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Understanding the Role of Exon Junction Complex-Dependent Nonsense Mediated Mrna Decay in Zebrafish Embryonic Development Disser

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Understanding the Role of Exon Junction Complex-Dependent Nonsense Mediated Mrna Decay in Zebrafish Embryonic Development Disser Understanding the Role of Exon Junction Complex-dependent Nonsense Mediated mRNA Decay in Zebrafish Embryonic Development Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Pooja Gangras, B.Tech. Graduate Program in Molecular Genetics The Ohio State University 2019 Dissertation Committee Dr. Sharon L. Amacher, Advisor Dr. Guramrit Singh, Co-Advisor Dr. Robin P. Wharton Dr. Christine Beattie (deceased) Dr. Anita K. Hopper © Copyright by Pooja Gangras 2019 ii Abstract Post-transcriptional control of gene expression is essential for proper development and is achieved largely by RNA-binding proteins. My graduate research has focused on understanding the role of one such RNA binding protein complex, the Exon Junction Complex (EJC) in development. EJC is deposited about 24 nts upstream of exon-exon junctions during splicing. EJC influences many aspects of post-transcriptional regulation such as mRNA splicing, export, localization and nonsense mediated mRNA decay (NMD). EJC-dependent NMD is recognized as one mode of rapidly regulating gene expression of normal mRNAs that contain NMD-inducing features such as 3′UTR intron or an upstream ORF. How regulation of gene expression via EJC-dependent NMD influences development of specific tissues is unknown. My work utilizes a strong developmental and genetic model system, zebrafish, to understand how EJC-dependent NMD shapes development. In order to address my scientific questions, I utilized the RNA-Seq technique. Our lab has a custom RNA-Seq library preparation protocol which I wanted to improve in order to increase the efficiency of isolating sample cDNA after reverse transcription of RNA fragments (Chapter 2). To improve the protocol, I incorporated biotinylated dNTPs in the RT reaction so that cDNA generated from RNA fragments could be extracted using streptavidin beads. This amendment to the library preparation protocol efficiently selects for extended RT product and avoids ligation of the unextended adapter and generation of insert-lacking cDNAs in the library. iii To study EJC function during development (Chapter 3), I generated zebrafish mutants in EJC core protein genes rbm8a and magoh. Homozygous rbm8a and magoh mutants (EJC mutants) are paralyzed and have muscle and neural defects. As expected, RNA profiling revealed that annotated aberrant and normal NMD targets are significantly upregulated in EJC mutants. An mRNA is targeted for NMD by the key NMD-regulator Upf1 when an exon-exon junction, marked by the EJC, is bound ≥ 50 nts downstream of the terminated ribosome. Surprisingly, I discovered that some upregulated normal transcripts contain a conserved proximal 3′ UTR intron (3′UI) < 50 nts downstream of the stop codon. These ‘proximal 3′UI-containing NMD targets’ are similarly up-regulated in Upf1-deficient and NMD inhibitor-treated embryos, suggesting that this subset of rbm8a- and magoh-regulated transcripts is regulated via NMD. The same trend is observed in Upf1-deficient mammalian cells. One proximal 3′UI+ NMD target which is upregulated in EJC mutants and upf1 morphants encodes Foxo3b. I find that heterozygous and homozygous knockout of foxo3b partially rescues motor neuron outgrowth defects in EJC mutants. My work establishes zebrafish as a system to study NMD, characterizes zebrafish embryonic muscle and neural defects associated with loss of EJC and identifies proximal 3′UTR intron-containing genes a new class of NMD targets. iv Dedication This work is dedicated to Maithili, I hope you aspire to be the best version of yourself that you can be… v Acknowledgments I have been able to reach this point in my life and career due to the help, guidance and support of several people. Firstly, I would like to thank my advisors Dr. Sharon Amacher and Dr. Guramrit Singh for being amazing mentors and truly warm people. Sharon and Amrit have been instrumental in teaching me several techniques and the scientific method of thinking. I have been very fortunate to have mentors who have always supported my ideas. I am grateful to the MolGen Graduate Student Advisor and my committee member, Dr. Robin Wharton, who really helped me find my way to this collaborative project in my first year when I was completely clueless. I would like to thank all my committee members Dr. Robin Wharton, Dr. Christine Beattie (deceased) and Dr. Anita Hopper for their support throughout graduate school. I would also like to thank my undergraduate mentors Dr. Akihiro Ikeda (UW-Madison) and Dr. Barbara Kloeckener-Gruissem (Univerity of Zurich) who had faith in my ability to do science. I would also like