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Genetic interaction mapping reveals a role for the SWI/SNF nucleosome remodeler in spliceosome activation in fission yeast.

Patrick KL, Ryan CJ, Xu J, Lipp JJ, Nissen KE, Roguev A, Shales M, Krogan NJ, Guthrie C - PLoS Genet. (2015)

Bottom Line: Overexpression of SF3 components in ΔSWI/SNF cells led to inefficient splicing of many fission yeast introns, predominantly those with non-consensus splice sites.Deletion of SWI/SNF decreased recruitment of the splicing ATPase Prp2, suggesting that SWI/SNF promotes co-transcriptional spliceosome assembly prior to first step catalysis.Importantly, defects in SWI/SNF as well as SF3 overexpression each altered nucleosome occupancy along intron-containing genes, illustrating that the chromatin landscape both affects--and is affected by--co-transcriptional splicing.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, California, United States of America.

ABSTRACT
Although numerous regulatory connections between pre-mRNA splicing and chromatin have been demonstrated, the precise mechanisms by which chromatin factors influence spliceosome assembly and/or catalysis remain unclear. To probe the genetic network of pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe, we constructed an epistatic mini-array profile (E-MAP) and discovered many new connections between chromatin and splicing. Notably, the nucleosome remodeler SWI/SNF had strong genetic interactions with components of the U2 snRNP SF3 complex. Overexpression of SF3 components in ΔSWI/SNF cells led to inefficient splicing of many fission yeast introns, predominantly those with non-consensus splice sites. Deletion of SWI/SNF decreased recruitment of the splicing ATPase Prp2, suggesting that SWI/SNF promotes co-transcriptional spliceosome assembly prior to first step catalysis. Importantly, defects in SWI/SNF as well as SF3 overexpression each altered nucleosome occupancy along intron-containing genes, illustrating that the chromatin landscape both affects--and is affected by--co-transcriptional splicing.

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Related in: MedlinePlus

Nucleosome occupancy is altered at intron-containing genes by deletion of snf5 and overexpression of sap145.(A.) Schematic representation of nucleosome scanning assay as adapted from [45]. (B.) qPCR analysis of nucleosome occupancy at rps3 and dbp2 (intron-containing genes) in WT, Δsnf5, Δsnf5sap145DAmP strains. Graph is represented as mononucleosome signal/undigested DNA signal at each primer set. Gene representation below is aligned by basepair. Circles represent approximate nucleosome location. Bold circles indicate nucleosomes downstream of introns. (C.) qPCR analysis of nucleosome occupancy at the silenced mat3_Mm gene (untranscribed control) in WT, Δsnf5, Δsnf5sap145DAmP strains. (D.) ChIP performed using anti-RNAPII CTD antibody (8WG16) in WT, Δsnf5, and Δsnf5sap145DAmP strains. Gene diagram below indicates approximate location of qPCR amplicons and basepairs are shown. Capped error bars are ±SEM of n = 3 biological replicates. (E.) qPCR analysis of nucleosome occupancy at rps3, dbp2 and act1 (non-intron containing) for WT and sap145DAmP strains.
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pgen.1005074.g006: Nucleosome occupancy is altered at intron-containing genes by deletion of snf5 and overexpression of sap145.(A.) Schematic representation of nucleosome scanning assay as adapted from [45]. (B.) qPCR analysis of nucleosome occupancy at rps3 and dbp2 (intron-containing genes) in WT, Δsnf5, Δsnf5sap145DAmP strains. Graph is represented as mononucleosome signal/undigested DNA signal at each primer set. Gene representation below is aligned by basepair. Circles represent approximate nucleosome location. Bold circles indicate nucleosomes downstream of introns. (C.) qPCR analysis of nucleosome occupancy at the silenced mat3_Mm gene (untranscribed control) in WT, Δsnf5, Δsnf5sap145DAmP strains. (D.) ChIP performed using anti-RNAPII CTD antibody (8WG16) in WT, Δsnf5, and Δsnf5sap145DAmP strains. Gene diagram below indicates approximate location of qPCR amplicons and basepairs are shown. Capped error bars are ±SEM of n = 3 biological replicates. (E.) qPCR analysis of nucleosome occupancy at rps3, dbp2 and act1 (non-intron containing) for WT and sap145DAmP strains.

Mentions: SWI/SNF is generally thought to deposit and remodel nucleosomes on a DNA template in order to regulate RNAPII transcription. To test whether nucleosome positioning and/or occupancy could be altered in ΔSWI/SNF cells, we performed a nucleosome-scanning assay, as described in Infante et al., 2012 [45]. Following micrococcal nuclease (MNase) treatment, mononucleosome-protected DNA fragments (~150bp) were excised from an agarose gel and tiling qPCR was performed along several fission yeast genomic loci (Fig. 6A). At all expressed genes examined, we saw a striking decrease in signal from protected DNA in Δsnf5 and Δsnf5sap145DAmP cells, implicating Snf5 in depositing or maintaining the position of nucleosomes along coding regions (Fig. 6B, replicate experiment in S6B, C, D Fig). Both the Δsnf5 single mutant and the Δsnf5sap145DAmP double mutant displayed lower nucleosome occupancy at intron-containing genes, although certain nucleosomes were differentially protected; generally, nucleosomes downstream of introns exhibited the biggest changes in nucleosome occupancy relative to wild type (Fig. 6B, bold circles). We propose that a certain level/distribution of nucleosomes is required for optimal recruitment of Prp2 and that SWI/SNF contributes to creation and/or maintenance of this nucleosome environment at intron-containing genes.


