The Yeast SR-Like Protein Npl3 Links Chromatin Modification to mRNA Processing

Abstract

Eukaryotic gene expression involves tight coordination between transcription and pre–mRNA splicing; however, factors responsible for this coordination remain incompletely defined. Here, we explored the genetic, functional, and biochemical interactions of a likely coordinator, Npl3, an SR-like protein in Saccharomyces cerevisiae that we recently showed is required for efficient co-transcriptional recruitment of the splicing machinery. We surveyed the NPL3 genetic interaction space and observed a significant enrichment for genes involved in histone modification and chromatin remodeling. Specifically, we found that Npl3 genetically interacts with both Bre1, which mono-ubiquitinates histone H2B as part of the RAD6 Complex, and Ubp8, the de-ubiquitinase of the SAGA Complex. In support of these genetic data, we show that Bre1 physically interacts with Npl3 in an RNA–independent manner. Furthermore, using a genome-wide splicing microarray, we found that the known splicing defect of a strain lacking Npl3 is exacerbated by deletion of BRE1 or UBP8, a phenomenon phenocopied by a point mutation in H2B that abrogates ubiquitination. Intriguingly, even in the presence of wild-type NPL3, deletion of BRE1 exhibits a mild splicing defect and elicits a growth defect in combination with deletions of early and late splicing factors. Taken together, our data reveal a connection between Npl3 and an extensive array of chromatin factors and describe an unanticipated functional link between histone H2B ubiquitination and pre–mRNA splicing.

Histone H3K36 methylation regulates pre-mRNA splicing inSaccharomyces cerevisiae

“…With the interpretation that the phenotypes observed are mediated through the loss of the histone modifications normally found at these residues, our work confirmed roles for H3 acetylation by Gcn5, 27,28 H2BK123 ubiquitination by Bre1, 29,30 and support a role for the previously identified modification enzymes Set1, Set2 29 and Bdf1 and Vps72 64 in RNA splicing. Importantly, we uncovered novel players in splicing regulation.…”

“…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.…”

“…For example, deletion of the Bre1 E3 ubiquitin ligase, as well as deletion of Ubp8 deubiquitinase, which both modulate H2B ubiquitination levels, cause a reduction in RNA splicing. 30 Furthermore, H3 acetylation by Gcn5 and deacetylation by Hos2/3 are required for RNA splicing. 27,28 Here we show that deletion of the H3K36me1/2 demethylase Jhd1 has modest positive genetic interactions with splicing factor genes (e.g.,, jhd1D ist3D; Fig.…”

“…29,30 Set2 methylates nucleosomal H3K36, and generates mono-, di-, and tri-methylated forms. 36 Studies show that Set2 is associated with the elongating form of RNA Pol II and mediates H3K36me2/me3 to recruit a number of chromatin-modifying complexes (Rpd3S and Isw1b) that maintain a repressive chromatin environment that is resistant to pervasive transcription in the coding regions of genes.…”