H3K36 methylation and the chromodomain protein Eaf3 are required for proper co-transcriptional spliceosome assembly

In the eukaryotic cell, spliceosomes assemble onto pre-mRNA cotranscriptionally. The fact that spliceosome assembly takes place in the context of a dynamic chromatin environment suggests that the state of the chromatin may affect splicing. The molecular details and mechanisms through which chromatin regulates RNA splicing, however, are still unclear. Here, we show a widespread role for the histone methyltransferase Set2 and its histone modification, H3K36 methylation, in pre-mRNA splicing through high-throughput sequencing. Moreover, we find that the effect of H3K36 methylation on pre-mRNA splicing is not dependent on changes in RNA polymerase II elongation, but are driven by the chromodomain protein Eaf3. We find that Eaf3 is recruited to intron-containing genes and that Eaf3 physically interacts with the splicing factor Prp45. Eaf3 acts with Prp45 and Prp19 after formation of the pre-catalytic B complex around the time of splicing activation revealing the step in splicing that is regulated by H3K36 methylation. These studies support a model whereby H3K36 facilitates recruitment of an “adapter protein” to support efficient, constitutive splicing. Overall design: RNA sequence data of 4 samples with two biological replicates each were analyzed, including wildtype, set2 deletion, eaf3 deletion, and H3K36A point mutation. ChIP sequence of RNA polymerase II S2P of three samples, wildtype with 2 technical replicates, H3K36A point mutation without replication, and eaf3 deletion without replication were also analyzed along with ChIP-seq of wildtype H3K36 trimethylation without replication. For ChIP sequencing, we analyzed as compared to three input libraries, including one replicate each for wildtype, H3K36A, and eaf3 deletion.

8

Leung CS, Douglass SM, Morselli M, Obusan MB, Pavlyukov MS, Pellegrini M, Johnson TL