UCLA

In the nucleus of the eukaryotic cell, spliceosome assembly and the subsequent catalytic steps of precursor RNA (pre-mRNA) splicing occur cotranscriptionally in the context of a dynamic chromatin environment. A key challenge in the field has been to understand the role that chromatin and, more specifically, histone modification plays in coordinating transcription and splicing. Despite previous work to decipher the coordination between these processes, a mechanistic understanding of the role that chromatin modification plays in spliceosome assembly has remained elusive.

Histone H3K36 trimethylation (H3K36me3) is a highly conserved histone mark found in the body of actively transcribed genes, making it a particularly attractive candidate for having a role in the regulation of pre-mRNA splicing. Although there have been reports that H3K36me3 influences alternative splicing, the underlying mechanisms have remained elusive. Moreover, despite the prevalence of this mark on actively transcribed genes across eukaryotes, there has not been a mechanistic dissection of its potential role in constitutive splicing. Here we describe how we have addressed these questions and uncovered a core role for H3K36 methylation (H3K36me) in splicing in Saccharomyces cerevisiae (S. cerevisiae).

RNA-seq analyses of yeast strains deleted of the histone methyltransferase SET2 or harboring a mutation at the residue lysine 36 revealed widespread and overlapping splicing changes. Through a combination of RNA-seq, ChIP-seq, and biochemical analyses, we determined that the H3K36me effects on splicing are not due defects in transcription, but rather to loss of binding of the chromodomain protein Eaf3. We further show that Eaf3 interacts with the splicing factor Prp45, the human ortholog of SKIP. Consistent with this, Eaf3 is required for the cotranscriptional recruitment of the Prp Nineteen Complex (NTC), which is required for spliceosome activation. Hence, loss of H3K36 methylation has a direct and crucial role in spliceosome assembly. We demonstrate a central role for a highly conserved histone modification in constitutive pre-mRNA splicing. We also show that the reciprocal is true—splicing affects chromatin state.

Although it is established that spliceosome assembly occurs cotranscriptionally, a question that remains is how the activities of the proteins that regulate the dynamic rearrangements of the spliceosome are modulated by transcription and the state of the chromatin. Prp43, an ATPase involved in spliceosome disassembly, has been implicated at numerous steps in the splicing cycle and is therefore an interesting target in the regulation of splicing. Our study aims to determine whether Prp43 plays a role in directing spliceosome rearrangements via chromatin interactions and how interactions between Prp43 and chromatin may affect splicing outcomes. We show that Prp43 associates with chromatin in an RNA-independent manner, but splicing-dependent manner. Furthermore, we observed that Prp43 interacts with H3K36me3, possibly for its role in spliceosome disassembly.