Splicing mainly occurs co-transcriptionally, suggesting that transcription and premRNA splicing could be synchronized. The nature of this phenomenon suggests that transcription elongation rate may influence splicing outcomes and, indeed, there is evidence for effects on alternative splicing in mammals. To elucidate potential effects of transcription rate on splicing efficiency and fidelity, splicing of nascent transcripts was investigated in fast and slow elongating RNA polymerase II (RNAPII) mutants in Saccharomyces cerevisiae. High kinetic resolution 4-thio Uracil labelling of nascent RNA reveals that fast RNAPII accumulates unspliced pre-mRNA that represents reduced co-transcriptional splicing. Conversely, low levels of unspliced pre-mRNA were detected in the slow mutant due to increased co-transcriptional splicing. The highly stable association of nascent transcripts with elongating RNAPII permits co-transcriptional splicing to be measured by analysis of transcripts that co-purify with RNAPII. Measuring co-precipitation of the spliced mRNA and excised intron that are associated with RNAPII demonstrates that splicing is mostly co-transcriptional with the slow mutant, and the fast mutant reduces co-transcriptional splicing. How elongation rate affects splicing fidelity in budding yeast and whether faster and slower transcription have the opposite effect on splicing fidelity as might be predicted by the kinetic coupling model is an open question. Using deep RNA sequencing, splicing fidelity was determined in yeast transcription elongation mutants. Results show that both fast and slow transcription reduce splicing fidelity mainly in ribosomal protein coding transcripts. Analysis reveals that splicing fidelity depends largely on intron length, secondary structure and splice site score. These analyses also provide new insights regarding the effect of altering transcription rate on selection of transcription start sites. Together, these results indicate that optimal splicing efficiency and fidelity require finely-tuned transcription speed.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:743664 |
| Date | January 2017 |
| Creators | Aslanzadeh, Vahid |
| Contributors | Beggs, Jean ; Granneman, Sander |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/29593 |
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