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RSR-2, the Caenorhabditis elegans ortholog of human spliceosomal component SRm300/SRRM2, regulates development by influencing the transcriptional machinery.

Fontrodona L, Porta-de-la-Riva M, Morán T, Niu W, Díaz M, Aristizábal-Corrales D, Villanueva A, Schwartz S, Reinke V, Cerón J - PLoS Genet. (2013)

Bottom Line: We took advantage of mutants and RNA interference (RNAi) to study rsr-2 functions in C. elegans, and through genetic epistasis analysis found that rsr-2 is within the germline sex determination pathway.Intriguingly, transcriptome analyses of rsr-2(RNAi) animals did not reveal appreciable splicing defects but instead a slight global decrease in transcript levels.Proteomic analyses identified proteins associated with RSR-2 that are involved in different gene expression steps, including RNA metabolism and transcription with PRP-8 and PRP-19 being the strongest interacting partners.

View Article: PubMed Central - PubMed

Affiliation: Cancer and Human Molecular Genetics, Bellvitge Biomedical Research Institute - IDIBELL, L'Hospitalet de Llobregat, Barcelona, Spain.

ABSTRACT
Protein components of the spliceosome are highly conserved in eukaryotes and can influence several steps of the gene expression process. RSR-2, the Caenorhabditis elegans ortholog of the human spliceosomal protein SRm300/SRRM2, is essential for viability, in contrast to the yeast ortholog Cwc21p. We took advantage of mutants and RNA interference (RNAi) to study rsr-2 functions in C. elegans, and through genetic epistasis analysis found that rsr-2 is within the germline sex determination pathway. Intriguingly, transcriptome analyses of rsr-2(RNAi) animals did not reveal appreciable splicing defects but instead a slight global decrease in transcript levels. We further investigated this effect in transcription and observed that RSR-2 colocalizes with DNA in germline nuclei and coprecipitates with chromatin, displaying a ChIP-Seq profile similar to that obtained for the RNA Polymerase II (RNAPII). Consistent with a novel transcription function we demonstrate that the recruitment of RSR-2 to chromatin is splicing-independent and that RSR-2 interacts with RNAPII and affects RNAPII phosphorylation states. Proteomic analyses identified proteins associated with RSR-2 that are involved in different gene expression steps, including RNA metabolism and transcription with PRP-8 and PRP-19 being the strongest interacting partners. PRP-8 is a core component of the spliceosome and PRP-19 is the core component of the PRP19 complex, which interacts with RNAPII and is necessary for full transcriptional activity. Taken together, our study proposes that RSR-2 is a multifunctional protein whose role in transcription influences C. elegans development.

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RSR-2 is associated with chromatin and modifies RNAPII distribution.(A) Snapshot of the genome browser (chromosome IV) showing ChIP-Seq data. Chromatin-binding profiles of RNAPII and RSR-2 are similar. RNAPII peaks are represented in black, RSR-2 peaks are represented in red, and input samples are represented in blue. Upon rsr-2 RNAi, the RNAPII peak profile at phy-2 locus shifts from the 3′ to the 5′end. RSR-2 peaks disappear upon rsr-2 RNAi. Data was visualized with the Integrated Genome Browser (IGB) software. (B) ChIP-qPCR showing how RNAPII occupancy changes upon rsr-2 RNAi at the phy-2 locus. Black bars are a scaled representation of the regions covered by the primer pairs used in this experiment. (C) Distribution of RNAPII and RSR-2 ChIP peaks along five zones within an averaged gene. Statistically called peaks from a single ChIP-Seq experiment were classified with respect to their position relative to the nearest TSS. Y axis indicates the frequency of peaks within each zone. rsr-2 RNAi slightly modifies the distribution of RNAPII along an averaged gene.
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pgen-1003543-g005: RSR-2 is associated with chromatin and modifies RNAPII distribution.(A) Snapshot of the genome browser (chromosome IV) showing ChIP-Seq data. Chromatin-binding profiles of RNAPII and RSR-2 are similar. RNAPII peaks are represented in black, RSR-2 peaks are represented in red, and input samples are represented in blue. Upon rsr-2 RNAi, the RNAPII peak profile at phy-2 locus shifts from the 3′ to the 5′end. RSR-2 peaks disappear upon rsr-2 RNAi. Data was visualized with the Integrated Genome Browser (IGB) software. (B) ChIP-qPCR showing how RNAPII occupancy changes upon rsr-2 RNAi at the phy-2 locus. Black bars are a scaled representation of the regions covered by the primer pairs used in this experiment. (C) Distribution of RNAPII and RSR-2 ChIP peaks along five zones within an averaged gene. Statistically called peaks from a single ChIP-Seq experiment were classified with respect to their position relative to the nearest TSS. Y axis indicates the frequency of peaks within each zone. rsr-2 RNAi slightly modifies the distribution of RNAPII along an averaged gene.

Mentions: We generated a rabbit polyclonal antibody against RSR-2 (Q5092) and confirmed its specificity by western blots and in immunofluorescence assays (Figure S5A and S5C). Interestingly, anti-RSR-2 staining overlapped with chromatin as indicated by confocal images of germ nuclei (Figure 4E). We next performed ChIP-Seq experiments to investigate the association of RSR-2 with chromatin at the L4 stage using RNAPII as a control. Surprisingly, the anti-RSR-2 antibody was capable of precipitating chromatin in a ChIP-Seq pattern that resembled that of anti-RNAPII (8WG16) (Figure 5A and S10). As expected, RSR-2 peaks disappeared in worms treated with rsr-2(RNAi) (Figure 5A).


