenous mRNA substrates of Cue2 and Slh1……….…………………….…………..……………30 2.3 Discussion…………………….…………………….……………………..31 Chapter III: Deletion screens for factors involved in mRNA decay and translation suppression in response to ribosome stalling…………………….………………...75 3.1 Introduction…………………….…………………….………………….…76 3.2 Results…………………….…………………….…………………….……76 3.2.1 Deletion screens to identify factors involved in mRNA decay and translation repression/activation on NGD substrates…………76 3.2.2 Temperature sensitive mutant screens……….………………78 3.2.3 Deletion screen candidates……….…………………….……..78 3.2.4 Transfer RNA modification genes are drastically enriched in NGD screens……….…………………….……….……………….…..78 3.2.5 Translation repressor GIGYF2 homologs Smy2 and Syh1 act on NGD mRNAs……….…………………….……….………………..79 3.2.6 Ribosome occupancy and mRNA decay……….………….…80 ix 3.2.7 Deubiquitinases involved in NGD……….………………….…82 3.3 Discussion…………………….…………………….……………………..83 Chapter IV: Materials and Methods…………………….…………………….….…117 Chapter V: Conclusion…………………….…………………….…………………..132 References…………………….…………………….……………………….……….135 Appendices…………………………………………………………………...135 Bibliography…..………………………………………………………………136 Biographical Statement…………………….…………………….……………….…151 x List of Tables Table 1: OPT reporter OE screen……….…………………….……….……….……57 Table 2: NGD-CGA reporter OE screen……….…………………….……….……..63 Table 3: NGD-AAA reporter OE screen……….…………………….……….……..69 Table 4: OPT reporter deletion screen……….…………………….……….………93 Table 5: NGD-CGA reporter deletion screen……….…………………….………...98 Table 6: NGD-AAA reporter deletion screen……….…………………….……….103 Table 7: OPT reporter temperature sensitive screen……….…………………....109 Table 8: NGD-CGA
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