Saccharomyces cerevisiae
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA106303 | ENA |
REPOSITORIES: ENA
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Project description:Mammalian SR proteins are a family of reversibly phosphorylated RNA binding proteins primarily studied for their roles in alternative splicing. While budding yeast lack alternative splicing, they do have three SR-like proteins: Npl3, Gbp2, and Hrb1. However, these have been primarily studied for their roles in mRNA export, leaving their potential roles in splicing largely unexplored. Here we combined high-density genetic interaction profiling and genome-wide splicing-sensitive microarray analysis to demonstrate that a single SR-like protein, Npl3, is required for efficient splicing of a large set of pre-mRNAs in Saccharomyces cerevisiae. We tested the hypothesis that Npl3 promotes splicing by facilitating co-transcriptional recruitment of splicing factors. Using chromatin immunoprecipitation, we showed that mutation of NPL3 reduces the occupancy of U1 and U2 snRNPs at Npl3-stimulated genes. This provides the first evidence that an SR protein can promote recruitment of splicing factors to chromatin.
2008-12-31 | GSE11491 | GEO
Project description:Mammalian SR proteins are a family of reversibly phosphorylated RNA binding proteins primarily studied for their roles in alternative splicing. While budding yeast lack alternative splicing, they do have three SR-like proteins: Npl3, Gbp2, and Hrb1. However, these have been primarily studied for their roles in mRNA export, leaving their potential roles in splicing largely unexplored. Here we combined high-density genetic interaction profiling and genome-wide splicing-sensitive microarray analysis to demonstrate that a single SR-like protein, Npl3, is required for efficient splicing of a large set of pre-mRNAs in Saccharomyces cerevisiae. We tested the hypothesis that Npl3 promotes splicing by facilitating co-transcriptional recruitment of splicing factors. Using chromatin immunoprecipitation, we showed that mutation of NPL3 reduces the occupancy of U1 and U2 snRNPs at Npl3-stimulated genes. This provides the first evidence that an SR protein can promote recruitment of splicing factors to chromatin. Splicing-specific microarrays were used to assay changes to splicing in single and double deletion mutants of non-essential SR proteins, in a deletion mutant of a non-essential component of the nonsense-mediated decay pathway, and in a double deletion mutant of in an SR protein plus a non-sense mediated decay factor in Saccharomyces cerevisiae. The data includes both samples obtained at the permissive temperature and also shifts to the non-permissive temperature for some mutants, as well as dye-flipped technical replicates.
2008-12-30 | E-GEOD-11491 | biostudies-arrayexpress
Project description:The conserved SR-like protein Npl3 promotes the splicing of diverse pre-mRNAs. However, the RNA sequence(s) recognized by the RNA Recognition Motifs (RRM1 & RRM2) of Npl3 during the splicing reaction remain elusive. Here, we developed a split-iCRAC approach in vivo to determine the consensus sequence bound to each RRM in yeast. High-resolution NMR structures show that RRM2 recognizes a 5´-GNGG-3´ motif leading to an unusual mille-feuille topology. In addition, our data indicate a non-specific interaction of the RS domain with RNA. These structures reveal how RRM1 preferentially interacts with a CC-dinucleotide upstream of this motif, and how the inter-RRM linker and the region C-terminal to RRM2 contributes to cooperative RNA-binding. Structure-guided studies show that Npl3 genetically interacts with U2 snRNP specific factors and we provide evidence that Npl3 melts U2 snRNA stem-loop I, a prerequisite for U2/U6 duplex formation within the catalytic centre of the Bact spliceosomal complex. Our findings provide a mechanistic role for Npl3 during spliceosome active site formation.
2022-12-01 | E-MTAB-11736 | biostudies-arrayexpress
Project description:Yeast Npl3 is a highly abundant RNA binding protein, related to metazoan SR proteins, with reported functions including transcription elongation, splicing and RNA 3’ end processing. To identify direct targets and functions for Npl3, we used UV crosslinking and analysis of cDNA (CRAC) to map precise RNA binding sites. Npl3 binds diverse RNA species, at sites indicative of roles in both early pre-mRNA processing and 3’ end formation on mRNAs and ncRNAs. Consistent with this, tiling array and RNAPII binding data revealed 3’ extended mRNA and snoRNA transcripts in the absence of Npl3. This reflected transcriptional readthrough by RNAPII, and extension and stabilization of cryptic unstable transcript (CUT) long noncoding RNAs. Transcription readthrough was widespread, often resulting in down-regulation of neighboring genes. We conclude that Npl3 is required for the formation of a termination-competent RNA, affecting both coding and noncoding RNAs.
