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The Dynamic Fate of the Exon Junction Complex

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The Dynamic Fate of the Exon Junction Complex The Dynamic Fate of the Exon Junction Complex Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Robert Dennison Patton, B.S. Graduate Program in Physics The Ohio State University 2020 Dissertation Committee Dr. Ralf Bundschuh, Advisor Dr. Guramrit Singh, Co-Advisor Dr. Michael Poirier Dr. Enam Chowdhury 1 © Copyrighted by Robert Dennison Patton 2020 2 Abstract The Exon Junction Complex, or EJC, is a group of proteins deposited on mRNA upstream of exon-exon junctions during splicing, and which stays with the mRNA up until translation. It consists of a trimeric core made up of EIF4A3, Y14, and MAGOH, and serves as a binding platform for a multitude of peripheral proteins. As a lifelong partner of the mRNA the EJC influences almost every step of post-transcriptional mRNA regulation, including splicing, packaging, transport, translation, and Nonsense-Mediated Decay (NMD). In Chapter 2 I show that the EJC exists in two distinct complexes, one containing CASC3, and the other RNPS1. These complexes are localized to the cytoplasm and nucleus, respectively, and a new model is proposed wherein the EJC begins its life post- splicing bound by RNPS1, which at some point before translation in the cytoplasm is exchanged for CASC3. These alternate complexes also take on distinct roles; RNPS1- EJCs help form a compact mRNA structure for easier transport and make the mRNA more susceptible to NMD. CASC3-EJCs, on the other hand, cause a more open mRNA configuration and stabilize it against NMD. Following the work with the two alternate EJCs, in Chapter 3 I examine why previous research only found the CASC3-EJC variant. Using the EJC as a case study which contains both a directly binding peripheral protein (CASC3) and indirectly binding peripheral protein (RNPS1), I show that CLIP-Seq, a photo crosslinking method widely used to study RNA Binding Proteins (RBPs) is ill suited to researching proteins which do not make direct contact with RNA, or otherwise photo crosslink poorly (RBP-Associated iii Factors, or RAFs). I propose that chemical crosslinking methods such as RIPiT-Seq be used instead when aiming to study RAFs. Finally, in Chapter 4 I consider another EJC peripheral protein, PYM, thought to aid in disassembly of the EJC co-transcriptionally. By inhibiting translation as well as PYM-EJC binding through a mutation on MAGOH, PYM is found to be unnecessary for EJC removal but does inhibit non-canonically located EJCs. I lay the groundwork for future work regarding PYM and NMD by creating a new NMD target transcript list which gives promising results when examining data from a previous study on NMD. iv Dedication I stand at the seashore, alone, and start to think. There are the rushing waves, mountains of molecules Each stupidly minding its own business Trillions apart, yet forming white surf in unison Ages on ages, before any eyes could see Year after year, thunderously pounding the shore as now For whom, for what? On a dead planet, with no life to entertain Never at rest, tortured by energy Wasted prodigiously by the sun, poured into space A mite makes the sea roar Deep in the sea, all molecules repeat the patterns Of one another till complex new ones are formed They make others like themselves And a new dance starts Growing in size and complexity Living things, masses of atoms, DNA, protein Dancing a pattern ever more intricate Out of the cradle onto the dry land Here it is standing Atoms with consciousness, matter with curiosity Stands at the sea, wonders at wondering I, a universe of atoms An atom in the universe - Richard P. Feynman v Acknowledgments First and foremost, I would like to thank my advisor Dr. Ralf Bundschuh and co- advisor Dr. Guramrit Singh. Ralf has been an incredible mentor and his guidance has helped me to grow as scientist more than I ever could have imagined. And though Amrit works in another department, he has always made me feel like one of his own and a true member of his group. Most of all I am thankful to both of them for their incredible patience. As someone from a pure physics background coming abruptly into the world of molecular genetics there are many mistakes to be made, and I have made them all at least twice, but my advisors have been kind and understanding teachers through the entire process. I would also like to thank my committee members Dr. Michael Poirier and Dr. Enam Chowdhury. Michael, whose group we share group meetings with, has played a prominent role throughout my graduate research career. Enam similarly played a large role as one of my undergraduate research collaborators and mentors, back when I still did AMO. And I would like to thank my group members