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.
In Barrass et al. (2015) we describe the use of metabolic labelling with 4-thio-uracil for very short times to isolate newly synthesised precursor RNAs and perform RNA-seq. In this way we compared the relative speed of splicing of different pre-mRNAs, observing that, on average, ribosomal protein gene transcripts are spliced faster than most other intron- containing transcripts. Moreover, splicing is faster for introns with secondary structures that are predicted to be less stable. In Wallace and Beggs (2017) we compared data from several sources, finding that ribosomal transcripts are also spliced more efficiently (i.e. more pre-mRNA gets spliced) and more co-transcriptionality (more splicing happens while the RNA is still associated with polymerase) compared to other intron-containing transcripts.
To investigate how speed of transcription elongation affects splicing, we measured the efficiency, the co-transcriptionality and the fidelity (accuracy of correct splice site use) of splicing in yeast strains with wild-type (WT) RNA polymerase II (Pol II), or with mutant Pol II that elongates faster or slower. We show that slow Pol II elongation increases both co-transcriptional splicing and splicing efficiency and that faster elongation reduces co- transcriptional splicing and splicing efficiency in budding yeast, suggesting that splicing is more efficient when co-transcriptional. Moreover, we demonstrate that altering the Pol II elongation rate in either direction compromises splicing fidelity (e.g. Figure 1). These effects are notably stronger for the highly expressed ribosomal protein coding transcripts, which are spliced with much higher fidelity than other intron-containing transcripts (Figure 2). We propose that transcription by RNA polymerase II is tuned to optimize the efficiency and accuracy of ribosomal protein gene expression, while allowing flexibility in splice site choice with the non-ribosomal protein transcripts.
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.
Aslanzadeh, V., Huang, Y., Sanguinetti, G. and Beggs, J.D. (2018). Transcription Rate Strongly Affects Splicing Fidelity and Co- transcriptionality in Budding Yeast. Genome Res. 28, 203-213.