Histone H3K36 methylation regulates pre-mRNA splicing in Saccharomyces cerevisiae

Co-transcriptional splicing takes place in the context of a highly dynamic chromatin architecture, yet the role of chromatin restructuring in coordinating transcription with RNA splicing has not been fully resolved. To further define the contribution of histone modifications to pre-mRNA splicing in Saccharomyces cerevisiae, we probed a library of histone point mutants using a reporter to monitor pre-mRNA splicing. We found that mutation of H3 lysine 36 (H3K36) – a residue methylated by Set2 during transcription elongation – exhibited phenotypes similar to those of pre-mRNA splicing mutants. We identified genetic interactions between genes encoding RNA splicing factors and genes encoding the H3K36 methyltransferase Set2 and the demethylase Jhd1 as well as point mutations of H3K36 that block methylation. Consistent with the genetic interactions, deletion of SET2, mutations modifying the catalytic activity of Set2 or H3K36 point mutations significantly altered expression of our reporter and reduced splicing of endogenous introns. These effects were dependent on the association of Set2 with RNA polymerase II and H3K36 dimethylation. Additionally, we found that deletion of SET2 reduces the association of the U2 and U5 snRNPs with chromatin. Thus, our study provides the first evidence that H3K36 methylation plays a role in co-transcriptional RNA splicing in yeast.

共转录剪接（co-transcriptional splicing）发生于高度动态的染色质结构（chromatin architecture）环境中，然而染色质重塑（chromatin restructuring）在协调转录与RNA剪接过程中的作用尚未完全阐明。为进一步明确酿酒酵母（Saccharomyces cerevisiae）中组蛋白修饰（histone modifications）对前体mRNA（pre-mRNA）剪接的贡献，我们利用可监测前体mRNA剪接的报告基因，对一组组蛋白点突变体（histone point mutants）文库开展了探究。我们发现，H3赖氨酸36（H3K36）——该位点在转录延伸（transcription elongation）过程中被Set2甲基化——的突变所呈现的表型，与前体mRNA剪接突变体的表型相似。我们鉴定出编码RNA剪接因子（RNA splicing factors）的基因，与编码H3K36甲基转移酶Set2和去甲基化酶Jhd1的基因之间存在遗传互作，同时还发现了可阻断甲基化（block methylation）的H3K36点突变。与该遗传互作结果一致，敲除SET2基因、修饰Set2催化活性（catalytic activity）的突变，或是H3K36点突变，均显著改变了本研究中报告基因的表达水平，并降低了内源内含子的剪接效率。上述效应依赖于Set2与RNA聚合酶II（RNA polymerase II）的结合，以及H3K36二甲基化（H3K36 dimethylation）。此外，我们发现敲除SET2基因会降低U2和U5小核糖核蛋白颗粒（snRNPs）与染色质的结合能力。因此，本研究首次证实，H3K36甲基化在酵母的共转录RNA剪接过程中发挥着关键作用。