Dynamic histone acetylation is critical for cotranscriptional spliceosome assembly and spliceosomal rearrangements

Abstract

Assembly of the spliceosome onto pre-mRNA is a dynamic process involving the ordered exchange of snRNPs to form the catalytically active spliceosome. These ordered rearrangements have recently been shown to occur cotranscriptionally, while the RNA polymerase is still actively engaged with the chromatin template. We previously demonstrated that the histone acetyltransferase Gcn5 is required for U2 snRNP association with the branchpoint. Here we provide evidence that histone acetylation and deacetylation facilitate proper cotranscriptional association of spliceosomal snRNPs. As with GCN5, mutation or deletion of Gcn5-targeted histone H3 residues leads to synthetic lethality when combined with deletion of the genes encoding the U2 snRNP components Lea1 or Msl1. Gcn5 associates throughout intron-containing genes and, in the absence of the histone deacetylases Hos3 and Hos2, enhanced histone H3 acetylation is observed throughout the body of genes. Deletion of histone deacetylaces also results in persistent association of the U2 snRNP and a severe defect in the association of downstream factors. These studies show that cotranscriptional spliceosome rearrangements are driven by dynamic changes in the acetylation state of histones and provide a model whereby yeast spliceosome assembly is tightly coupled to histone modification.pre-mRNA splicing | Saccharomyces cerevisiae R emoval of noncoding sequences from premessenger RNA is achieved by the activity of a dynamic ribonucleoprotein complex, the spliceosome. As the spliceosomal snRNPs sequentially recognize sequences in the pre-mRNA, the spliceosome undergoes ATP-dependent rearrangement of its RNA and protein components. Recently, it has been recognized that splicing can occur cotranscriptionally, while the RNA polymerase is still actively engaged with the chromatin template.The monomeric units of chromatin, nucleosomes, are comprised of DNA wrapped around the core histones H3, H4, H2A, and H2B. Histones undergo extensive posttranslational modification on their N-terminal tails that affect compaction of DNA and binding of regulatory factors. Although it is known that splicing occurs cotranscriptionally, it is far less well understood how changes in this chromatin template affect the reaction. Analysis of tiling array data has suggested that nucleosomes and, according to several of these studies, specific histone modifications are enriched in exon sequences, suggesting that there may be specific histone "marks" that are associated with splicing signals (1-7). Additionally, proteins that bind to methylated histones (H3K4me3 and H3K36me3) have been shown to facilitate the recruitment of snRNPs to the nascent transcript and influence efficiency of splicing and alternative splicing (8, 9). The chromatin-bound mammalian SWI/SNF complex associates with components of the spliceosome and affects alternative splicing (10, 11). Although these studies suggest a role for histone modifications in mammalian alternative splicing, there has been little analysis of their roles in constitu...

Histone H3K36 methylation regulates pre-mRNA splicing inSaccharomyces cerevisiae

“…Recent genomewide analysis in both metazoa 25 and in yeast 26 reveal that the presence of certain histone modifications differs between DNA sequences encoding exons and those encoding introns, leading to the emerging paradigm that histone modification can modulate RNA splicing. 11 This paradigm is supported by several recent studies showing that both histone H3 acetylation 27,28 and histone H2B-K123 ubiquitination 29,30 enhance splicing efficiency in yeast. Furthermore, several histone modifications have recently been implicated in co-transcriptional recruitment of splicing factors, providing evidence for the recruitment model of coupling transcription with RNA splicing.…”

“…The splicing defects we observe are similar in magnitude to those observed when other histone modifications are perturbed. [27][28][29][30]64 In addition, gene-specific splicing defects in budding yeast have been described previously [12][13][14] and could possibly be a result of features of the gene or intron such as the length of the gene, and/or its transcription frequency. Nevertheless, we do observe a trend of decreased splicing efficiency upon removal of Set2, as compared to the wild-type strain ( Fig.…”

“…This similar reporter phenograph and clustering behavior was expected, as Gcn5 is known to acetylate the tail of H3 48 and contribute to co-transcriptional spliceosome recruitment. 27,28 The YAF9 deletion mutant, also clustering in this small clade, is a component of the SWR1 complex that replaces histone H2A (HTA1/HTA2) with H2A.Z (HTZ1). 49 Additionally, Yaf9 is a component of the NuA4 histone acetyltransferase complex, known to acetylate the N-terminal lysines of H4, 50 consistent with the clustering behavior of the 2 H4 mutants H4K16A and H4K12Q (Fig.…”

“…The intron-containing genes we analyzed included DBP2, TEF5, and RPS21B, which have previously been analyzed in studies investigating co-transcriptional splicing. 14,[27][28][29]64 Yeast were grown at the permissive temperature prior to RNA extraction and the splicing mutant snu66D served as a positive control.…”

“…Furthermore, several histone modifications have recently been implicated in co-transcriptional recruitment of splicing factors, providing evidence for the recruitment model of coupling transcription with RNA splicing. 10,11 For example, histone H2B ubiquitination by the Bre1 E3 ubiquitin ligase 29 and Gcn5 histone acetyltransferase activity 27,28 facilitate splicing by recruiting splicing factors to splicing substrates in yeast. In metazoa, depletion of SETD2, the chromatin modification enzyme that tri-methylates H3K36 (see below), changes alternative splicing patterns and both tri-methylated H3K4 and tri-methylated H3K36 interact with splicing proteins to recruit them during transcription.…”