Grant: Discovery Grants Program - Individual

In eukaryotes, three DNA-directed RNA polymerases are responsible for the transcription of different types of genes: RNA polymerase (RNAP) I transcribes ribosomal RNAs (rRNAs), RNAPII transcribes messenger RNAs (mRNAs) and a variety of noncoding RNAs (ncRNAs), and RNAPIII transcribes transfer RNAs (tRNAs). Before becoming functional mature RNAs, newly-synthesized transcripts have to go through key processing steps. In contrast to the textbook view of RNA processing, which is generally depicted as a series of post-transcriptional events, it is now widely accepted that most RNA processing reactions occur co-transcriptionally as the nascent transcript is being synthesized. Recent studies have in fact underlined the critical role of transcription speed in the control of co-transcriptional RNA processing, regulating the production of alternative splice variants and the length of 3' untranslated regions. However, factors and mechanisms that control co-transcriptional RNA processing by modulating transcription elongation rates remain elusive. Our own studies using the fission yeast S. pombe have significantly contributed to this field by showing that the evolutionarily conserved RNA-binding protein Seb1 influences RNA processing by controlling the progression of RNA polymerases I and II. Accordingly, the GENERAL OBJECTIVE of this proposal is to determine the mechanism by which Seb1 controls co-transcriptional RNA processing. Our CENTRAL HYPOTHESIS, which is based on strong preliminary findings, is that Seb1 promotes polymerase pause/slow states during transcription elongation, which influence co-transcriptional RNA processing decisions. We will test our hypothesis and accomplish the objective of this proposal by pursuing the following SPECIFIC AIMS: 1. Determine the mechanism underlying Seb1-dependent RNA processing. We will use in vitro transcription assays and in vivo genomics approaches to test the hypothesis that Seb1 is a pause-promoting factor during transcription elongation. 2. Delineate how phosphorylation of the RNAPII C-terminal domain (CTD) contributes to co-transcriptional RNA processing. We have recently generated phosphorylation-deficient versions of the RNAPII CTD in fission yeast and will assess how they affect the transcriptome. The ORIGINALITY of this application relies on the use of a unique model system and complementary expertise to provide a better understanding of how coupling between transcription and RNA processing is controlled in eukaryotic cells. Students trained under this research program will acquire training in multidisciplinary approaches, including areas of molecular genetics, cellular biology, biochemistry, genomics, and proteomics. Students without distinction as to minorities will develop critical skills, allowing them to gain the status of highly trained RNA scientists for the Canadian biotech sector and the academic research community.