Acetylation by the Transcriptional Coactivator Gcn5 Plays a Novel Role in Co-Transcriptional Spliceosome Assembly

Abstract

In the last several years, a number of studies have shown that spliceosome assembly and splicing catalysis can occur co-transcriptionally. However, it has been unclear which specific transcription factors play key roles in coupling splicing to transcription and the mechanisms through which they act. Here we report the discovery that Gcn5, which encodes the histone acetyltransferase (HAT) activity of the SAGA complex, has genetic interactions with the genes encoding the heterodimeric U2 snRNP proteins Msl1 and Lea1. These interactions are dependent upon the HAT activity of Gcn5, suggesting a functional relationship between Gcn5 HAT activity and Msl1/Lea1 function. To understand the relationship between Gcn5 and Msl1/Lea1, we carried out an analysis of Gcn5's role in co-transcriptional recruitment of Msl1 and Lea1 to pre-mRNA and found that Gcn5 HAT activity is required for co-transcriptional recruitment of the U2 snRNP (and subsequent snRNP) components to the branchpoint, while it is not required for U1 recruitment. Although previous studies suggest that transcription elongation can alter co-transcriptional pre-mRNA splicing, we do not observe evidence of defective transcription elongation for these genes in the absence of Gcn5, while Gcn5-dependent histone acetylation is enriched in the promoter regions. Unexpectedly, we also observe Msl1 enrichment in the promoter region for wild-type cells and cells lacking Gcn5, indicating that Msl1 recruitment during active transcription can occur independently of its association at the branchpoint region. These results demonstrate a novel role for acetylation by SAGA in co-transcriptional recruitment of the U2 snRNP and recognition of the intron branchpoint.

Histone H3K36 methylation regulates pre-mRNA splicing inSaccharomyces cerevisiae

“…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.…”

“…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.…”

“…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.…”