University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2017 Killing The Messenger: Exploring Novel Triggers For Messenger Rna Decay In Eukaryotes Lee Elliott Vandivier University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Bioinformatics Commons, Biology Commons, and the Genetics Commons Recommended Citation Vandivier, Lee Elliott, "Killing The Messenger: Exploring Novel Triggers For Messenger Rna Decay In Eukaryotes" (2017). Publicly Accessible Penn Dissertations. 2621. https://repository.upenn.edu/edissertations/2621 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/2621 For more information, please contact [email protected]. Killing The Messenger: Exploring Novel Triggers For Messenger Rna Decay In Eukaryotes Abstract The lifecycle of messenger RNAs is regulated by multiple layers beyond their primary sequence. In addition to carrying the information for protein synthesis, mRNAs are decorated with RNA binding proteins, marked with covalent chemical modifications, and fold into intricate secondary structures. However, the full set of information encoded by these “epitranscriptomic” layers is only partially understood, and is often only characterized for select transcripts. Thus, it is crucial to develop and apply transcriptome-wide analytical tools to probe the location and functional relevance of epitranscriptome features. In this dissertation, I focus on applying such methods toward better understanding determinants of mRNA stability, through using 1) High Throughput Annotation of Modified Nucleotides, 2) nuclease- mediated probing of RNA secondary structure, and 3) detection of partial mRNA degradation from RNA sequencing. I observe that chemical modifications tend ot mark uncapped and small RNA fragments derived from mRNAs in plants and humans, suggesting a link between modifications and mRNA stability. I then show this link is direct through showing differential stability at Arabidopsis transcripts that change modification status during long-term salt stress. By probing secondary structure, I show a link between structure, smRNA production, and co-translational RNA decay. Finally, I develop a new in silico method to detect partial RNA degradation in mouse oocytes, and identify sequence elements that appear to block complete exonucleolytic transcript cleavage during meiosis. I then identify putative RNA binding proteins that might mediate this partial decay. In summary, I apply transcriptome-wide sequencing-based methods to survey the effects of covalent modifications, secondary structure, and RNA binding proteins on mRNA stability. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Brian D. Gregory Subject Categories Bioinformatics | Biology | Genetics This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/2621 KILLING THE MESSENGER: EXPLORING NOVEL TRIGGERS FOR MESSENGER RNA DECAY IN EUKARYOTES Lee E. Vandivier A DISSERTATION in Cell and Molecular Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2017 Supervisor of Dissertation _________________________ Brian Gregory, Ph.D. Associate Professor of Biology Graduate Group Chairperson _________________________ Dan Kessler, Ph.D. Associate Professor of Cell and Developmental Biology Dissertation Committee Doris Wager, Ph.D. Professor of Biology (Chairperson) R. Scott Poethig, Ph.D. John H. and Margaret B. Fassitt Professor of Biology Zissimos Mourelatos, M.D. Professor of Pathology and Laboratory Medicine Blake Meyers, Ph.D. Professor, Division of Plant Sciences, University of Missouri KILLING THE MESSENGER: EXPLORING NOVEL TRIGGERS FOR MESSENGER RNA DECAY IN EUKARYOTES COPYRIGHT 2017 Lee E. Vandivier This work is licensed under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 License To view a copy of this license, visit https://creativecommons.org/licenses/by-nc-sa/3.0/us/ ii ACKNOWLEDGMENTS Research is both an edifying and humbling experience, and I am grateful to everyone who helped me walk on this exciting but uncertain path. I must first thank my thesis mentor Brian Gregory, who I first encountered as an undergraduate in search of coursework in the natural sciences that I might truly enjoy. I did, decided to give up an English major, and eventually would meet Brian during my graduation ceremony – giving a pitch to my whole family as to why I should rotate in his lab. You have been a consistent supporter of my research, in all its successes and failures. You helped me push beyond my boundaries of expertise and learn genomics and computational biology. I have grown as a scientist under your guidance, and I am happy to have chosen your lab despite, in