How cell stress and 3' end alterations control the metabolism of a cellular non-coding RNA

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PROJECT SUMMARY RNA Polymerase II (Pol II) canonically acts as a DNA-dependent RNA polymerase (DdRP), using double stranded DNA to synthesize protein-encoding mRNAs and some non-coding RNAs. Pol II also has RNA-dependent RNA polymerase activity (RdRP), which uses an RNA as a template to synthesize RNA. An example of Pol II RdRP activity is the 3’ end extension of the non-coding B2 RNA to generate extended B2 (eB2) RNA. B2 RNA is encoded by Short Interspersed Elements (SINEs), which exist in over 350,000 copies in the mouse genome due to retrotransposition. Random insertion of these elements into the genome could be deleterious, depending on which region or gene is disrupted, and in some cases can cause disease. Upon cellular stress, transcription of non-coding B2 RNAs from B2 SINEs is greatly increased, thereby increasing the likelihood of retrotransposition of B2 SINEs. The RdRP activity of Pol II could control the levels of B2 RNA post-transcriptionally by generating eB2 RNA to promote its degradation. The proposed work will identify all forms of 3’ modified B2 RNAs in cells, measure their stabilities, and determine how 3’ end extension of B2 RNA changes its intracellular localization and retrotransposition. B2 RNA binds to Pol II, globally represses transcription, and undergoes a Pol II-dependent 18 nucleotide RdRP extension to form eB2 RNA. Furthermore, eB2 RNA has a drastically reduced half-life as compared to B2 RNA. The formation of eB2 RNA from B2 RNA is thought to be an autoregulatory mechanism to overcome transcriptional repression and promote the dissociation and degradation of eB2 RNA. To date, the majority of the characterization of B2 RNA has been through biochemical experiments, which do not encompass the complexity of a cellular system that could alter the efficacy of Pol II RdRP activity. This proposal aims to understand how an RNA polymerase controls RNA metabolism post-transcriptionally. To identify cellular 3’ modified B2 RNA species due to extension, processing, and determine how stress affects these species, a B2 RNA-specific sequencing scheme (B2 RNA-seq) will be developed. To quantify the stabilities of B2 RNA species, B2 RNA-seq will be coupled to bromouridine pulse-chase. Furthermore, to observe the localization of B2 and eB2 RNAs in cells before and after stress, RNA Fluorescence in situ hybridization (FISH) will be utilized. FISH will be coupled to immunofluorescence experiments to determine colocalization between RNAs and potential processing machinery. Additionally, to determine how 3’ extension and cellular stress affect the ability of B2 RNA to retrotranspose, a cell-culture model of retrotransposition will be used. Altogether, these studies will provide an understanding of the interplay between B2 RNA expression, Pol II RdRP extension, RdRP-regulated degradation, cellular localization, and cellular stress on the life cycle of this ncRNA.