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RNA Regulation in the Nervous System: Circrna Expression University of Nevada, Reno RNA regulation in the nervous system: CircRNA expression changes during aging, and function of the Calm1 extended 3′ UTR isoform. A Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Cellular and Molecular Biology by Hannah N. Gruner Dr. Pedro Miura/Advisor August, 2018 THE GRADUATE SCHOOL We recommend that the dissertation prepared under our supervision by Hannah N. Gruner be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Pedro Miura, Ph. D., Advisor Bradley Ferguson, Ph. D., Committee Member Wei Yan, Ph. D., Committee Member Grant Mastick, Ph. D., Committee Member Sue Liebman, Ph. D., Graduate School Representative David W. Zeh, Ph. D., Dean, Graduate School August, 2018 i Abstract Post-transcriptional regulation provides opportunities to affect RNA dynamics after transcription. As most cells within an organism have the same genome, regulation of RNA plays an important role in creating, and maintaining, different cell types such as epithelial cells and neurons. The nervous system is a complicated structure with a correspondingly complex transcriptome, composed of both coding and non-coding RNAs. One type of RNA adding to the complex neural transcriptome are circular RNAs (circRNAs). These enigmatic transcripts are found in many organisms, ranging from prokaryotes to humans, but their prevalence was not recognized until recently. CircRNAs are stable, generated from protein coding genes via splicing, and regulate cellular processes post-transcriptionally. We found that hundreds of circRNAs accumulated in the aging mouse brain, but not in the heart. As this trend was specific for the nervous system, we speculate that circRNAs are regulated by neural factors, and may play a functional role in the brain. Another component of the transcriptome are messenger RNAs (mRNAs), which are often regulated by cis- and trans-factors acting on the 3′ UTR. Sequences contained within the 3′ UTR can influence translation, stability, or localization of a given mRNA. Through the process of alternative cleavage and polyadenylation (APA), an individual gene can make more than one possible 3′ UTR, including short and long 3′ UTR isoforms. The brain is particularly enriched for long 3′ UTRs compared to other tissues, however there have been few in vivo examples demonstrating the function of long 3′ UTRs. To better understand what role these long 3′ UTRs play in the nervous system, we focused on one example, Calmodulin1. Using CRISPR/Cas9 genome editing we created an allelic series of deletion mutants targeting the long 3′ UTR of Calm1 in mice. We found the extended 3′ UTR is important for proper development of several neural populations, including the dorsal root ganglion (DRG) and ii the vagus nerve. Together, these studies have found new trends and roles for neurally enriched RNAs. Our work showed circRNA expression levels are elevated in the aging mouse brain, which may suggest circRNAs play a role in age-related decline of the nervous system. Additionally, we found an in vivo role for a long 3′ UTR isoform in the developing brain. Our study demonstrates the importance of studying long 3′ UTRs in vivo, and indicates that further research into the in vivo functions of APA events in the nervous system are warranted. iii Acknowledgements Thanks to the past and current members of the Miura lab; with special thanks to Bongmin Bae, Mariela Cortés-López, Maebh Lynch, Vicente Gapuz III, Henry Ng, Daphne Cooper, Ryan Peterson, Kevin So, and of course Dr. Miura. Thanks to Grant Mastick, Farnaz Shoja-Taheri, Minkyung Kim, and Katie Weller for the introduction to lab work and coincident support. Thanks to my husband, Matt Gruner, for teaching me how to change the filter on a serological pipette after I accidently sucked liquid into it multiple times, on a weekend, as an undergraduate. iv Table of Contents Table of Contents .......................................................................................................... iv List of Figures ............................................................................................................... vii List of Tables ................................................................................................................. ix List of Abbreviations...................................................................................................... ix 1. INTRODUCTION .................................................................................................................. 1 BIOGENESIS OF RNA ..................................................................................................... 1 CO-TRANSCRIPTIONAL PROCESSING OF RNA .................................................................... 1 1.2.1. 5′ END CAPPING ......................................................................................................... 1 1.2.2. SPLICING .................................................................................................................. 2 1.2.3. CLEAVAGE AND POLYADENYLATION ............................................................................. 6 SEQUENCING RNA ........................................................................................................ 9 1.3.1. RNA DETECTION AND EXPRESSION ANALYSIS USING RNA-SEQ ..................................... 10 1.3.2. ANALYSIS OF RNA-SEQ DATA .................................................................................... 12 THE NERVOUS SYSTEM TRANSCRIPTOME ........................................................................ 16 CIRCULAR RNAS ......................................................................................................... 19 1.5.1. DETECTION AND VALIDATION OF CIRCRNAS ................................................................ 20 ANNOTATION OF CIRCRNAS FROM RNA-SEQ DATA...................................................... 22 CONFIRMATION OF CIRCRNAS .................................................................................. 23 1.5.2. CIRCRNA BIOGENESIS ............................................................................................. 24 CIRCRNA BIOGENESIS MEDIATED VIA INTRON ELEMENTS .............................................. 25 DOUBLE-LARIAT MEDIATED BACK-SPLICING ................................................................. 27 RBP REGULATION OF CIRCRNA BIOGENESIS .............................................................. 29 FUNCTIONS OF CIRCRNAS ............................................................................................ 30 CIRCRNAS IN THE NERVOUS SYSTEM............................................................................. 34 1.7.1. CIRCRNAS AND SYNAPSES ....................................................................................... 35 v 1.7.2. NEURAL FUNCTIONS OF CIRCRNAS ............................................................................ 36 AGING AND CIRCRNAS ................................................................................................. 37 POST-TRANSCRIPTIONAL REGULATION VIA 3′ UTRS ......................................................... 41 1.9.1. MIRNA—3′ UTR POST-TRANSCRIPTIONAL REGULATION ............................................... 41 1.9.2. 3′ UTR—RBP REGULATION ...................................................................................... 42 1.9.3. STRUCTURAL ELEMENTS WITHIN 3′ UTRS ................................................................... 44 ALTERNATIVE CLEAVAGE AND POLYADENYLATION ........................................................... 45 PREVALENCE AND TRENDS OF APA ............................................................................... 46 LONG 3′ UTRS IN THE NERVOUS SYSTEM ........................................................................ 47 1.12.1. GENOME WIDE NEURAL APA TRENDS ......................................................................... 47 1.12.2. FUNCTION OF NEURAL 3′ UTR LENGTHENING .............................................................. 51 RNA REGULATION IN AXON GUIDANCE ............................................................................ 53 1.13.1. POST-TRANSCRIPTIONAL REGULATION WITHIN NEURITES .............................................. 55 1.13.2. LOCAL TRANSLATION WITHIN GROWTH CONES ............................................................. 55 1.13.3. SUBCELLULAR LOCALIZATION OF TRANSCRIPTS IN AXONS ............................................. 56 1.13.4. LONG 3′ UTRS AND SUBCELLULAR LOCALIZATION ........................................................ 57 NEURAL DEVELOPMENT AND EXTENDED 3′ UTRS ............................................................ 60 1.14.1. CALCIUM AND CALMODULIN IN AXON GUIDANCE............................................................ 60 THE ROLE OF CALM1 IN NEURAL DEVELOPMENT ....................................................... 61 2. STATEMENT OF PROBLEM, HYPOTHESES, AND OBJECTIVES ................................................... 63 3. CIRCRNA ACCUMULATION IN THE AGING MOUSE BRAIN ........................................................ 65 ABSTRACT ............................................................................................................... 66 INTRODUCTION ....................................................................................................... 66 RESULTS .................................................................................................................. 69 3.3.1. MAPPING CIRCRNAS
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