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. 2014 Apr;106(4):126-38.
doi: 10.1111/boc.201400003. Epub 2014 Feb 25.

H2B ubiquitylation modulates spliceosome assembly and function in budding yeast

Lucas Hérissant  1 , Erica A Moehle, Diego Bertaccini, Alain Van Dorsselaer, Christine Schaeffer-Reiss, Christine Guthrie, Catherine Dargemont
Affiliations

H2B ubiquitylation modulates spliceosome assembly and function in budding yeast

Lucas Hérissant et al. Biol Cell. 2014 Apr.

Abstract

Background information: Commitment to splicing occurs co-transcriptionally, but a major unanswered question is the extent to which various modifications of chromatin, the template for transcription in vivo, contribute to the regulation of splicing.

Results: Here, we perform genome-wide analyses showing that inhibition of specific marks - H2B ubiquitylation, H3K4 methylation and H3K36 methylation - perturbs splicing in budding yeast, with each modification exerting gene-specific effects. Furthermore, semi-quantitative mass spectrometry on purified nuclear mRNPs and chromatin immunoprecipitation analysis on intron-containing genes indicated that H2B ubiquitylation, but not Set1-, Set2- or Dot1-dependent H3 methylation, stimulates recruitment of the early splicing factors, namely U1 and U2 snRNPs, onto nascent RNAs.

Conclusions: These results suggest that histone modifications impact splicing of distinct subsets of genes using distinct pathways.

Keywords: H2B ubiquitylation; Histone marks; Pre-mRNA Splicing; snRNP.

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Figures

Figure 1
Figure 1. Defects in Ub-H2B promote splicing defects
(A) Schematic of probes contained on the microarray for each intron-containing gene. (B) Heat maps of log2 ratios of each gene feature in bre1Δ or htb1K123R compared to isogenic WT strains after a 3-hour shift to 39°C. Gene order is different for each sub-panel. (C) RT-qPCR measurements of unspliced mRNAs using single-locus RT-qPCR. Percent unspliced RNA is represented as fold change compared to wild-type.
Figure 2
Figure 2. Defects in Ub-H2B, H3K4me, H3K36me cause introns to accumulate for distinct subsets of transcripts
(A, B) Heat maps of log2 ratios of each gene feature in set2Δ or set1Δ strains compared to isogenic WT strains after a 3-hour shift to 39°C. Gene order is different for each sub-panel. (C) Histogram of number of genes exhibiting log2 (Intron/Exon) ratio greater than 0.3 or less than -0.3. Heat map within bar shows degree of splicing change of those genes. (D) Venn diagram of genes from histograms for comparison of each genotype directly. Note: set1Δ and htb1K123R have 1 gene overlap which cannot be shown. The circle sizes are representative of the numbers of genes with log2I/E>0.3 but the overlaps are not to scale.
Figure 3
Figure 3. Preventing ubiquitylation of H2B alters the recruitment of early splicing factors to CBC-associated mRNPs
(A) Nuclear mRNPs were purified from Cbc2-TAP-tagged WT or htb1K123R cells after a 3-hour shift to 39°C and associated proteins were analyzed by MS-MS after SDS-PAGE. In order to highlight relative differences in the protein composition, the number of unique peptides for each indicated protein has been considered as index of abundance (Merz et al., 2007). Complete identification of the CBC proteomes is shown in Table S4. (B) Components of the U1 and U2 snRNPs associated with nuclear mRNPs were semi-quantified using a spectral counting approach. The mean ± SD of spectral counts corresponding to three injections are indicated. Significance of the differences observed between both strains was evaluated using Student t-test (*P 0.01–0.05; ***P <0.001). Significant differences are indicated in bold. (C) Nuclear mRNPs were purified from WT or htb1K123R cells expressing Cbc2-TAP and Prp42-HA or Lea1-HA. Co-purifying proteins were detected by Western Blot with anti-HA or anti-Mex67 antibodies (left panel). The ratio of mRNP-associated proteins relative to the WT cells and to the immuno-purified Cbc2-TAP was determined from at least 3 independent experiments (mean ± SD) (right panel). Significance of the differences observed between both strains was evaluated using Student t-test (**P 0.001–0.01).
Figure 4
Figure 4. Loss of Set1-dependent methylation does not affect the recruitment of U1 snRNP to CBC-associated mRNPs
Nuclear mRNPs were purified from WT or set1Δ cells expressing Cbc2-TAP and Prp42-HA. Co-purifying proteins were detected by Western Blot with anti-HA or anti-Mex67 antibodies (left panel). The ratio of mRNP-associated proteins relative to the WT cells and to the immuno-purified Cbc2-TAP was determined from at least 3 independent experiments (mean ± SD) (right panel). Significance of the differences observed between both strains was evaluated using Student t-test (**P 0.001–0.01).
Figure 5
Figure 5. Loss of H2B ubiquitylation, but not Set1-, Set2- or Dot1- dependent methylation of H3, impairs recruitment of early splicing factors to transcribing genes
ChIP experiments were performed on extracts prepared from the indicated HA-tagged strains after a 3-hour shift to 39°C, using anti-RNAPII or -HA antibodies. Four intron-containing genes were considered, 3 ribosome protein genes RPS21B, RPL14B and RPL34B and the non-ribosome protein gene HNT1 (Supplementary Table S1). Histograms depict the mean and standard deviations of at least three independent experiments. The significance of the differences of recruitment observed between WT and mutant cells was evaluated using Student t-test (*P 0.01–0.05; **P 0.001–0.01).
Figure 6
Figure 6. H2B ubiquitylation promotes to RNAPII Ser2 phosphorylation and Npl3 recruitment on a subset of intron-containing genes
(A) Phosphorylation of RNAPII Ser2 on indicated intron-containing genes was analyzed by ChIP assay in WT and htb1K123R cells using antibodies to RNAPII and phosphoSer2 specific antibodies. The RNAPII Ser2P/RNAPII ratio is shown. (B) Recruitment of Npl3 on indicated intron-containing genes was analyzed by ChIP assay in WT and htb1K123R cells using anti Npl3 antibodies and normalized to the intergenic region. Significance of the differences observed between both strains was evaluated using Student t-test (*P 0.01– 0.05; **P 0.001–0.01).
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