Investigating Roles of Unconventional RNA Primary, Secondary, and Tertiary Motifs in Regulation of the Protein Kinase PKR

Open Access
Author:
Toroney, Rebecca
Graduate Program:
Chemistry
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
October 07, 2010
Committee Members:
  • Philip C. Bevilacqua, Dissertation Advisor
  • Philip C. Bevilacqua, Committee Chair
  • Nicholas Winograd, Committee Member
  • Scott A Showalter, Committee Member
  • Craig Eugene Cameron, Committee Member
Keywords:
  • Hepatitis C Virus
  • RNA-protein interactions
  • IRES
  • PKR
  • innate immunity
  • triphosphate
Abstract:
Much is known about the importance of protein folding and structure in human health. Like protein, RNA is a biopolymer essential to cellular function that is known to adopt multiple alternative structures, yet much less is known about the possible involvement of RNA folding in human disease. The protein kinase PKR is a component of the innate immune response in humans that functions by sensing molecular patterns in RNA, specifically long stretches of double-stranded RNA (dsRNA) that accumulate in the cell during viral infection. Activation of PKR ultimately leads to the shut down of cellular translation and thus viral proliferation. In this way, the ability of PKR to recognize and be activated by a wide variety of RNA structural elements is crucial to maintaining the first line of defense against viral infection. The objective of this thesis is to characterize the structural basis of PKR activation and inhibition by various RNA elements at all three levels of the RNA folding hierarchy: primary, secondary, and tertiary. This research may ultimately provide a connection between RNA structure and human illness. At the primary structural level, experiments were performed to establish the RNA structural determinants for PKR activation by single-stranded RNAs (ssRNA) with an essential triphosphate at the 5’-end. The requirement of 5’-triphosphate, which is not present in cellular RNA, for PKR activation by these ssRNAs is a novel discovery and was shown to be reproducible in vitro and in human cells. Activation assays, structure mapping techniques, and binding studies were performed which established that in addition to the dependency on triphosphate––and not diphosphate, monophosphate, or 7’-methylguanosine, at the 5’-end––PKR activation by these ssRNAs requires short, ~5 bp stem loops located optimally near the center of the RNA. Additional experiments were conducted to characterize the binding pocket of this 5’-triphosphate in PKR. Competitive PKR activation assays and a variety of biophysical techniques including fluorescence competition, isothermal titration calorimetry (ITC), and fluorescence stopped-flow indicated that PKR binds the 5’-triphosphates of RNA at a site that is unique from the catalytic ATP binding site. Results from these studies also demonstrated that the 5’-triphosphate binding site is weaker in affinity and lower in specificity than the catalytic site, which potentially increases the ability of PKR to recognize a wider variety of pathogenic RNA 5’-end signatures. At the secondary and tertiary structural levels, PKR regulation by various elements of hepatitis C virus (HCV IRES) RNA was investigated. Conflicting reports in the literature were resolved by performing activation and inhibition assays of the full-length IRES at a range of RNA concentrations, which established the role of the full-length HCV IRES as both an activator and inhibitor of PKR. Additionally, individual elements of the IRES secondary structure were identified as activators, with particular emphasis on domain II, which functions as a potent activator despite its limited number of canonical basepairs. Footprinting and mutational analyses as well as modeling were utilized to establish that noncanonical regions within domain II are critical for activation through mimicry of dsRNA structure. The role of IRES tertiary structure was also investigated through analysis of PKR activation by mutant IRES RNAs that lack this tertiary structure. Results obtained suggest that while tertiary structure promotes optimal PKR activation, the IRES still activates well in its absence. Finally, a focused examination of the role of one specific RNA secondary motif, the tandem GA mismatch, was conducted. Tandem GA mismatches adopt one of two distinct conformations with disparate representation in biological RNA. The ability of PKR to distinguish between these two conformers was investigated through activation and inhibition assays of various model RNAs containing one or more mismatches of each tandem mismatch conformer. Experiments indicated a slight preference for PKR regulation by the biologically rare conformer, suggesting that small changes in RNA structure may serve as recognition motifs of foreign RNA for PKR.