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Loss of the yeast SR protein Npl3 alters gene expression due to transcription readthrough

ABSTRACT: 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.

ORGANISM(S): Saccharomyces cerevisiae S288c

SUBMITTER: Chenchen Zhu

PROVIDER: E-MTAB-3642 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Loss of the yeast SR protein Npl3 alters gene expression due to transcription readthrough

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

The SR-like protein Npl3 transfers mRNP components from the transcription site onto the mRNA

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

Unphosphorylated SR-like Protein Npl3 Stimulates RNA Polymerase II Elongation

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

A single SR-like protein, Npl3, promotes pre-mRNA splicing in budding yeast

2008-12-31 | GSE11491 | GEO

A single SR-like protein, Npl3, promotes pre-mRNA splicing in budding yeast

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

Npl3 stabilizes R-loops at telomeres to prevent accelerated replicative senescence

Project description:Telomere shortening rates must be regulated to prevent premature replicative senescence. TERRA R-loops become stabilized at critically short telomeres to promote their elongation through homology-directed repair (HDR), thereby counteracting senescence onset. Using a non-bias proteomic approach to identify telomere binding factors, we identified Npl3, an RNA-binding protein previously implicated in multiple RNA biogenesis processes. Using Chromatin- and RNA immunoprecipitation, we demonstrate that Npl3 interacts with TERRA and telomeres. Furthermore, we show that Npl3 associates to telomeres in an R-loop dependent manner, as changes in R-loop levels, e.g. at short telomeres, modulate the recruitment of Npl3 to chromosome ends. Through a series of genetic and biochemical approaches we demonstrate that Npl3 binds to TERRA and stabilizes R-loops at short telomeres, which in turn promotes HDR and prevents premature replicative senescence onset. This may have implications for diseases associated with excessive telomere shortening.

2020-01-31 | PXD016772 | Pride

Npl3 functions in mRNP assembly by recruitment of mRNP components to the transcription site and their transfer onto the mRNA

Project description:Assessing the function of the RNA-binding activity of RNA-binding proteins (RBPs) is challenging. Here, Keil et al. use a targeted XL-ms approach to identify RNA-binding sites in components of nuclear mRNPs in S. cerevisiae. The analysis of RNA-binding mutants of one selected example, the SR-like protein Npl3, uncovers two novel functions of Npl3, namely in the recruitment of mRNP components to transcribed protein-coding genes and their transfer onto the mRNA. This data set applies label-free protein quantification to assess protein abundancies in NPL3 or CBC2 affinity purification experiments from cellular lysates of NPL3-WT and NPL3-P196D/A197D double-mutant expressing cells.

2023-03-11 | PXD034656 | Pride

The Nrd1-like protein Seb1 coordinates co-transcriptional 3â end processing and polyadenylation site selection

Project description:Termination of RNAPII transcription is associated with RNA 3â end formation. For coding genes, termination is initiated by the cleavage/polyadenylation machinery. In contrast, a majority of noncoding transcription events in S. cerevisiae do not rely on RNA cleavage for termination, but instead terminate via a pathway that requires the Nrd1-Nab3-Sen1 (NNS) complex. Here we show that the S. pombe ortholog of Nrd1, Seb1, does not function in NNS-like termination, but promotes polyadenylation site selection of coding and noncoding genes. We found that Seb1 associates with 3â end processing factors, is enriched at the 3â end of genes, and binds RNA motifs downstream of cleavage sites. Importantly, a deficiency in Seb1 resulted in widespread changes in 3â UTR length as a consequence of increased alternative polyadenylation. Given that Seb1 levels affected the recruitment of conserved 3â end processing factors, our findings indicate that the conserved RNA-binding protein Seb1 co-transcriptionally controls alternative polyadenylation. Two biological replicates of Seb1 and Control (parental strain) CRAC experiments

2016-07-22 | E-GEOD-82326 | biostudies-arrayexpress

ChIP-chip data of Npl3-Myc and Rrm3-Flag in wild-type and npl3∆ cells

Project description:Transcription is a major obstacle for replication fork progression and a cause of genome instability. Such instability increases in mutants with a suboptimal assembly of the nascent messenger ribonucleo-protein particle (mRNP), as THO/TREX and some heterogeneous nuclear ribonucleoproteins (hnRNPs) mutants. Here we show that yeast npl3∆ cells show genome-wide replication obstacles as determined by accumulation of the Rrm3 helicase. Such obstacles preferentially occur at long and highly expressed genes, to which Npl3 is preferentially bound in wild-type cells.

2013-11-15 | GSE50185 | GEO

Npl3 functions in mRNP assembly by recruitment of mRNP components to the transcription site and their transfer onto the mRNA

Project description:RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate several functions at once and likely impact the structural integrity of the large mRNP assemblies, these proteins often function in. Thus, the function of the RNA-binding activity of RBPs is often unknown. Using UV-induced RNA-protein crosslinking and subsequent mass spectrometric (MS) analysis, we first identified more than 100 in vivo RNA crosslinks in 16 nuclear mRNP components. For functional analysis, we chose Npl3, for which we identified crosslinks in its two RNA recognition motifs (RRMs) as well as in the flexible linker connecting the two. Both RRM domains and the linker uniquely contribute to RNA recognition. Interestingly, mutations in each of these three regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains of Npl3. Notably, the npl3-Linker mutant strongly impairs recruitment of some mRNP components to chromatin and the incorporation of other mRNP components into nuclear mRNPs, establishing a function of Npl3 in mRNP assembly. Taken together, we determined the specific function of the RNA-binding activity of a nuclear mRNP component, an approach that can be applied to many RBPs in any RNA metabolic process.

2023-03-11 | PXD035153 | Pride

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