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UNIVERSITY of CALIFORNIA, SAN DIEGO the Role of Nonsense

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UNIVERSITY of CALIFORNIA, SAN DIEGO the Role of Nonsense UNIVERSITY OF CALIFORNIA, SAN DIEGO The Role of Nonsense-mediated RNA Decay in Arsenic Toxicity A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Neurosciences by Alexandra E. Goetz Committee in Charge: Professor Miles Wilkinson, Chair Professor Nicola Allen Professor Jonathan Lin Professor Jens Lykke-Andersen Professor Pamela Mellon 2018 Signature Page The Dissertation of Alexandra E. Goetz is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair University of California, San Diego 2018 iii DEDICATION Dedication This work would not have been possible without the support of my family members- my mother, my father, and my brother Matt. Their support to follow my passions, even when the going got tough, helped me finish graduate school, lab change and all. I also want to dedicate this to my husband, Nadav, who was the best shoulder to cry on, cheerleader, and partner to celebrate with through graduate school. iv TABLE OF CONTENTS Table of Contents Signature Page .......................................................................................................................... iii Dedication .................................................................................................................................. iv Table of Contents ........................................................................................................................ v List of Abbreviations ................................................................................................................. viii List of Figures ............................................................................................................................ ix List of Tables ............................................................................................................................. xi Acknowledgements ................................................................................................................... xii Vita .......................................................................................................................................... xiv Abstract of the Dissertation ...................................................................................................... xvi Introduction ................................................................................................................ 1 Aims of this dissertation ..................................................................................................... 6 ACKNOWLEDGEMENTS ...................................................................................................... 7 REFERENCES ...................................................................................................................... 7 Impact of UPF3B on arsenic-induced toxicity ............................................................12 ABSTRACT ..........................................................................................................................13 INTRODUCTION ..................................................................................................................14 Arsenic .............................................................................................................................14 NMD protects cells from stress-induced apoptosis ...........................................................16 NMD degrades specific RNAs to protect cells from apoptosis ..........................................17 MATERIALS AND METHODS ..............................................................................................23 Mouse neural stem cell generation, growth and arsenic exposure ....................................23 HeLa cell culture...............................................................................................................23 Mouse arsenic exposure ..................................................................................................23 RNA isolation ...................................................................................................................24 DNA isolation and genotyping ..........................................................................................24 Cell Cycle and Apoptosis FACS analysis .........................................................................26 Mouse brain isolation and immunohistochemistry .............................................................26 RNA sequencing analysis .................................................................................................27 RESULTS .............................................................................................................................29 Role of UPF3B in arsenic-induced apoptosis and cell cycle arrest in vitro ........................29 Role of UPF3B in arsenic-induced changes to cell cycle in vivo .......................................36 Evidence that UPF3B protects specific brain regions from apoptosis and cell stress ........38 Identification of arsenic-regulated genes in mNSCs .........................................................49 Identification of arsenic-induced transcripts whose expression pattern is modulated by UPF3B .............................................................................................................................51 Identification of arsenic-regulated genes in mouse frontal cortex ......................................56 DISCUSSION .......................................................................................................................63 Effects of UPF3B on arsenic-induced changes in cell cycle and apoptosis .......................63 Effects of Arsenic on Gene Expression ............................................................................64 Effects of UPF3B on Arsenic-induced changes in Gene Expression.................................66 ACKNOWLEDGEMENTS .....................................................................................................68 REFERENCES .....................................................................................................................68 v NMD Raises the Threshold for Cellular Stress .........................................................80 ABSTRACT ..........................................................................................................................81 INTRODUCTION ..................................................................................................................82 The UPR ..........................................................................................................................82 NMD suppresses the UPR pathway .................................................................................86 UPR transcripts are targeted for decay by the NMD pathway ...........................................89 NMD suppression has differential effects in mammals and C. elegans .............................92 Other Stress Signals ........................................................................................................95 Arsenic UPR Activation ....................................................................................................95 Stress Granules and RNA Degradation ............................................................................96 NMD and Autophagy ...................................................................................................... 100 MATERIALS AND METHODS ............................................................................................ 104 U2OS Cell culture ........................................................................................................... 104 Environmental toxicants ................................................................................................. 104 Immunostaining .............................................................................................................. 104 RNA isolation and analysis ............................................................................................. 104 RESULTS ........................................................................................................................... 107 Some Environmental Toxicants Trigger the Unfolded Protein Response ........................ 107 Inorganic arsenic upregulates UPR gene expression in HeLa cells ................................ 109 Sodium arsenite upregulates UPR gene expression earlier and for a prolonged time in NMD-deficient HeLa cells or mNSCs .............................................................................. 111 NMD deficient HeLa cells form stress granules at higher rates than wild-type cells ........ 111 NMD deficient U2OS form stress granules at higher rates and lower thresholds than control cells .................................................................................................................... 114 DISCUSSION ..................................................................................................................... 117 ACKNOWLEDGEMENTS ................................................................................................... 120 REFERENCES ................................................................................................................... 120 Arsenic Suppresses the NMD Pathway .................................................................. 130 ABSTRACT .......................................................................................................................
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