Histone H3K36 methylation regulates pre-mRNA splicing in Saccharomyces cerevisiae

Abstract

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.

Keywords: Chromatin; co-transcriptional pre-mRNA splicing; coupling; histones; methylation; snRNPs; transcription.

Figure 1.

Specific histone mutants phenocopy loss of functionally relevant gene expression factors. Gene expression reporter phenograph data from the collection of histone point mutants were integrated with the deletion collection data set and hierarchically clustered with complete linkage and a centered absolute correlation similarity metric, resulting in a small number of histone point mutants clustering with deletion mutants within gene expression pathways (A). Nodes are annotated with the colors indicated in the legend. Global clustering behavior of the histone mutants is depicted in Fig. S1. Gene expression reporter phenograph overlays for deletion and histone mutants of interest (B-D) are depicted. Mutants are shown in pseudo-color on the left and middle panels and overlaid onto a grayscale wild-type phenograph for comparison. For the rightmost panel the phenograph for the deletion mutant (blue) and histone mutant (red) are overlaid to demonstrate similarity. The H3KtailR mutant is H3K9,14,18,23R. Note: there are 2 vps72Δ strains in the deletion mutant collection. (E). Gene Expression reporter schematic. Expression is driven by the constitutive TDH3 promoter. The sequence features of the pre-mRNA are modeled based on the inefficiently spliced CYH2 gene. The intron is marked by the dashed lines. The mCherry ORF is in frame with exon I, thus production of mCHerry results from mRNAs that have retained an intron. The GFP is in frame with exon 2 driving GFP expression only when the intron is removed.

Figure 2.

Mutations that alter H3K36 methylation state are genetically implicated in RNA splicing. RNA splicing factors display genetic interactions with SET2 and JHD1 (A and B). Deletion of SET2 combined with a deletion of a gene encoding a splicing factor leads to a range of synthetic growth defects (A). Deletion of JHD1 partially suppresses the growth defect of select strains harboring a deletion of a splicing factor gene (B). (C and D) RNA splicing factors display synthetic genetic interactions with H3K36R (C) and H3K36A (D). Serial dilutions of WT, single and double mutant strains grown on rich media at 31°C and 37°C. Plates photographed after 48 hours growth (note that the ist3Δ 37°C panel was photographed after 72 hours growth).

Figure 3.

Genetic modulation of H3K36 methylation state results in gene-specific pre-mRNA splicing defects. RNA was harvested from the indicated yeast strains at early/mid log phase. RT-qPCR analysis was performed, determining the pre-mRNA and total levels of the indicated intron containing transcripts. Reported values were calculated using a standard curve calculations and are relative to wild-type ratio; ANOVA p-values and Tukey p-values for gene-specific pair-wise strain comparisons reported in Table S4 (A). The data represent biological triplicates and error bars represent one standard error of measurement. * indicates p <0.01 and ˆ indicates p<0.05 when comparing pre-mRNA/total in the given mutant strain to wild-type. For other pair-wise comparisons see Table S4 (B) The data are also shown in heat map form using Cluster and TreeView.

Figure 4.

Synthetic-genetic effects on the splicing of endogenous introns. Heat map representation of RT-qPCR data for the strains designated to the right of each heat map for the introns labeled on the top (A). Splicing efficiency/intron accumulation was calculated as a ratio of pre-mRNA/total relative to wild-type and described on a green to red color scale, with black being set at a value of 1.0 (equal with wild-type). Values less than one indicate increased splicing efficiency while values greater than 1 indicate a splicing defect. Values represent the average from 3 biological replicates. Legend shown in the bottom right reflects the colors and values. ANOVA p-values and Tukey p-values for gene-specific pair-wise strain comparisons reported in Table S5 (B). Asterisks represent the p-values for the comparison of the splicing factor deletion mutant to the double mutant (i.e., ist3Δ versus set2Δ ist3Δ; gray asterisk indicates p < 0.05. white asterisk indicates p < 0.01). (B) Hierarchical clustering of the RT-qPCR data for all the strains is shown.

Figure 5.

Identification of pre-mRNA splicing and transcription elongation defects in set2 mutants. (A). Expression of the gene expression reporter in a set2Δ strain carrying plasmids with set2 mutants (blue) compared to set2Δ covered by a wild-type SET2 plasmid (red). A shift towards the mCherry axis is indicative of a pre-mRNA splicing defect. (B) RT-qPCR analysis of the levels of four endogenous introns. Values shown are the ratio of pre-mRNA to total and are made relative to the wild-type (WT) SET2 strain. The average of 3 biological triplicates is used and error bars represent one SEM; ANOVA p-values and Tukey p-values for gene-specific pair-wise strain comparisons reported in Table S6. * indicates p < 0.01 and ˆ indicates p<0.05 when comparing pre-mRNA/total in the given mutant strain to SET2 (WT) strain. For other pair-wise comparisons see Table S6. (C and D) Improper transcription elongation in a set2Δ does not explain all the splicing defects observed in a set2Δ. Five-fold serial dilution of indicated strains were spotted and incubated at higher temperature (C) or spotted on 6-AU (transcription elongation inhibitor) (D) to monitor how various mutations of Set2 influence set2Δ phenotypes. Catalysis by Set2 is important for both resistance to 6-AU and suppressing the bypass of spt16-11, while interaction with RNAPII is dispensable for suppressing 6-AU resistance.

Figure 6.

Deletion of SET2 impacts the recruitment of the U2 snRNP and U5 snRNP. Schematic diagram of the RPS21B, SRB2 and DBP2 genes and the primer sets used in the chromatin immunoprecipitation (ChIP) assays (A). Chromatin immunoprecipitations were carried out using α-HA or α-RNA pol II antibodies in a Lea1-HA strain strain (“wild-type”) or a Lea1-HA set2Δ strain (set2Δ) (B) or Snu114-HA (“wild-type”) or a Snu114-HA set2Δ strain (set2Δ) (C) strain grown at 30°C then shifted to 37°C for 30 min. Shown are the average of HA-tagged protein or RNA polymerase bound relative to input at the indicated regions (note that each IP sample was normalized to an averaged relative amount for an intergenic region). Error bars represent +/− SEM for each strain and primer set, n = 3–4 biological replicates (see Materials and Methods for details). * indicates p < 0.05 and ˆ and # indicate approaching significance (ˆ p < 0.07, # p < 0.09) comparing the set2Δ strain to wild-type.