To investigate mammalian RNA-dependent RNA polymerase (RdRP) activity, RdRP-derived RNAs must be enriched from human HEK-293 and mouse NIH-3T3 cells cells using the bromouridine immunoprecipitation assay, and potential RdRP substrates were confirmed using qRT-PCR. The specific RNAs analyzed were Alu and B2 RNAs. These RNAs are encoded by a short interspersed nuclear elements (SINEs) and both inhibit DNA-dependent RNA polymerase activity in human and mouse cells, respectively. B2 RNA, found in mice cells, has been shown to have an inhibitory effect on Pol II transcription. Interestingly, while studying the inhibitory effects of B2 RNA, our laboratory discovered a novel RNA-dependent RNA polymerase (RdRP), function of Pol II (2 from proposal), which extends B2 RNA in a templated manner by 18 nt. RdRP activity has been shown to have an effect on gene expression within bacteria and nematodes (5,6), however mammalian genomes have not been shown to have an encoded RdRP enzyme (7). Mammalian RdRP activity is just beginning to be elucidated, with mouse B2 RNA as the only confirmed Pol II RdRP-derived RNA. Mammalian RdRP activity could be a previously unrecognized form of posttranscriptional regulation. Based upon the results, 3’ RdRP extension of B2 RNA decreased upon heat shock within mouse cells, we hypothesize that RdRP activity may act to regulate B2 RNA steady state levels under various cellular conditions, as the RdRP 3’ extension of B2 RNA changed in response to stress. The studies presented here are the first to study human Alu RNA as a substrate for RdRP activity, and showed that Alu RNA does not appear to be a significant substrate for RdRP extension within human cells under normal or heat shock conditions.

Investigating mammalian RNA-dependent RNA polymerase activity in human HEK-293 and mouse NIH-3T3 cells

251st National Meeting (2016)

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