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IN VIVO ANALYSIS of METAZOAN TRNA INTRON SPLICING Casey IN VIVO ANALYSIS OF METAZOAN TRNA INTRON SPLICING Casey Alexandra Schmidt A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum in Genetics and Molecular Biology in the School of Medicine. Chapel Hill 2019 Approved by: Robert Duronio Amy Maddox William Marzluff A. Gregory Matera Mark Peifer ©2019 Casey Alexandra Schmidt ALL RIGHTS RESERVED ii ABSTRACT Casey Alexandra Schmidt: In vivo analysis of metazoan tRNA intron splicing (Under the direction of A. Gregory Matera) Pre-tRNA processing is an essential step in generating a supply of functional mature tRNAs. In some instances, this processing event includes removal of an intron. Recent work from our lab has shown that these introns are cut out of pre-tRNAs and ligated into circular RNAs, called tricRNAs, in the fruit fly Drosophila melanogaster. To study the mechanism of tricRNA biogenesis in Drosophila, I generated a series of splicing reporters adapted for expression in human and fly cells. Using these reporters, I discovered specific cis-acting elements important for proper spicing, including a conserved base pair found in all metazoan pre-tRNAs to date. I also identified candidate tRNA processing factors in Drosophila via sequence homology to human factors, and verified these candidates using an in vivo cellular splicing assay. My results show that Drosophila use the “direct ligation”-type tRNA ligation pathway also found in archaea and human cells. I also examined Drosophila tRNA processing factors in animals. Using available stocks, I observed striking neurological phenotypes when mutating or depleting these enzymes. These data correlate with a known human disease called Pontocerebellar hypoplasia (PCH), caused by mutations in human tRNA processing factors. I further found a tissue-specific requirement for the CLP1 ortholog cbc in both viability and locomotion, consistent with previous data from a mouse model. In addition to my cellular and animal work, I also developed a method to express circular RNAs using tRNA splicing. This method utilizes standard molecular biology iii techniques and can generate circular RNAs for a variety of functions. There are many potential applications to this technology. My work is the first characterization of tricRNA splicing in a metazoan model organism. My analysis of cis-acting elements and trans-acting factors yielded findings that are consistent with previously published data. Furthermore, the method I have developed has a wide range of potential uses, and my analysis of processing factor mutants provides new context to understanding a human neurological disease. Overall, my investigation of tricRNA biogenesis adds a new dimension to an established field. iv To my parents, my brother Jeff, and my dear friends Caitlin and Amanda v ACKNOWLEDGEMENTS First and foremost I want to thank my parents. They always encouraged me to pursue my passion for science even though they were unfamiliar with the field. They have been an incredible source of support during my years in graduate school and helped me get through some of the most difficult times, never doubting my ability to finish even though I sometimes doubted myself. I also wish to thank my brother Jeff. Somehow he has always been able to make me laugh, no matter what my mood. Phone calls and text messages shared with him were always a source of joy. Living near my grandmother for the first time in my adult life has been a treat; I appreciate the many lunches and dinners we’ve shared, and I’ve truly enjoyed building a relationship with her. I am lucky to have an amazing family. I next want to thank my advisor Greg for helping me develop into the scientist I am today. I have always enjoyed our scientific conversations, and I appreciate the writing and manuscript-reviewing opportunities Greg gave me. I am an independent researcher and a much better scientific writer because of him. Furthermore, members of my committee have been invaluable to my growth as a scientist. Dr. Amy Maddox, Dr. Bob Duronio, Dr. Bill Marzluff, and Dr. Mark Peifer have all given me good advice over the years and collectively they have guided me toward a project that I’m very proud of. I am especially grateful to my labmates, past and present. They have given me scientific opportunities, listened to my rants, celebrated my high points with me, and pulled me up when I was feeling low. Thank you for helping me get through graduate school. vi Finally, I owe sincere thanks to my incredible friends. Their love and support have meant the most to me during this long and challenging period of my life. Thank you for being wonderful people. vii TABLE OF CONTENTS LIST OF FIGURES ............................................................................................... xii LIST OF TABLES ................................................................................................ xiv CHAPTER 1: INTRODUCTION ............................................................................. 1 Introns are a feature of certain tRNA genes ................................................................... 1 tRNA intron removal requires specific cis-acting elements and trans-acting factors ...... 4 Cis-acting elements important for tRNA splicing ........................................................ 4 Trans-acting factors involved in tRNA splicing ........................................................... 7 Fate of tRNA introns ..................................................................................................... 11 Direct ligation pathway ............................................................................................. 11 Healing and sealing pathway .................................................................................... 12 Function of tRNA introns .............................................................................................. 13 Connecting tRNA splicing to human disease ............................................................... 14 Identifying new regulators of tRNA processing ............................................................ 16 Summary ...................................................................................................................... 18 CHAPTER 2: A METHOD FOR EXPRESSING AND IMAGING ABUNDANT, STABLE, CIRCULAR RNAS IN VIVO USING TRNA SPLICING ......................... 19 Introduction ................................................................................................................... 19 Circular RNAs ........................................................................................................... 19 Engineering and imaging circRNAs .......................................................................... 20 Design and generation of tricRNA vectors ................................................................... 21 Isolation of parental tRNA gene ................................................................................ 22 Notes on the vector .................................................................................................. 22 viii Mutagenesis of parental tRNA gene ......................................................................... 25 Notes on choosing restriction sites ........................................................................... 26 Addition of external promoters .................................................................................. 28 Notes on pol III promoters ........................................................................................ 28 Notes on cloning ....................................................................................................... 28 In vivo expression of tricRNAs ..................................................................................... 31 Transfection .............................................................................................................. 32 Notes on cell line ...................................................................................................... 32 Notes on transfection reagent .................................................................................. 32 RNA isolation ............................................................................................................ 34 Analysis of products ................................................................................................. 34 Northern blotting ....................................................................................................... 34 RT-PCR .................................................................................................................... 35 Sequencing ............................................................................................................... 36 In-gel imaging of tricRNAs ............................................................................................ 36 Cellular imaging of tricRNAs ........................................................................................ 42 Concluding remarks ..................................................................................................... 44 Acknowledgements ...................................................................................................... 45 CHAPTER 3: MOLECULAR DETERMINANTS OF METAZOAN TRICRNA BIOGENESIS ...................................................................................... 46 INTRODUCTION .........................................................................................................
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