to specially thank Dr. Shubha Tole (TIFR, Mumbai) for all her valuable advice. I would like to thank Dr. Jared Talbot who taught me everything I know about working with zebrafish, I came in with no experience and he was very patient in training me. I would also like to thank my lab mates Lauren, Justin, Zhongxia and Kiel. My lab mates created a wonderful atmosphere of laughter and friendship which made me want to come to lab even through the hard times in graduate school. I am grateful to Lauren for being my companion through every phase of graduate school and for being my sounding board through all the hard times. I am thankful to Zhongxia for keeping my love for science alive in my last year of graduate school with his excitement for all the newly vi published cool papers. I have made some of the best conference memories with Justin and Kiel, they both helped me settle into the Singh and Amacher labs respectively and I am grateful for that. I am grateful to Robert Patton and Michael Parthun for all their help with bioinformatics. I would like to thank the center of RNA biology for supporting my project with a seed grant and with the graduate student fellowship. I would also like to thank the Amacher lab managers Zachary Morrow and Danielle Pvirre who worked with their army of undergraduates to always keep the fish facility functioning which allowed me to pursue my research. I would also like to thank all the undergraduates who helped out with the lab chores in Singh lab. Lastly, I would like to thank my mother Devyani, sister Maithili and husband Abhijoy for all the emotional support they have provided over the last 5 years. Talking to my mom and sister every weekend was the best therapy I could ask for. They both always made me laugh and encouraged me to push through everything. I am so thankful to have met Abhijoy in my first week in Columbus. Abhijoy has been my biggest cheerleader throughout graduate school and I can’t even imagine working through graduate school without him. Meeting Abhijoy every day after work and walking home together as we discuss the joys and sorrows of the day will continue to be one of my most cherished memories of the last 5 years. vii Vita Education Bachelor of Technology (B.Tech.) | 2010-2014 | SRM University, Chennai, India · Major: Genetic Engineering; Bachelor thesis project, University of Zurich, Switzerland Publications 1. Gangras, P.*, Dayeh, D.M., Mabin, J.W., Nakanishi, K., Singh, G., 2018. Cloning and Identification of Recombinant Argonaute-Bound Small RNAs Using Next- Generation Sequencing. Methods Mol. Biol. Methods article 2. Woodward L.A., Mabin J., Gangras P.*, Singh G.,2016. The Exon Junction Complex: A Lifelong Guardian of mRNA Fate. WIREs RNA. Advanced review article 3. Woodward L.A., Gangras P.*, Singh G. 2019. Identification of footprints of RNA:protein complexes via RNA Immunoprecipitation in Tandem followed by sequencing (RIPiT-Seq). J. of Visul. Experiments. Fields of Study Major Field: Molecular Genetics viii Table of Contents Abstract .......................................................................................................................... iii Dedication ....................................................................................................................... v Acknowledgments.......................................................................................................... vi Vita ............................................................................................................................... viii Publications .................................................................................................................. viii Table of Contents ........................................................................................................... ix List of Tables ................................................................................................................ xii List of Figures .............................................................................................................. xiii Chapter 1 Introduction ........................................................................................................ 1 1.1 RNA-binding proteins in human disease .............................................................. 1 1.2 Role of the Exon Junction Complex in RNA metabolism ................................... 4 1.3 EJC-dependent Nonsense Mediated mRNA Decay ............................................. 7 1.4 Role of core EJC proteins, eIF4AIII, RBM8A and MAGOH in development and disease ................................................................................................................... 11 1.5 Defects associated with Nonsense mediated mRNA Decay ............................... 19 1.6 Brief overview of zebrafish muscle development ..............................................
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