Regulation of splicing and functional links between splicing, transcription and chromatin
Transcription and RNA splicing are at the centre of gene expression in eukaryotes, controlling gene expression levels and removing introns from primary transcripts. The mechanisms and machineries involved in both transcription and RNA splicing are highly conserved throughout eukaryotes, and the budding yeast Saccharomyces cerevisiae makes an excellent model system, permitting the application of genetic approaches in parallel with molecular studies. Our current focus is on links between RNA splicing and other metabolic processes, especially transcription and chromatin. Our approaches include: quantitative RT-PCR, ChIP-seq, RNA- seq, biochemical analyses and molecular genetics.
To investigate links between transcription and splicing, we performed high resolution kinetic ChIP experiments to follow the recruitment of RNA polymerase II (Pol II) and splicing factors to inducible reporter genes. We found that, soon after the initiation of transcription, Pol II pauses transiently near the 3’ end of an intron in response to splicing. The carboxy-terminal domain (CTD) of the paused Pol II large subunit is hyper-phosphorylated, suggesting regulation through phosphorylation (Alexander et al., 2010). We propose that this represents a novel splicing–dependent transcription checkpoint that may be associated with the quality control activities of splicing ATPases, such as Prp5 (Figure 1A). Supporting this hypothesis, mutations (e.g. prp5-1) that block pre-spliceosome formation cause a transcription defect, with phosphorylated Pol II accumulating on introns (Figure 1B). In the prp5-1 mutant,U2-associated Cus2p is thought to remain in a defective transcription/splicing complex, but deletion of CUS2 suppresses the transcription defect. We propose that Cus2p is a potential checkpoint factor that signals the status of pre-spliceosome formation to the transcription machinery (Chathoth et al., 2014). Ongoing work includes investigating the mechanism by which splicing and transcription affect each other, and how coupling these processes benefits gene expression.
To facilitate studies of how other processes affect splicing, we developed a procedure for labelling RNA in vivo with 4-thio-uracil (4tU) for very short periods. In this way, short-lived precursor RNAs can be sequenced during a brief time course of labelling (Barrass et al., 2015). We have compared the speed of splicing of different pre-mRNAs obtained by this method, with the efficiency of co-transcriptional splicing derived from nascent RNA-seq analysis (Harlen et al., 2016) and the distance (nt) of Pol II from 3’ splice sites when splicing is 90% complete (Carrillo Oesterreich at al., 2016), finding remarkable agreement that highlights a striking difference between the co-transcriptionality, speed and efficiency of splicing ribosomal protein transcripts compared to other intron-containing transcripts (Figure 2).
Alexander, R., Innocente, S., Barrass, J.D. and Beggs, J.D. (2010). Splicing causes RNA polymerase pausing in yeast. Mol Cell 40, 582-593. Chathoth, K., Barrass, J.D., Webb, S. and Beggs, J.D. (2014) A splicing-dependent transcriptional checkpoint associated with pre- spliceosome formation. Mol Cell 53, 779-790.
Barrass, J.D., Reid, J.E.A., Huang, Y., Hector, R.D., Sanguinetti, G., Beggs, J.D. and Granneman, S. (2015) Transcriptome-wide RNA processing kinetics revealed using extremely short 4tU labeling. Genome Biol 16,282.
Wallace, E. and Beggs, J.D. (2017) Extremely fast and incredibly close: co-transcriptional splicing in budding yeast. RNA 23, doi: 10.1261/rna.060830