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Molecular Basis of the Dexh-Box RNA Helicase RNA Helicase a (RHA/DHX9) in Eukaryotic Protein Synthesis

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Molecular Basis of the Dexh-Box RNA Helicase RNA Helicase a (RHA/DHX9) in Eukaryotic Protein Synthesis Molecular basis of the DExH-box RNA helicase RNA helicase A (RHA/DHX9) in eukaryotic protein synthesis DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Sarah Elizabeth Fritz, B.S. Graduate Program in Integrated Biomedical Science Program The Ohio State University 2015 Dissertation Committee: Dr. Kathleen Boris-Lawrie, Advisor Dr. Joanna Groden Dr. Jeffrey Parvin Dr. Dawn Chandler Copyright by Sarah Elizabeth Fritz 2015 Abstract Protein synthesis is a fundamental molecular event of cell biology. Its enactment requires the intricate choreographing of RNA-protein assemblies. These ribonucleoprotein (RNP) complexes are the performers of translation that function to regulate ribosome dynamics upon a target transcript. The outcome is distinct regulation of protein production. Deregulated translation activity is associated with cancer, neurological diseases and disorders, neurodegeneration, growth defects, and innate immune disorders. This dissertation aimed to enhance our understanding of the eukaryotic translation process by elucidating the molecular role of RNA helicase A (RHA/DHX9) in protein synthesis. RHA is a cellular protein and member of the DExH/D-box RNA helicase family that is necessary for the translation of pathogenic retroviral transcripts and the cellular proto-oncogene junD. The known characteristic of RHA-mediated protein synthesis is the select recognition and association of RHA with the distinguishing 5' termini of retroviral and junD transcripts. These mRNAs harbor the distinct 5' RNA motif known as the posttranscriptional control element (PCE). The PCE functions in cis to stimulate translation activity via the canonical cap-dependent scanning mechanism that defines eukaryotic protein synthesis. The main gap in knowledge is how RHA, as the host effector of the PCE, engages a RNP complex that facilitates translation activity. ii In this study, RHA was identified to engage a unique RNP that confers a novel role for its activity in cap-dependent translation during cell stress. Here RHA was demonstrated to selectively interact with the non-canonical CBP80/20 cap-binding protein in the cell cytoplasm and on polysomes. Notably, this interaction was maintained during serum deprivation, torin 1-mediated mTOR inhibition and HIV-1 expression, all mechanisms that evoke cell stress and suppression of the canonical eIF4E cap-dependent translation. This observed effect correlated with sustained interactions between RHA, CBP80/20, and the target PCE transcript HIV-1 gag on polysomes. The outcome was maintained cap-dependent retroviral PCE translation. This novel molecular finding proposed a new paradigm for eukaryotic translation control in that the RHA-CBP80/20 RNP maintains selective cap-dependent protein synthesis during cell stress. This dissertation also identified novel significance for RHA as a post-initiation effector of protein synthesis. DExH/D-box RNA helicases are known to mediate the initial translation events of 5' ribosome binding and scanning. Through a reverse genetics approach, the N-terminal double-stranded RNA binding domains of RHA were shown to be critical for these canonical family activities. Notably, a role for the C-terminal arginine-glycine-rich domain of RHA was identified in 80S ribosome stabilization. This post-initiation translation activity was fundamental to the engagement of RHA with polyribosomes and completion of the translation process. These outcomes proposed a new paradigm for the molecular act of DExH/D-box RNA helicases in translation control that encompass both initiation and post-initiation translation activity. iii A third major finding of this dissertation was the identification of RHA to engage multiple RNP states that regulate its translation activity. Here RHA was shown to self- associate, a homopolymeric binding event that impaired translation cofactor binding. This result indicated a role for RHA self-association in the regulation of its RNP formation that facilitates translation activity. In addition, a select interaction between RHA and related DExH-box RNA helicase DHX30 was identified. This heteropolymeric-binding event was distinct in that it characterized a RHA RNP engaged with polyribosomes. This result indicated a role for a RHA-DHX30 association in the direct regulation of RHA translation during active protein synthesis. Together, these findings proposed novel significance for RHA RNP dynamics in the control of its translation activity. Collectively, the data obtained from this dissertation research provided novel and significant insight into the eukaryotic translation process. By elucidating the