| Description |
Protein kinase RNA-activated (PKR) is a key a mediator of the innate immune system in response to viral infections. Upon sensing long doublestranded RNA (dsRNA), PKR is potently activated and triggers a stress-response pathway culminating in translation repression and apoptosis. Lower levels of PKR activation are associated with the cell-cycle, Alzheimer's Disease, and metabolic disorders. Importantly, this low-level PKR activity occurs in the absence of any pathogen, and therefore exogenous dsRNA. Only recently has the pool of endogenous dsRNA been interrogated as potentially linking diseased states with PKR activity. In this dissertation, I investigate endogenous RNAs that associate with PKR during metabolic stress and examine the structural features of those RNAs that contribute to PKR activation. First, in Chapter 2, we identify small nucleolar RNAs (snoRNAs) as the major class of RNA associated with PKR in a palmitic acid (PA)- and RNA-binding-motif-dependent manner. To understand how a protein commonly described as cytosolic interacts with nucleolar RNAs, we examined the localization patterns for PKR and snoRNAs. We found that while neither PKR nor snoRNAs relocalize after PA treatment, a fraction of PKR is nuclear. We then demonstrated that in vitro transcribed snoRNAs bind and activate PKR in vitro. These results suggest a novel role for snoRNAs in the regulation of PKR activity during metabolic stress. Previous iv studies, including our own, suggested that the presence of a 5'-triphosphate is required for PKR activation by RNAs with regions of single-stranded and doublestranded RNA. In Chapter 3, the importance of the 5'-triphosphate was called into question using multiple methods to generate a snoRNA, SNORD113, with different 5'-ends. Native polyacrylamide gel electrophoresis revealed that in vitro transcribed SNORD113 adopts three predominant conformers. Only one conformer of SNORD113 activated PKR after purification from native PAGE; importantly, this species contains antisense SNORD113 generated by T7 RNA polymerase during in vitro transcription. This study casts doubt on the expanding repertoire of PKR ligands which are generated by in vitro transcription. In Chapter 4, I discuss additional questions raised by the results presented in this dissertation and propose experiments for future studies. |
