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Mechanistic Insights Into Translational Modulation of Selected Rnas by RNA Helicase A

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Mechanistic Insights Into Translational Modulation of Selected Rnas by RNA Helicase A Mechanistic insights into translational modulation of selected RNAs by RNA helicase A DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Arnaz K Ranji, M.Sc. Graduate Program in Molecular Genetics The Ohio State University 2011 Dissertation Committee Dr. Kathleen Boris-Lawrie, Advisor Dr. Stephen A Osmani Dr. Mamuka Kvaratskhelia Dr. Harold Fisk Copyright by Arnaz K Ranji 2011 Abstract The 5' untranslated region (UTR) of mRNAs is the entry gate for the translation machinery. Features of the 5’ UTR control the efficiency of mRNA translation. Relatively little is known about the specialized proteins that modulate the entry of the translation machinery. Our previous research has identified a redundant structural element in the 5’ UTR of many retroviruses and select cellular RNAs that facilitates access of scanning ribosomes. The ribosome loading is facilitated by interaction with RNA helicase A (RHA). This dissertation describes important open questions. Biochemical, genetic and biophysical approaches were employed to define residues that are necessary for selective recognition of target mRNAs and that tether the ATP- dependent helicase for rearrangement of the complex 5’ UTR. The first of the five chapters of this dissertation introduces the present fundamental understanding and key open issues of the process of mRNA translation. Proper control of translation is essential for healthy cell growth and response to infectious agents. The initiation stage of mRNA translation determines the activity of an mRNA as a productive mRNA template or the alternative outcome as a triaged mRNA that is not a productive mRNA template. Chapter 1 describes the discovery and characterization of the retroviral structural element (PCE) and its effector protein RHA. RHA interacts with ii PCE at the 5’ UTR and is necessary for the translation of virion proteins of avian, bovine, simian and human retroviruses and the JunD proto-oncogene. RHA also modulates viral RNA replication and gene expression in two other virus families. The final section of Chapter 1 describes the emerging roles of RHA and other RNA helicases in virus replication. Three experimental studies that define the mechanism of RHA translational regulation of target mRNA are presented in Chapters 2, 3 and 4. Chapter 2 defined the residues of RNA helicase A required for the selective interaction of RNA helicase A with its cognate mRNA. Results of mutagenesis, biochemical and biophysical assays defined three lysine residues (K54,K55 and K236) within the 300 amino acid amino-terminal domain are necessary. Exogenous expression of this domain is sufficient to block translation activity of endogenous RHA and the lysine mutations eliminated the squelching effect on endogenous RHA. In sum, residues within the amino-terminal double-stranded RNA binding domains confer selective interaction of RHA with PCE RNA that tethers the ATP-dependent helicase for rearrangement of the complex 5' UTR. Experiments in Chapter 3 determined residues of RHA that are necessary for translation activity of target RNAs. Sucrose density gradient analysis, localization studies, RNA immunoprecipitations, and translation assays in cells identified roles for the amino-terminal RNA binding domains, central ATP-binding pocket and arginine-rich iii carboxy-terminal domains of RHA in recognition and translation of PCE-containing mRNAs. The N-terminal residues required for binding of the isolated N-terminal domain to PCE RNA are also important for binding of full-length RHA to PCE RNA. The N- terminal and helicase domains are required for translation initiation that culminates in polysome loading. Carboxy-terminal residues are required for productive completion of the translation process. RHA mutants that are defective in translation initiation become sequestered in stress granules, irrespective of PCE RNA binding ability. RHA is not a core component of stress granules, but accumulates in stress granules during oxidative stress. The arginine-rich carboxy-terminal residues are necessary for oxidative stress- dependent shuttling of RHA to these cytoplasmic triage granules. This indicates that all three of these RHA domains are required for productive translation of RHA target RNAs. In Chapter 4 I identified the residues of RHA that interact with PCE RNA in cells. The study established a protein footprinting and mass spectrometric protocol to compare residues protected by RHA expressed in E. coli or immunoprecipitated from mammalian cells. The results identified that the conformation of the N-terminal domain of RHA does not differ in the context of the