molecular basis of RHA in protein synthesis, several new paradigms were identified for the eukaryotic translation process. These include: a molecular basis for maintained cap- dependent translation during cell stress, significance of DExH/D-box RNA helicases in post-initiation translation control, and a role for distinct homo- and hetero-polymeric binding events in the regulation of RNP dynamics that control the translation process. We posit that these findings afford molecular significance for the association of RHA with breast, prostate and lung cancer, its identification as the major auto-antigen in systemic lupus erythematosus patients, and the role for RHA as a major stimulator of pathogenic viruses that infect and cause disease within animal and human hosts. Future studies are aimed to connect the molecular findings of this dissertation research with the clinical iv significance of RHA. The objective is to provide a greater understanding of the relationship between RHA, eukaryotic protein synthesis, and cell biology that informs animal and human health and disease. v Dedication Dedicated to my grandmother and forever kindred spirit, Mary Louise Fritz vi Acknowledgments To my advisor, Dr. Kathleen Boris-Lawrie, thank you for being my greatest advocate and supporter. You always believed in me when I doubted myself, a characteristic of a true mentor. To all members of the Boris-Lawrie lab, both past and present, I appreciate all of the help, guidance, and support you provided. Each of you went above and beyond to offer assistance at any moment, and I very much appreciated it. Arnaz, Marcela, Amit, Amy, Justin, Jon, Alaina, Ioana, Gati, Allison, Greeshma and all others that I have worked alongside, thank you for being incredible colleagues and friends. To my committee members, Dr. Joanna Groden, Dr. Jeffrey Parvin, and Dr. Dawn Chandler, thank you for working together with Dr. Boris-Lawrie and I to make this moment possible. Your support and encouragement made the entire Ph.D. journey a rewarding experience. To all colleagues in the Center for Retrovirus Research, you are incredible scientists, people, and friends. It has been a pleasure working alongside each and every one of you. Amanda, Jacob, Krissy, Cory, Alice, Nathan, Corine, Suresh, Jenna, Fei Fei, Becca, and Raj, keep being your amazing selves. And a special thanks to Tim Vojt for believing in our message and working very efficiently to help create the amazing figures that provided us that voice. vii To the Gettysburg College Biology Department, I am forever indebted to your continuous support and encouragement. Your passion for science and commitment to inspiring your students is, in itself, a true inspiration. I aspire to follow in your footsteps. To the Columbus Running Company, thank you for making Columbus home. Star, Stephanie, Caroline, Katie, Melissa, Paige, Liz T., Liz M., Beth, Anna, Mary, Kerri, Megan, Tayler, and all the many other incredible individuals I have shared runs with over these past five years, you are the greatest of training partners, role models, and friends. Together, you make every marathon possible. To my fellow Buckeyes, Julia, Hans, Adam, and Christian, and the amazing people you brought into my life, Matt, Isabel, Angela, and Ashley, our days in Ohio will always be ones that I treasure. It has been a true honor to go through this Ph.D. process with you and the experience would not have been the same or as meaningful without all of you as a part of it. To my former Gettysburg teammates, thank you for continuing to be the greatest of role models, supporters, and friends. Especially to my dear friend Kerrin, I would not be the person I am today without your friendship, support, and encouragement. And lastly, to my parents, thank you for letting me become the woman I am today. You never question nor do you ever doubt any of my decisions. You simply encourage me to choose my own path. There is nothing more I could ever ask for. I am eternally grateful. viii Vita 2006................................................................Ledyard High School 2010 ...............................................................B.S. Biochemistry and Molecular Biology, Gettysburg College 2010 to present ..............................................Ph.D. candidate, Integrated Biomedical Sciences, The Ohio State University Fields of Study Major Field: Integrated Biomedical Science Program Translation control RNA biology Molecular virology ix Table of Contents Abstract .............................................................................................................................. ii Dedication ......................................................................................................................... vi Acknowledgments ..........................................................................................................
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