2015-12-02 | E-MTAB-3642 | biostudies-arrayexpress
Project description:Yeast Npl3 is a highly abundant RNA binding protein, related to metazoan SR proteins, with reported functions including transcription elongation, splicing and RNA 3’ end processing. To identify direct targets and functions for Npl3, we used UV crosslinking and analysis of cDNA (CRAC) to map precise RNA binding sites. Npl3 binds diverse RNA species, at sites indicative of roles in both early pre-mRNA processing and 3’ end formation on mRNAs and ncRNAs. Consistent with this, tiling array and RNAPII binding data revealed 3’ extended mRNA and snoRNA transcripts in the absence of Npl3. This reflected transcriptional readthrough by RNAPII, and extension and stabilization of cryptic unstable transcript (CUT) long noncoding RNAs. Transcription readthrough was widespread, often resulting in down-regulation of neighboring genes. We conclude that Npl3 is required for the formation of a termination-competent RNA, affecting both coding and noncoding RNAs.
2015-12-03 | GSE70191 | GEO
Project description:Eukaryotic cells have to prevent the export of unspliced pre-mRNAs until intron removal is completed to avoid the expression of aberrant and potentially harmful proteins. Only mature RNAs associate with the export receptor Mex67 (mammalian TAP) and enter the cytoplasm. The underlying nuclear quality control mechanisms are still unclear. Here we show that two shuttling SR-proteins Gbp2 and Hrb1 are key surveillance factors for the selective export of spliced mRNAs in yeast. Their absence leads to the significant leakage of unspliced pre-mRNAs into the cytoplasm. They bind to pre-mRNAs and the spliceosome during splicing, where they are necessary for the surveillance of splicing and the stable binding of the TRAMP-complex to the spliceosome-bound transcripts. Faulty transcripts are marked for their degradation at the nuclear exosome. On correct mRNAs the SR-proteins recruit Mex67 upon completion of splicing to allow a quality controlled nuclear export. Altogether, these data identify a role for shuttling SR-proteins in mRNA surveillance and nuclear mRNA quality control. 6 samples, i.e. 2 replicates per protein Gbp2, Hrb1 and Npl3
2013-12-04 | E-GEOD-52808 | biostudies-arrayexpress
Project description:The production of a functional mRNA is regulated at every step of transcription. An area not well-understood is the transition of RNA polymerase II from elongation to termination. The S. cerevisiae SR-like protein Npl3 functions to negatively regulate transcription termination by antagonizing the binding of polyA/termination proteins to the mRNA. In this study, Npl3 is shown to interact with the CTD and have a direct stimulatory effect on the elongation activity of the polymerase. The interaction is inhibited by phosphorylation of Npl3. In addition, Casein Kinase 2 was found to specifically phosphorylate Npl3 and affect its ability to compete against Rna15 (Cleavage Factor I) for binding to polyA signals. Our results suggest that phosphorylation of Npl3 promotes its dissociation from the mRNA/RNAP II, and contributes to the association of the polyA/termination factor Rna15. This work defines a novel role for Npl3 in elongation and its regulation by phosphorylation. Keywords: Gene expression on a tiling array
2008-09-06 | GSE12677 | GEO
Project description:Loss of the yeast SR protein Npl3 alters gene expression due to transcription readthrough
| PRJNA287824 | ENA
Project description:In order to determine specifically the RNA-binding function of proteins involved in nuclear mRNP assembly, we first determined the amino acids involved in RNA-binding by RNPXL. We identified about 100 amino acids cross-linked to RNA in vivo in the nuclear mRNP components Npl3, Nab2, Tho1, Mex67-Mtr2 and the TREX complex. Second, we can now specifically elucidate the function of the RNA-binding activity of these proteins by mutation of the identified amino acids. Npl3 is an SR-like protein with functions in transcription elongation, splicing, 3’ end processing, mRNP assembly and nuclear mRNA export. The middle part of Npl3 consists of two RNA recognition motif (RRM) domains, RRM1 and RRM2, connected by an eight amino acid long flexible linker. In order to analyze the function of the RNA-binding activity of Npl3, we mutated several amino acids that cross-linked to RNA. We generated three npl3 mutants, one in the RRM1, one in the linker and a third in the RRM2 domain, and elucidated the functional consequences of these mutations. Interestingly, these three npl3 mutants show different phenotypes. Thus, abrogation of mRNA-binding in different regions of Npl3 has different functional outcomes. Furthermore, analysis of the npl3-Linker mutant revealed a novel function of Npl3. Npl3 functions in the transfer of nuclear mRNP components from the site of transcription onto the mRNA. Taken together, we identify the in vivo RNA-binding sites of nuclear mRNA-binding proteins involved in mRNP assembly and nuclear mRNA export. In addition, we show that abrogation of RNA-binding in different regions of the protein Npl3 has specific and surprisingly different functional consequences. Furthermore, we uncovered a novel function of Npl3 in nuclear mRNP assembly.
2022-11-26 | GSE160709 | GEO
Project description:Unphosphorylated SR-like Protein Npl3 Stimulates RNA Polymerase II Elongation
| PRJNA112753 | ENA