Pre-mRNA splicing and other RNA processing events occur co-transcriptionally. High resolution kinetic studies performed in our lab showed splicing-dependent RNA Pol II (RNA polymerase II) pausing near the 3’ splice site of a reporter gene. Pausing requires splicing, as mutations that block splicing lead to loss of pausing, and restoring splicing restores pausing. It was proposed that RNA Pol II pausing may occur at splicing-dependent transcriptional checkpoints. In this study, I aimed to search for splicing helicases that might couple splicing with transcription. The ts alleles prp5-1 and prp16-2 were found to cause transcription defects. These genes encode RNA helicases that were reported to act as fidelity factors during splicing. In vivo RNA labelling and RT-qPCR experiments performed with these temperature-sensitive mutants demonstrated reduced transcription coinciding with the splicing defect at restrictive temperature. Furthermore, RNA Pol II ChIP analysis showed polymerase accumulating over intron-containing genes in both mutants. ChIP analysis using antibodies specific to the phosphorylation status of the CTD (Carboxy Terminal Domain) of RNA Pol II, revealed that the apparently stalled polymerase is hyper-phosphorylated at serine 5. Intriguingly, prp8-R1753K, a ts allele of PRP8, a non-helicase splicing factor mutant also showed reduced nascent RNA synthesis but no RNA Pol II accumulation. To elucidate the reason for the observed RNA Pol II accumulation and to identify a possible splicing-dependent transcriptional checkpoint factor, prp5-1 was investigated further. RNA Pol II ChIP-Seq analysis verified that maximum enrichment genome-wide occurred on introns at restrictive conditions in prp5-1, supporting the earlier observation. Furthermore, the double mutant strain cus2Δprp5-1 abolished the RNA Pol II accumulation observed in prp5-1 at restrictive temperature and restored transcription. Recreating a stalled spliceosome in a U2 mutant strain also showed RNA Pol II accumulation in the presence of Cus2p, as observed in prp5-1. My observations suggest a link between transcription and monitoring of splicing and indicate that Cus2p, a U2 snRNP associated protein, could be a checkpoint factor in transcription prior to pre-spliceosome formation. I speculate that fidelity factors may impose transcriptional checkpoints at different stages of splicing.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:699873 |
| Date | January 2013 |
| Creators | Thelakkad Chathoth, Keerthi |
| Contributors | Beggs, Jean ; West, Steven |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/17547 |
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