Limits...
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.

Show MeSH

Related in: MedlinePlus

Reduction of transcript levels in rsr-2(RNAi) L4 animals.(A) Comparison of the average signal intensities of transcripts, exons and introns in tiling arrays between gfp(RNAi) and rsr-2(RNAi) animals. Error bars represent standard error of the mean (SEM). (B) mRNA levels of several sex determination genes upon rsr-2 RNAi. qPCR expression data was normalized to transcript levels of tbb-2. mRNA levels in rsr-2(RNAi) animals are represented relative to the expression in gfp(RNAi) control animals (arbitrary value of 1.0). Three separate experiments were analyzed. Bars represent the standard deviation within each data set. Student's independent samples t-test was used to study significantly different gene expression between the two conditions: one, two and three asterisks indicate p<0.05, p<0.01 and p<0.001, respectively. (C) A subset of germline-related genes are correctly spliced upon rsr-2 inactivation. Semiquantitative RT-PCR analysis of germline transcripts in wild type and smg-5(r680) NMD mutants treated with control gfp RNAi and rsr-2 RNAi. rpl-12 was used as a control for defective NMD pathway. act-1 was used as an endogenous control.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3675011&req=5

pgen-1003543-g003: Reduction of transcript levels in rsr-2(RNAi) L4 animals.(A) Comparison of the average signal intensities of transcripts, exons and introns in tiling arrays between gfp(RNAi) and rsr-2(RNAi) animals. Error bars represent standard error of the mean (SEM). (B) mRNA levels of several sex determination genes upon rsr-2 RNAi. qPCR expression data was normalized to transcript levels of tbb-2. mRNA levels in rsr-2(RNAi) animals are represented relative to the expression in gfp(RNAi) control animals (arbitrary value of 1.0). Three separate experiments were analyzed. Bars represent the standard deviation within each data set. Student's independent samples t-test was used to study significantly different gene expression between the two conditions: one, two and three asterisks indicate p<0.05, p<0.01 and p<0.001, respectively. (C) A subset of germline-related genes are correctly spliced upon rsr-2 inactivation. Semiquantitative RT-PCR analysis of germline transcripts in wild type and smg-5(r680) NMD mutants treated with control gfp RNAi and rsr-2 RNAi. rpl-12 was used as a control for defective NMD pathway. act-1 was used as an endogenous control.

Mentions: To shed light on the molecular functions of rsr-2 during C. elegans development, we used Affymetrix tiling arrays to examine not only transcript levels, but also intron retention events. We purified total RNA from synchronized L4 animals, when the sperm/oocyte switch takes place, grown on gfp and rsr-2 dsRNA-expressing bacteria. We used two biological replicates for each condition, and raw data (in CEL files) were analyzed using the Tiling Analysis Software (TAS) developed by Affymetrix. This analysis provided information about the levels of 30,430 transcripts. To estimate gene expression in gfp(RNAi) and rsr-2(RNAi) animals, we plotted mean signal intensities for transcripts, exons and introns by chromosomes (Figure 3A). These analyses indicated that the rsr-2 RNAi produced a slight overall reduction in transcript, exon and intron levels in all chromosomes, with the exception of the X chromosome. Mean signal intensities and normalized values for transcripts, exons and introns are shown in Table S1.


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)

Reduction of transcript levels in rsr-2(RNAi) L4 animals.(A) Comparison of the average signal intensities of transcripts, exons and introns in tiling arrays between gfp(RNAi) and rsr-2(RNAi) animals. Error bars represent standard error of the mean (SEM). (B) mRNA levels of several sex determination genes upon rsr-2 RNAi. qPCR expression data was normalized to transcript levels of tbb-2. mRNA levels in rsr-2(RNAi) animals are represented relative to the expression in gfp(RNAi) control animals (arbitrary value of 1.0). Three separate experiments were analyzed. Bars represent the standard deviation within each data set. Student's independent samples t-test was used to study significantly different gene expression between the two conditions: one, two and three asterisks indicate p<0.05, p<0.01 and p<0.001, respectively. (C) A subset of germline-related genes are correctly spliced upon rsr-2 inactivation. Semiquantitative RT-PCR analysis of germline transcripts in wild type and smg-5(r680) NMD mutants treated with control gfp RNAi and rsr-2 RNAi. rpl-12 was used as a control for defective NMD pathway. act-1 was used as an endogenous control.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003543-g003: Reduction of transcript levels in rsr-2(RNAi) L4 animals.(A) Comparison of the average signal intensities of transcripts, exons and introns in tiling arrays between gfp(RNAi) and rsr-2(RNAi) animals. Error bars represent standard error of the mean (SEM). (B) mRNA levels of several sex determination genes upon rsr-2 RNAi. qPCR expression data was normalized to transcript levels of tbb-2. mRNA levels in rsr-2(RNAi) animals are represented relative to the expression in gfp(RNAi) control animals (arbitrary value of 1.0). Three separate experiments were analyzed. Bars represent the standard deviation within each data set. Student's independent samples t-test was used to study significantly different gene expression between the two conditions: one, two and three asterisks indicate p<0.05, p<0.01 and p<0.001, respectively. (C) A subset of germline-related genes are correctly spliced upon rsr-2 inactivation. Semiquantitative RT-PCR analysis of germline transcripts in wild type and smg-5(r680) NMD mutants treated with control gfp RNAi and rsr-2 RNAi. rpl-12 was used as a control for defective NMD pathway. act-1 was used as an endogenous control.
Mentions: To shed light on the molecular functions of rsr-2 during C. elegans development, we used Affymetrix tiling arrays to examine not only transcript levels, but also intron retention events. We purified total RNA from synchronized L4 animals, when the sperm/oocyte switch takes place, grown on gfp and rsr-2 dsRNA-expressing bacteria. We used two biological replicates for each condition, and raw data (in CEL files) were analyzed using the Tiling Analysis Software (TAS) developed by Affymetrix. This analysis provided information about the levels of 30,430 transcripts. To estimate gene expression in gfp(RNAi) and rsr-2(RNAi) animals, we plotted mean signal intensities for transcripts, exons and introns by chromosomes (Figure 3A). These analyses indicated that the rsr-2 RNAi produced a slight overall reduction in transcript, exon and intron levels in all chromosomes, with the exception of the X chromosome. Mean signal intensities and normalized values for transcripts, exons and introns are shown in Table S1.

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