Genetic interaction mapping reveals a role for the SWI/SNF nucleosome remodeler in spliceosome activation in fission yeast.

Patrick KL, Ryan CJ, Xu J, Lipp JJ, Nissen KE, Roguev A, Shales M, Krogan NJ, Guthrie C - PLoS Genet. (2015)

Nucleosome occupancy is altered at intron-containing genes by deletion of snf5 and overexpression of sap145.(A.) Schematic representation of nucleosome scanning assay as adapted from [45]. (B.) qPCR analysis of nucleosome occupancy at rps3 and dbp2 (intron-containing genes) in WT, Δsnf5, Δsnf5sap145DAmP strains. Graph is represented as mononucleosome signal/undigested DNA signal at each primer set. Gene representation below is aligned by basepair. Circles represent approximate nucleosome location. Bold circles indicate nucleosomes downstream of introns. (C.) qPCR analysis of nucleosome occupancy at the silenced mat3_Mm gene (untranscribed control) in WT, Δsnf5, Δsnf5sap145DAmP strains. (D.) ChIP performed using anti-RNAPII CTD antibody (8WG16) in WT, Δsnf5, and Δsnf5sap145DAmP strains. Gene diagram below indicates approximate location of qPCR amplicons and basepairs are shown. Capped error bars are ±SEM of n = 3 biological replicates. (E.) qPCR analysis of nucleosome occupancy at rps3, dbp2 and act1 (non-intron containing) for WT and sap145DAmP strains.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4380400&req=5

pgen.1005074.g006: Nucleosome occupancy is altered at intron-containing genes by deletion of snf5 and overexpression of sap145.(A.) Schematic representation of nucleosome scanning assay as adapted from [45]. (B.) qPCR analysis of nucleosome occupancy at rps3 and dbp2 (intron-containing genes) in WT, Δsnf5, Δsnf5sap145DAmP strains. Graph is represented as mononucleosome signal/undigested DNA signal at each primer set. Gene representation below is aligned by basepair. Circles represent approximate nucleosome location. Bold circles indicate nucleosomes downstream of introns. (C.) qPCR analysis of nucleosome occupancy at the silenced mat3_Mm gene (untranscribed control) in WT, Δsnf5, Δsnf5sap145DAmP strains. (D.) ChIP performed using anti-RNAPII CTD antibody (8WG16) in WT, Δsnf5, and Δsnf5sap145DAmP strains. Gene diagram below indicates approximate location of qPCR amplicons and basepairs are shown. Capped error bars are ±SEM of n = 3 biological replicates. (E.) qPCR analysis of nucleosome occupancy at rps3, dbp2 and act1 (non-intron containing) for WT and sap145DAmP strains.
Mentions: SWI/SNF is generally thought to deposit and remodel nucleosomes on a DNA template in order to regulate RNAPII transcription. To test whether nucleosome positioning and/or occupancy could be altered in ΔSWI/SNF cells, we performed a nucleosome-scanning assay, as described in Infante et al., 2012 [45]. Following micrococcal nuclease (MNase) treatment, mononucleosome-protected DNA fragments (~150bp) were excised from an agarose gel and tiling qPCR was performed along several fission yeast genomic loci (Fig. 6A). At all expressed genes examined, we saw a striking decrease in signal from protected DNA in Δsnf5 and Δsnf5sap145DAmP cells, implicating Snf5 in depositing or maintaining the position of nucleosomes along coding regions (Fig. 6B, replicate experiment in S6B, C, D Fig). Both the Δsnf5 single mutant and the Δsnf5sap145DAmP double mutant displayed lower nucleosome occupancy at intron-containing genes, although certain nucleosomes were differentially protected; generally, nucleosomes downstream of introns exhibited the biggest changes in nucleosome occupancy relative to wild type (Fig. 6B, bold circles). We propose that a certain level/distribution of nucleosomes is required for optimal recruitment of Prp2 and that SWI/SNF contributes to creation and/or maintenance of this nucleosome environment at intron-containing genes.

Bottom Line: Overexpression of SF3 components in ΔSWI/SNF cells led to inefficient splicing of many fission yeast introns, predominantly those with non-consensus splice sites.Deletion of SWI/SNF decreased recruitment of the splicing ATPase Prp2, suggesting that SWI/SNF promotes co-transcriptional spliceosome assembly prior to first step catalysis.Importantly, defects in SWI/SNF as well as SF3 overexpression each altered nucleosome occupancy along intron-containing genes, illustrating that the chromatin landscape both affects--and is affected by--co-transcriptional splicing.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, California, United States of America.

ABSTRACT
Although numerous regulatory connections between pre-mRNA splicing and chromatin have been demonstrated, the precise mechanisms by which chromatin factors influence spliceosome assembly and/or catalysis remain unclear. To probe the genetic network of pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe, we constructed an epistatic mini-array profile (E-MAP) and discovered many new connections between chromatin and splicing. Notably, the nucleosome remodeler SWI/SNF had strong genetic interactions with components of the U2 snRNP SF3 complex. Overexpression of SF3 components in ΔSWI/SNF cells led to inefficient splicing of many fission yeast introns, predominantly those with non-consensus splice sites. Deletion of SWI/SNF decreased recruitment of the splicing ATPase Prp2, suggesting that SWI/SNF promotes co-transcriptional spliceosome assembly prior to first step catalysis. Importantly, defects in SWI/SNF as well as SF3 overexpression each altered nucleosome occupancy along intron-containing genes, illustrating that the chromatin landscape both affects--and is affected by--co-transcriptional splicing.

Show MeSH
Related in: MedlinePlus