isolated recombinant N-terminal domain or mammalian cells. PCE RNA protects two carboxy residues of RHA (K806 and K1048). Mutagenesis and RNA immunoprecipitation assays in transfected cells determined that these residues are not essential for PCE RNA binding. However, mutation of K806 hampered the iv recruitment of RHA to stress granules during oxidative stress, which indicates that this residue could be important for interactions with co-factors that are required for stress granule recruitment. The results demonstrate that our proteomic approach using immunoprecipitated protein can help identify novel residues that are important for RHA function. The final chapter considers the significance of this research and essential future questions. In closing, this dissertation has determined the molecular basis for the activation of translation by RHA involves selective recognition of target mRNAs by the N-terminal domains, and regulation of both the initiation and post-initiation stages of translation. The dual molecular functions of RNA helicase A in the process of translation provides a potent strategy to fine tune expression of retroviruses and JunD. This process is essential for healthy control of cell growth and virus replication. v Dedication Dedicated to my family, without whom this would not be possible. vi Acknowledgements I would like to thank my advisor Dr. Kathleen Boris-Lawrie for her guidance and help throughout the duration of my PhD. I appreciated all the effort that went into making me the scientist I am today. Even with her busy schedule she has always found time to meet with me and help me through my latest experimental crisis. I am forever grateful that she took a chance on me and allowed me to join her lab and I will miss her company. I would also like to thank my committee members Dr. Kvaratskhelia, Dr. Osmani and Dr. Fisk. They have been a pleasure to work with and have looked out for my best interests through the years. I would like to separately acknowledge Dr. Kvaratskhelia who spent several hours going over my data and making brilliant suggestions that have resulted in a fruitful collaboration between the Boris-Lawrie and Kvaratskhelia labs and my first data publication. In addition, I would like to thank Dr. Shkiriabai for his help with protein purification and mass spectrometry. I also benefited from interactions with Dr. Karin Musier-Forsyth and her laboratory and would like to sincerely thank them all. In my own lab, I am indebted to several members who helped me through my time in this lab. Life would have been a lot less interesting without them. In particular I would like to acknowledge Dr. Cheryl Bolinger for her help and companionship as well as her data that I have used in this dissertation. I would also like to thank Dr. Marcela vii Hernandez who has guided me on numerous occasions and been very generous with her precious time. Finally I’d like to thank the members in the Lairmore, Green and Wu labs for being excellent information resources as well as fun lunch buddies. I will miss our interactions when I move on. Finally I would like to thank all the friends and family members who have played such an important role in getting me though this process. My parents have always supported me in every decision I made and I am thankful that they encouraged me to do what I really wanted to do in life. Together with my grandfather they were responsible for my being able to leave home to study, first for a masters and then for a PhD. I am very lucky to have the support of so many family members who are always encouraging and never doubted my abilities. So to Mumz, Dada, Masi, Nugi, Beba, Granny, Grandpa, Nonu and Katy… a big thank you. Also, a big thanks to my little sister Shernaz who has always enriched my life with her humor and support. Finally a big thanks to my nearest and dearest friends who have been like part of the family… Cookie, Ali, Zoee and Diloo, thanks for being there for me whenever I needed you. viii Vita 1998-2001………………………………. BSc, Life sciences and Biochemistry St Xavier’s College, Mumbai, India. 2001-2002………………………………..MSc, Medical Genetics Glasgow University, Scotland. 2002-2003………………………………..Medical Technical Officer 1 Duncan Guthrie Institute of Medical Genetics, Glasgow, Scotland. 2004-present……………………………...Graduate Research Associate Department of molecular genetics The Ohio State University Columbus, Ohio PUBLICATIONS Ranji, A ., Shkriabai, N., Kvaratskhelia, M., Musier-Forsyth, K. and Boris-Lawrie, K. Features of double-stranded RNA binding domains of RNA Helicase A are necessary for selective recognition and translation of complex mRNAs. Journal of Biological Chemistry, in press December 1, 2010. Ranji, A. and Boris-Lawrie, K. RNA Helicases: Emerging roles in viral replication and the host innate response. RNA biology Volume 7,
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