Links between splicing, transcription and chromatin in Saccharomyces cerevisiae

Abstract

There is increasing evidence from yeast to humans that splicing is mainly a co-transcriptional process, and it is becoming well established that splicing, transcription and chromatin are functionally coupled such that they influence one another. The present work explored the links between splicing and transcription and links between splicing and chromatin in the budding yeast Saccharomyces cerevisiae. Currently, there is little mechanistic insight into the contribution of the core transcription elongation machinery to co-transcriptional spliceosome assembly and splicing. To understand how members of the core transcription elongation machinery affect splicing, I used the auxin-inducible degron (AID) system to conditionally deplete essential and non-essential transcription elongation factors and I analysed the effects on RNA polymerase II, co-transcriptional spliceosome assembly and splicing. The transcription elongation factors that I analysed are all conserved from yeast to mammals and include: Spt5, Paf1, Ctk1, Bur1 and Bur2. Most notable were the effects of depletion of the transcription elongation factor Spt5, mutations in which were known to cause splicing defects. Here, Spt5 depletion resulted in reduced recruitment of the U5 snRNP to intron-containing genes, meaning proper co-transcriptional activation of the spliceosome was inhibited, explaining how loss or mutation of Spt5 results in splicing defects. This effect was not dependent on phosphorylation of Spt5, however, the unphosphorylated form of Spt5 enhanced co-transcriptional formation of the catalytically activated spliceosome. Together, these data indicate a two-part function for Spt5 in co-transcriptional spliceosome assembly in S. cerevisiae. Firstly, the physical presence of Spt5 is required for proper co-transcriptional recruitment or stable association of the U5 snRNP and B complex formation. Secondly, the loss of Bur1 kinase activity and at least the unphosphorylated form of Spt5 enhances co-transcriptional formation of the catalytically activated spliceosome and splicing. There is correlative and causative evidence that splicing affects chromatin structure and vice versa. Of particular interest to the present work are links between splicing and Histone 3 Lysine 4 trimethylation (H3K4me3), a chromatin mark associated with promoters of active genes. H3K4me3 has been shown to influence and be influenced by splicing in mammalian cells. However, the molecular basis of this is unknown. To further understand the links between splicing and H3K4me3, I used the AID system to conditionally deplete essential splicing factors that act at different stages of the splicing cycle and analysed the effects on H3K4me3. Whilst depletion of splicing factors that affect the first or second catalytic step of splicing reduces H3K4me3 on intron-containing genes, notably, depletion of the late-acting factor Prp22 reduces H3K4me3 in the absence of defects in splicing catalysis, suggesting a more direct role for Prp22. Prp22 is an RNA-dependent ATPase that proofreads to product of the second step of splicing and promotes mRNA release from the post-spliceosome. Interestingly, the effect of Prp22 on H3K4me3 is dependent on its ATPase activity. Furthermore, Prp22 and Set1 were found to interact in a pull-down assay and depletion of Prp22 results in reduced recruitment of Set1 to intron-containing genes. These data show a previously unknown link between Prp22, Set1 and H3K4me3 in S. cerevisiae. Collectively, these analyses provide new mechanistic insight into the links between splicing and transcription and links between splicing and chromatin in S. cerevisiae.