Poster abstracts

Poster number 20 submitted by Laurie Heinicke

Regulation of the protein kinase PKR by perfect and defect dsRNAs

Laurie A. Heinicke (Department of Chemistry, The Pennsylvania State University), Amy Diegelman-Parente (Division of Mathematics and Natural Sciences, Altoona College The Pennsylvania State University), Subba Rao Nallagatla (Department of Chemistry, The Pennsylvania State University), Philip Bevilacqua (Department of Chemistry, The Pennsylvania State University)

Abstract:
PKR is an RNA regulated protein kinase and is part of the innate immune response. PKR is phosphorylated in the presence of certain RNA structures with downstream effects including inhibition of translation and regulation of cell differentiation. PKR is activated by perfect double-stranded RNAs (dsRNAs) greater than 33 bp, but is also activated by minimally structured single-stranded RNAs (ssRNAs) containing a 5’-triphosphate and dsRNAs with numerous imperfections. In this study, three types of RNAs were tested in PKR activation assays; perfect dsRNAs, minimally structured ssRNAs, and biologically relevant Hepatitis Delta Virus (HDV) RNAs.

Time-course activation assays have been performed to investigate the mechanism of PKR activation by perfect 79 bp RNA and minimally structured RNA. In the presence of 79 bp RNA, a 1 to 2 minute lag is observed followed by an increase of activation to 10 minutes. Activation assays in the presence of urea and at various temperatures suggest that PKR is not very stable. Numerous cleavable and non-cleavable HDV RNA constructs have been tested for PKR activation. Short HDV constructs do not activate PKR, while most longer HDV constructs containing increasingly complex secondary and tertiary structures and less than 33 consecutive base pairs do activate PKR. Interestingly, two short constructs (-54/-1 and 1/99) do not activate PKR, whereas a non-cleavable form of the combined constructs (-54/99 C75U) does activate PKR. In addition, PKR activation is inhibited by a long non-cleavable construct (-54/226 C75U) that contains a stem-loop (13 bp) connected to a long defect 94 bp rod. Regulation of PKR by intermediate HDV RNA transcripts has biological implications due to the in vivo dynamics of RNA folding during transcription. These findings may be useful in identifying general inhibitory and activating elements in other viral RNAs.

Keywords: PKR