RSR-2, the Caenorhabditis elegans ortholog of human spliceosomal component SRm300/SRRM2, regulates development by influencing the transcriptional machinery.

Fontrodona L, Porta-de-la-Riva M, Morán T, Niu W, Díaz M, Aristizábal-Corrales D, Villanueva A, Schwartz S, Reinke V, Cerón J - PLoS Genet. (2013)

RSR-2 is associated with chromatin and modifies RNAPII distribution.(A) Snapshot of the genome browser (chromosome IV) showing ChIP-Seq data. Chromatin-binding profiles of RNAPII and RSR-2 are similar. RNAPII peaks are represented in black, RSR-2 peaks are represented in red, and input samples are represented in blue. Upon rsr-2 RNAi, the RNAPII peak profile at phy-2 locus shifts from the 3′ to the 5′end. RSR-2 peaks disappear upon rsr-2 RNAi. Data was visualized with the Integrated Genome Browser (IGB) software. (B) ChIP-qPCR showing how RNAPII occupancy changes upon rsr-2 RNAi at the phy-2 locus. Black bars are a scaled representation of the regions covered by the primer pairs used in this experiment. (C) Distribution of RNAPII and RSR-2 ChIP peaks along five zones within an averaged gene. Statistically called peaks from a single ChIP-Seq experiment were classified with respect to their position relative to the nearest TSS. Y axis indicates the frequency of peaks within each zone. rsr-2 RNAi slightly modifies the distribution of RNAPII along an averaged gene.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675011&req=5

pgen-1003543-g005: RSR-2 is associated with chromatin and modifies RNAPII distribution.(A) Snapshot of the genome browser (chromosome IV) showing ChIP-Seq data. Chromatin-binding profiles of RNAPII and RSR-2 are similar. RNAPII peaks are represented in black, RSR-2 peaks are represented in red, and input samples are represented in blue. Upon rsr-2 RNAi, the RNAPII peak profile at phy-2 locus shifts from the 3′ to the 5′end. RSR-2 peaks disappear upon rsr-2 RNAi. Data was visualized with the Integrated Genome Browser (IGB) software. (B) ChIP-qPCR showing how RNAPII occupancy changes upon rsr-2 RNAi at the phy-2 locus. Black bars are a scaled representation of the regions covered by the primer pairs used in this experiment. (C) Distribution of RNAPII and RSR-2 ChIP peaks along five zones within an averaged gene. Statistically called peaks from a single ChIP-Seq experiment were classified with respect to their position relative to the nearest TSS. Y axis indicates the frequency of peaks within each zone. rsr-2 RNAi slightly modifies the distribution of RNAPII along an averaged gene.
Mentions: We generated a rabbit polyclonal antibody against RSR-2 (Q5092) and confirmed its specificity by western blots and in immunofluorescence assays (Figure S5A and S5C). Interestingly, anti-RSR-2 staining overlapped with chromatin as indicated by confocal images of germ nuclei (Figure 4E). We next performed ChIP-Seq experiments to investigate the association of RSR-2 with chromatin at the L4 stage using RNAPII as a control. Surprisingly, the anti-RSR-2 antibody was capable of precipitating chromatin in a ChIP-Seq pattern that resembled that of anti-RNAPII (8WG16) (Figure 5A and S10). As expected, RSR-2 peaks disappeared in worms treated with rsr-2(RNAi) (Figure 5A).

Bottom Line: We took advantage of mutants and RNA interference (RNAi) to study rsr-2 functions in C. elegans, and through genetic epistasis analysis found that rsr-2 is within the germline sex determination pathway.Intriguingly, transcriptome analyses of rsr-2(RNAi) animals did not reveal appreciable splicing defects but instead a slight global decrease in transcript levels.Proteomic analyses identified proteins associated with RSR-2 that are involved in different gene expression steps, including RNA metabolism and transcription with PRP-8 and PRP-19 being the strongest interacting partners.

View Article: PubMed Central - PubMed

Affiliation: Cancer and Human Molecular Genetics, Bellvitge Biomedical Research Institute - IDIBELL, L'Hospitalet de Llobregat, Barcelona, Spain.

ABSTRACT
Protein components of the spliceosome are highly conserved in eukaryotes and can influence several steps of the gene expression process. RSR-2, the Caenorhabditis elegans ortholog of the human spliceosomal protein SRm300/SRRM2, is essential for viability, in contrast to the yeast ortholog Cwc21p. We took advantage of mutants and RNA interference (RNAi) to study rsr-2 functions in C. elegans, and through genetic epistasis analysis found that rsr-2 is within the germline sex determination pathway. Intriguingly, transcriptome analyses of rsr-2(RNAi) animals did not reveal appreciable splicing defects but instead a slight global decrease in transcript levels. We further investigated this effect in transcription and observed that RSR-2 colocalizes with DNA in germline nuclei and coprecipitates with chromatin, displaying a ChIP-Seq profile similar to that obtained for the RNA Polymerase II (RNAPII). Consistent with a novel transcription function we demonstrate that the recruitment of RSR-2 to chromatin is splicing-independent and that RSR-2 interacts with RNAPII and affects RNAPII phosphorylation states. Proteomic analyses identified proteins associated with RSR-2 that are involved in different gene expression steps, including RNA metabolism and transcription with PRP-8 and PRP-19 being the strongest interacting partners. PRP-8 is a core component of the spliceosome and PRP-19 is the core component of the PRP19 complex, which interacts with RNAPII and is necessary for full transcriptional activity. Taken together, our study proposes that RSR-2 is a multifunctional protein whose role in transcription influences C. elegans development.

Show MeSH
Related in: MedlinePlus