Regulation of RNA Metabolism by the RNA-dependent RNA Polymerase Activity of Mammalian RNA polymerase II

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PROJECT SUMMARY/ABSTRACT In mammalian cells, transcription of mRNA is catalyzed by RNA polymerase II (Pol II), a DNA- dependent RNA polymerase. Pol II also has RNA-dependent RNA polymerase (RdRP) activity; it can use RNA as a template to synthesize new RNA of defined sequence. The breadth of functions for the Pol II RdRP activity in mammalian cells is unknown, yet could have broad biological significance. Pol II RdRP activity is medically relevant; it is required for replication of hepatitis delta virus, which causes liver disease. Seminal experiments are proposed to characterize the RdRP activity of Pol II in human and mouse cells and to determine its role in controlling RNA metabolism and retrotransposition. In addition, the RdRPome – the RNAs made by RdRP activity – will be identified in human and mouse cells. This will be the first concerted effort to determine the extent to which RdRP activity affects the RNA population in mammalian cells, paving the way for future studies of the biological impacts and functions of RdRP-generated or modified RNAs. Aim 1. Understand how the Pol II RdRP activity couples with Drosha and Dicer to control SINE RNA metabolism and SINE retrotransposition. Pol II RdRP activity can extend the 3' ends of B2 and Alu RNAs, which are transcribed from short interspersed elements (SINEs). Preliminary data suggest that this extension allows the RNAs to be cleaved by Drosha and perhaps Dicer. The roles of the Pol II RdRP, Drosha and Dicer in regulating the degradation of SINE RNAs will be determined using biochemical and cell-based experiments. In addition, studies will be performed to understand the roles of these three activities in controlling SINE retrotransposition, which requires an RNA intermediate and can cause disease. Aim 2. Identify the human and mouse RdRPomes and determine the effect of the Pol II RdRP on the metabolism of select RNAs. The extent to which Pol II or other cellular polymerases function as RdRPs remains an open question, and no systematic method to identify mammalian RdRP-derived sequences has been devised. A technique will be developed that selectively labels RNA sequences made by RdRP activity in cells, purifies these RNAs, and identifies them via deep sequencing. The RdRP-generated RNAs made by Pol II will be defined, and functional studies will probe whether the Pol II RdRP activity regulates the stability of select RNAs in cells. The studies will test roles for Pol II that go beyond its function in the traditional central dogma. RdRPs have been shown to play important roles in non-mammalian systems; the proposed studies could reveal critical yet uncharacterized mechanisms of cellular regulation involving mammalian RdRP activity. The relative absence of studies aimed at understanding the prevalence and importance of RdRP activities in mammalian cells amplifies the timeliness and significance of the proposed research.