Mechanistic Investigation Of Activation Of The Innate Immune Sensor Pkr By Various Levels And Types Of Rna Structure

Open Access
Author:
Hull, Chelsea Michelle
Graduate Program:
Chemistry
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
February 24, 2016
Committee Members:
  • Philip C. Bevilacqua, Dissertation Advisor
  • Scott A Showalter, Committee Member
  • Christine Dolan Keating, Committee Member
  • Craig Eugene Cameron, Committee Member
Keywords:
  • PKR
  • RNA
  • bacterial RNA
  • riboswitch
  • ribozyme
  • innate immunity
Abstract:
The innate immune response is the first line of defense against invading pathogens, thus it is of very critical importance to human health and medicine. There are many dangerous pathogens and microorganisms that threaten the human cell at all times and without the innate immune response’s recognition of patterns from these pathogens life would not be the same, and perhaps not even possible for humans. In 2011, the Nobel Prize in medicine was awarded to Drs. Bruce Beutler and Jules Hoffmann for their discoveries on the activation of the innate immune system. The protein kinase PKR is an essential protein in the innate immune response that upon recognition of pathogenic patterns is activated and eventually leads to apoptosis of this infected cell. PKR recognizes molecular patterns through unique pathogenic RNA patterns, and classically by long stretches of double-stranded RNA that a virus or other pathogen could release upon infection. It is important that innate immune sensors detect broad non-specific patterns among these pathogens, and in recent years understanding of the mechanism of PKR’s regulation in the cell has grown tremendously. Although classical activation is by long double-stranded RNA, PKR recently has been shown to be more permissive, binding other functional, biological and non-conventional RNAs, as well as some proteins. RNA molecules can adopt an array of structures and perform many different functions in the cell. It is important to understand both sides of the interaction between PKR and RNA because they are both functional molecules. Some pathogens are not recognizable by the innate immune response, and it seems as though they have developed a way of disguising themselves from the innate immune sensors. The objective of this thesis is to provide a better understanding on how PKR recognizes a broad spectrum of RNAs and more importantly how it recognizes the difference between self and non-self RNA. Much is already known about how PKR recognizes pathogenic RNA from viruses, but recently PKR has been shown to be activated in cells infected with RNA from bacteria. There are many levels of RNA structure found in the bacterial transcriptome where much of these RNAs regulate genes and functions. It was our hypothesis that PKR activation by bacterial RNA was through these highly structured functional RNAs including riboswitches and ribozymes. Through this idea, I published the first study that a discrete functional bacterial RNA, the trp 5’UTR, which binds a protein (TRAP) and regulates L-tryptophan levels in Bacillus subtilis, potently activates PKR in both ligand (TRAP/L-trp) –free and –bound states. I found that many structural features of this RNA, which are common to functional bacterial RNAs, activated PKR in manners that are typical to PKR activation and that this activity was maintained when lowering Mg2+ concentrations to human physiological conditions, which are known to affect RNA structure. I also expanded this into a detailed mechanistic study that different types of functional bacterial RNAs with extensive tertiary structure activate PKR including a riboswitch, a riboswitch-ribozyme, and a ribozyme. I showed that these bacterial RNAs maintain tertiary structure upon binding to PKR and activate PKR as strongly as classical viral dsRNA. A structural investigation by ribonuclease mapping and protein footprinting revealed that PKR is able to dimerize onto the native structures of these bacterial RNAs. Overall, these studies show how PKR acts as a signaling protein for bacteria and suggests the apparent absence for such type of naked RNAs in the human genome in that they would activate innate immunity. Taking a step back and investigating PKR’s interaction with RNA at the primary and secondary levels of RNA structure led to some findings that helped us understand how PKR can detect a broad range of RNA structure. At the primary level, our lab earlier showed that PKR activation by small single-stranded RNAs that required a 5’-triphosphate. In my work, we hypothesized that PKR had an exclusive method of recognizing such RNAs, but instead our studies revealed that the binding site for 5-triphosphorylated ssRNA overlapped with that of double-stranded RNA but was mutually exclusive from the ATP binding site of PKR. And lastly, some recent discoveries in how PKR interacts with viral RNAs with long extended base-pairing regions and helical deformations, yet still led to PKR activation was further investigated by mechanistically looking at how much helical deformation PKR can handle. In recent years, much has been learned regarding how PKR interacts with many different types of RNA structure and even proteins; however, it is still not well understood how PKR functions in vivo. For this reason, I attempted crosslinking and immunoprecipitation (CLIP) to gain a library of total RNAs that PKR interacts with in healthy and infected cells, as well as to look at PKR activity in human cells infected with bacteria. Both of these results led to a foundation for how to develop methods to work with PKR in such in vivo systems that can be pursued by future studies in the lab. We made a shift to look at how PKR interacts with RNA in more “in-vivo-like” systems by adding in a molecular crowder or total RNA extracted from E. coli. I found that total E. coli RNA activates PKR potently and this activation surprisingly increases after degrading this RNA down into smaller fragments. My dissertation work has made advancements in the molecular understanding of how PKR and the innate immune system identify the difference between self- and non-self. The classical model of activation of PKR by long-dsRNA has been greatly expanded, and the mechanisms of activation by all levels of RNA in the hierarchy have been investigated. I have established novel ways in which bacterial RNA is recognized by PKR under in vitro and in vivo-like conditions, which lays the foundation for in vivo studies of these interactions.