your words, “the worst rotation in history.” I am equally thankful to the members of Brian’s lab who taught me how to code, analyze big datasets, troubleshoot everything, and set realistic expectations for research. To Ian Silverman and Matthew Willmann, for your guidance at the bench. To Fan Li, Qi Zheng, and Nathan Berkowitz for your patience in teaching a complete novice everything from how to type on a command line to how to develop and version-control custom pipelines. Your help has been invaluable. Likewise, the Gregory lab has grown, and it has been a pleasure to know and learn from Sager Gosai, Shawn Foley, Marianne Kramer, Lucy Shan, Xiang Yu, Steve Anderson, and Zach Anderson. Zach, I am thankful for your endless patience in triple- checking the HAMR pipeline. I have also been fortunate to mentor many talented students, including Jacky Buckley, Nickole Kanyuch, and Rafael Campos. You have taught me invaluable lessons about how to listen, manage, and teach. I must also thank my collaborators, whose expertise has enriched my research and my education. To Paul Ryvkin, Pavel Kuksa, and Li-San Wang, for developing HAMR. To Nur Selamoglu and Fevzi Daldal for all the mass spectrometry and proteomics work. To Richard Schultz and Jun Ma for having helped open my eyes to the world of mouse development, and for trusting me to contribute to your research. I am equally thankful to my committee members. To Scott Poethig and Doris Wagner, for your expertise in plant genetics and for your insights that have helped increase the relevance of my work. To Zissimos Mourelatos, for your expertise in all things RNA, and your encouragement to pursue difficult projects. To Blake Meyers, for helping to bridge the worlds of plant and RNA biology, and for continuing to support my work even after moving to the Danforth Center in St. Louis. And I course, I would be never have made it this far without my parents John and Wendy. Your unconditional love has allowed me to pursue my interests to wherever they take me, and you have given me the passion to learn. To my father, who has taught me patience and kindness and how to cook delicious food. To my mother, who has taught me with your sharp mind and artist’s eye and activist’s clarity. To my Grandmom and Zaydee, who have taught me diligence and the joy of eating a second dinner every night. To my Grandpa Van, whose intellectual spirit has inspired me. Most of all, I must thank my wife and true love, Minghua Hsiao. I have learned more from you than from all my years in school. You have supported me through every high and every low of graduate school. Your love is fearless, and I admire your courage as we live across nations. I am honored to live my life with you. iii ABSTRACT KILLING THE MESSENGER: EXPLORING NOVEL TRIGGERS FOR MESSENGER RNA DECAY IN EUKARYOTES Lee E. Vandivier Brian D. Gregory The lifecycle of messenger RNAs is regulated by multiple layers beyond their primary sequence. In addition to carrying the information for protein synthesis, mRNAs are decorated with RNA binding proteins, marked with covalent chemical modifications, and fold into intricate secondary structures. However, the full set of information encoded by these “epitranscriptomic” layers is only partially understood, and is often only characterized for select transcripts. Thus, it is crucial to develop and apply transcriptome-wide analytical tools to probe the location and functional relevance of epitranscriptome features. In this dissertation, I focus on applying such methods toward better understanding determinants of mRNA stability, through using 1) High Throughput Annotation of Modified Nucleotides, 2) nuclease-mediated probing of RNA secondary structure, and 3) detection of partial mRNA degradation from RNA sequencing. I observe that chemical modifications tend to mark uncapped and small RNA fragments derived from mRNAs in plants and humans, suggesting a link between modifications and mRNA stability. I then show this link is direct through showing differential stability at Arabidopsis transcripts that change modification status during long-term salt stress. By probing secondary structure, I show a link between structure, smRNA production, and co-translational RNA decay. Finally, I develop a new in silico method to detect partial RNA degradation in mouse oocytes, and identify sequence elements that appear to block complete exonucleolytic transcript cleavage during meiosis. I then identify putative RNA binding proteins that might mediate this partial decay. In summary, I apply transcriptome-wide sequencing-based methods to survey