Mechanistic insights into translational modulation of selected RNAs by RNA helicase A

Chapter 1 describes the discovery and characterization of the retroviral structural element (PCE) and its effector protein RHA. RHA interacts with 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 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 stressdependent 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 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. (Abstract shortened by UMI.)...