who preceded me, Dr. Billy Baez and Dr. Blythe Moreland, who were incredibly helpful in every conceivable way when I was a fresh graduate student. My current group members Elan Shatoff and Kyle Crocker have also been indispensable companions. Of course, I must also thank my collaborators in the Singh lab, especially Dr. Lauren Woodward, Justin Mabin, and Zhongxia Yi, who are responsible for nearly all of the experimental work done in bringing this thesis to fruition. vi Unfortunately, I cannot remember the name of every teacher I ever had, but I feel indebted to every person who has poured some knowledge into this brain. I am especially thankful for some of my undergraduate physics professors who played a large part in shaping my goals and interests, especially Dr. Robert Perry and Dr. Thomas Lemberger. And of course, my undergraduate advisor at OSU, Dr. Gregory Lafyatis, who was not only a gracious advisor but gave me my first experiences traversing a more biological realm. Finally, I would like to thank my mother, Jennifer and father, Bob for instilling in me the importance of curiosity from a young age, and always encouraging me towards lofty aspirations. And I would not be who I am today without my elder siblings Sara, Eric, and Courtney who only complained a little bit when I made them watch NOVA or took their books about science and physics without asking. vii Vita The Ohio State University Columbus, Ohio Bachelor of Science in Physics; Minor in Japanese May 2015 Thesis: "Optimization of Direct-Write 3D Photolithography in PMMA” Publications 1. Patton, R., Sanjeev, M., Woodward, L., Mabin, J., Bundschuh, R., & Singh, G. (2020). Chemical crosslinking enhances RNA immunoprecipitation for efficient identification of binding sites of proteins that photo-crosslink poorly with RNA. RNA, https://doi.org/10.1261/rna.074856.120 2. Gangras, P., Gallagher, T., Patton, R., Yi, Z., Parthun, M., Tietz, K., Deans, N., Bundschuh, R., Amacher, S., & Singh, G. (2020). Zebrafish rbm8a and magoh mutants reveal EJC developmental functions and suggest new rules for 3′UTR intron-dependent NMD. PLOS Genetics, https://doi.org/10.1101/677666 3. Mabin, J.*, Woodward, L.*, Patton, R.*, Yi, Z., Jia, M., Wysocki, V., Bundschuh, R., & Singh, G. (2018). The exon junction complex undergoes a compositional switch that alters mRNP structure and nonsense-mediated mRNA decay activity. Cell Rep., 25, 2431-2446. [* co-first authors] Fields of Study Major Field: Physics viii Table of Contents Abstract .............................................................................................................................. iii Dedication ........................................................................................................................... v Acknowledgments.............................................................................................................. vi Vita ................................................................................................................................... viii Table of Contents ............................................................................................................... ix List of Figures ................................................................................................................... xii Chapter 1 Introduction ........................................................................................................ 1 1.1 The Central Dogma ................................................................................................... 1 1.1.1 Physical structure of nucleic acids ..................................................................... 4 1.1.2 Physical structure of proteins ............................................................................. 6 1.1.3 The physics of RNA:Protein interactions .......................................................... 8 1.2 mRNA Binding Proteins and the Exon Junction Complex ..................................... 12 1.2.1 Splicing and the birth of mRNA ...................................................................... 13 1.2.2 The Exon Junction Complex ............................................................................ 15 1.2.3 EJC peripheral proteins .................................................................................... 18 1.2.4 Canonical EJCs ................................................................................................ 20 1.2.5 Non-Canonical EJCs ........................................................................................ 20 1.2.6 EJCs influence mRNP structure ....................................................................... 22 1.2.7 EJCs and Nonsense-Mediated Decay .............................................................. 22 1.3 Biophysics and
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