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Abstract
The RNA-dependent protein kinase (PKR) is an interferon-induced enzyme that phosphorylates the alpha subunit of the translation initiation factor eIF2, which causes translation to cease. In this dissertation, the minor groove of duplex RNA is site-specifically modified to regulate and study dsRBM containing proteins. Such site-specific modification in the minor groove of a fragment of the Epstein-Barr virus encoded RNA 1 leads to controlled inhibition of binding by PKR at the two different sites. A conventionally held belief was that the minimum length of the RNA required for the activation of PKR was 33bp. Recently, it has been reported that short interfering RNAs (19bp) activates PKR, contributing to the observed off-target effects during RNA interference (RNAi). Affinity cleavage experiments and the benzyl-modification of the minor groove of the siRNAs were used to understand the mode of PKR activation by short interfering RNAs (siRNAs). Based on these data a model for siRNA binding and activation of PKR is presented. In addition, incorporation of N2-benzylguanosine at positions 7, 9 and 14 of the sense strand of siRNA negatively regulates the activity of PKR, while maintaining the silencing activity. Our findings indicate that such minor groove base-modifications can be used to regulate other proteins, implicated in the off-target effects in RNAi. A surprising and exciting by-product of the study of siRNA mediated activation of PKR was the discovery of phosphorylation of the ADAR2 N-terminal fragment. ADAR2 is an adenosine deaminase that acts on RNA and has essential physiological functions, especially in the neurological systems. This is the first report in the literature of phoshporylation of ADAR2. NetphosK, a web-based phosphorylation prediction software, predicted T126 of ADAR2 as a potential phosphorylation site for PKC. Experiments with T126 mutants support this prediction and emphasize the specificity of PKC for this residue. A discussion on the potential regulation of ADAR2 activity by phosphorylation, a common posttranslational regulatory mechanism, is presented.
