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RNA Editing at Baseline and Following Endoplasmic Reticulum Stress

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RNA Editing at Baseline and Following Endoplasmic Reticulum Stress RNA Editing at Baseline and Following Endoplasmic Reticulum Stress By Allison Leigh Richards A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Human Genetics) in The University of Michigan 2015 Doctoral Committee: Professor Vivian G. Cheung, Chair Assistant Professor Santhi K. Ganesh Professor David Ginsburg Professor Daniel J. Klionsky Dedication To my father, mother, and Matt without whom I would never have made it ii Acknowledgements Thank you first and foremost to my dissertation mentor, Dr. Vivian Cheung. I have learned so much from you over the past several years including presentation skills such as never sighing and never saying “as you can see…” You have taught me how to think outside the box and how to create and explain my story to others. I would not be where I am today without your help and guidance. Thank you to the members of my dissertation committee (Drs. Santhi Ganesh, David Ginsburg and Daniel Klionsky) for all of your advice and support. I would also like to thank the entire Human Genetics Program, and especially JoAnn Sekiguchi and Karen Grahl, for welcoming me to the University of Michigan and making my transition so much easier. Thank you to Michael Boehnke and the Genome Science Training Program for supporting my work. A very special thank you to all of the members of the Cheung lab, past and present. Thank you to Xiaorong Wang for all of your help from the bench to advice on my career. Thank you to Zhengwei Zhu who has helped me immensely throughout my thesis even through my panic. Thank you to Jonathan Toung who taught me how to code and was so patient when I had trouble. Thank you to Alan Bruzel who helped me with protocols and made my whole experience more enjoyable. And thank you to everyone else in the Cheung lab for all of your fun and support. iii I would especially like to thank my friends who not only helped me with my lab work but also kept me sane: Michelle Nguyen-McCarty, Lauren Brady, Jillian Boden and Becky Glineburg. Michelle, thank you for the support and the distractions. Becky, thank you for introducing me to fun; it has served me well. Lauren, thank you for the understanding and the gossip. Jill, thank you for the lunches and steadying me when I get off-balance. And finally, I would like to thank my family. To my father for always believing in me and comforting me when all seemed lost. To my mother who always listened even when I repeated myself 20 times. Thank you to Deborah and Ted for your support. And thank you to Matt, who never let me give up. Without even one of you, I could never have accomplished anything. iv Table of Contents Dedication.......................................................................................................................................ii Acknowledgments…………………………………………………………………………………………………………………….iii List of Figures…………………………………………………………………………………………………………………………….ix List of Tables…………………………………………………………………………………………………………………………….x Abstract…………………………………………………………………………………………………………………………………….xi Chapter 1: Introduction………………………………………………………………………………………………………..……1 Identification of RNA Editing…………………………………………………………………………………………..2 RNA Editing in Plants………………………………………………………………………………………………………4 Identification of RNA Editing in Mammals………………………………………………………………………5 ADAR RNA Editing…………………………………………………………………………………………………………..7 Non-canonical Editing…………………………………………………………………………………………………..11 Endoplasmic Reticulum Stress………………………………………………………………………………………12 My Thesis Summary……………………………………………………………………………………………………..14 References……………………………………………………………………………………………………………………15 Chapter 2: Widespread RNA and DNA Sequence Differences in the Human Transcriptome…….26 Abstract……………………………………………………………………………………………………………………….26 Introduction…………………………………………………………………………………………………………………27 Samples………………………………………………………………………………………………………………………..28 Differences between RNA and corresponding DNA sequences………….……………………….30 RDD in expressed sequence tags………………………………………………………………………………..32 Sanger sequencing of B cells, skin, and brain………………………………………………………………..33 Proteomic evidence of RDD………………………………………………………………………………………….34 v Individual variation in abundance of RDD…………………………………………………………………..36 Characteristics of RDD sites………………………………………………………………………………………….37 RDD levels…………………………………………………………………………………………………………………….38 Conclusions………………………………………………………………………………………………………………….39 References……………………………………………………………………………………………………………………41 Tables and Figures………………………………………………………………………………………………………..46 Chapter 3: RNA-DNA Differences Are Generated in Human Cells within Seconds After RNA Exits Pol II…………………………………………………………………………………………………………………………………………52 Abstract……………………………………………………………………………………………………………………….52 Introduction…………………………………………………………………………………………………………………53 Nascent RNA from GRO-seq and PRO-seq…………………………………………………………………….55 RDDs in very nascent RNA…………………………………………………………………………………………….56 RDD formation occurs within seconds after transcription…………………………………………….59 RDD frequency is lower in cells from a patient with Senataxin mutation………………………61 A-to-G RDDs in very nascent RNA are not mediated by ADAR…………………………………..….62 Other characteristics of RDDs in very nascent RNA…………………………………………………....63 Conclusions………………………………………………………………………………………………………………….64 Experimental Procedures……………………………………………………………………………………………..66 References……………………………………………………………………………………………………………………70 Tables and Figures………………………………………………………………………………………………………..75 Chapter 4: Cis Regulation of RNA Editing in Human………………………………………………………………….82 Abstract……………………………………………………………………………………………………………………….82 Introduction…………………………………………………………………………………………………………………83 Identification of RNA editing and RDDs in human B-cells………………………..…………………...84 vi Characteristics of RNA editing and RDD………………………………………………………………………..85 Cis-Regulation of RNA editing levels……………………………………………………………………………..86 Individual variation in RNA editing levels………………………………………………………………………88 Effect of genomic variation on RNA editing………………………………………..………………………..89 SEC16A…………………………………………………………………………………………………………………………89 Conclusions…………………………………………………………………………………………………………………92 Methods……………………………………………………………………………………………………………………….93 References…………………………………………………………………………………………………………………100 Tables and Figures………………………………………………………………………………………………………102 Chapter 5: RNA Editing in Response to ER Stress in Human B-cells……………………………….……….122 Abstract……………………………………………………………………………………………………………………..122 Introduction……………………………………………………………………………………………………………….123 Induction of ER stress…………………………………………………………………………………………………124 Gene expression changes following ER stress…………………………………………………………….124 ADAR RNA editing levels following ER stress……………………………………….……..………………126 Validation of ADAR editing following ER stress……………………………………………………………128 ADAR editing and gene expression……………………………………………………………………………..129 Conclusions………………………………………………………………………………………………………………..131 Methods…………………………………………………………………………………………………………………….132 References………………………………………………………………………………………………………………….138 Tables and Figures………………………………………………………………………………………………………140 Chapter 6: Conclusion…………………………………………………………………………………………………………….146 Summary of Findings……………………………………………..…………………………………………………..146 vii Significance………………………………………………………………………………………………………………..148 On-going Work……………………………………………………………………………………………………………149 Future Directions………………………………………………………………………………………………………..151 In Summary………………………………………………………………………………………………………………..153 Methods…………………………………………………………………………………………………………………….154 References………………………………………………………………………………………………………………….155 Tables and Figures………………………………………………………………………………………………………160 viii List of Figures Figure 2.1: Characteristics of RDD sites……………………………………………………………………………………50 Figure 2.2: Identification of peptides coded by both RNA and DNA sequences………………………..51 Figure 3.1: GRO-seq and PRO-seq analysis……………………………………………………………………………….78 Figure 3.2: Analysis steps to identify RNA-DNA sequence differences………………………………………79 Figure 3.3: RNA-DNA differences in very nascent transcripts……………………………………………………80 Figure 3.4: Locations of RDD sites within sequence reads………………………………………………………..81 Figure 4.1: Characteristics of RNA editing sites and RDDs………………………………………………………102 Figure 4.2: Correlation of editing levels of sites in the same transcript…………………………………..103 Figure 4.3: Cis-association of editing levels…………………………………………………………………………….104 Figure 4.4: RNA Editing of SEC16A………………………………………………………………………………………….105 Figure S4.1: Analysis for identification of RDDs among ten individuals…………………………………..117 Figure S4.2: ATG14: Example of RNA folding and editing…………………………..…………………………..118 Figure S4.3: Editing of SEC16A in three tissues……………………………………………………………………….119 Figure S4.4: HuR RNA-IP of SEC16A……………………………………………………..…………………………………120 Figure S4.5: HuR RNA-IP by ddPCR……………………………………………………………..………………………….121 Figure 5.1: Gene expression changes following ER stress…………………………………..…………………..140 Figure 5.2: Editing level changes following ER stress……………………………..……………………………….141 Figure 5.3: Editing level and gene expression following ER stress…………………………………………..142 Figure S5.1: ADAR expression under ER stress conditions…………………………..………………………….145 Figure 6.1: Effect of HuR and ADAR knock-down on editing of SEC16A…………………..……………..160 ix List of Tables Table 2.1: Selected examples of sites that show RNA-DNA Differences in B-cells and EST clones………………………………………………………………………………………………………………………………………46 Table 2.2: Sanger sequencing of RDD sites……………………………………………………………………………….47 Table 2.3: Peptides encoded
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