The impact of Prp18p on splicing fidelity and efficiency in budding yeast
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA544962 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR9130290_1.fastq.gz | Fastqsanger.gz | |||
| SRR9130290_2.fastq.gz | Fastqsanger.gz | |||
| SRR9130291_1.fastq.gz | Fastqsanger.gz | |||
| SRR9130291_2.fastq.gz | Fastqsanger.gz | |||
| SRR9130292_1.fastq.gz | Fastqsanger.gz |
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Project description:Fidelity of 3´-splice site (3´SS) selection by the spliceosome is critical for proper gene expression but is a daunting task considering the low complexity of the 3´SS consensus YAG. Here we show that loss of the splicing factor Prp18p in budding yeast activates a diverse array of alternative 3´SS at more than half of all introns. Some alternative sites highly diverge from the YAG consensus, demonstrating a critical role for Prp18p in promoting spliceosome fidelity. Usage of alternative 3´SS is determined by distance from the branchpoint, local RNA secondary structures, upstream poly(U) content, and adenosine enrichment in exons. The 3´SS fidelity function of Prp18p can be genetically uncoupled from its role in splicing efficiency and is promoted by interactions with Slu7p and Prp8p. Taken together, these results provide a comprehensive mechanism into how the spliceosome achieves specificity of 3´SS selection and how it prevents aberrant activation of non-canonical splice sites.
2020-05-25 | GSE131797 | GEO
Project description:The functional consequences for alternative splicing of altering the transcription rate have been the subject of intensive study in mammalian cells but less is known about effects on splicing of changing the transcription rate in yeast. We present several lines of evidence showing that slow RNA polymerase II elongation increases both co-transcriptional splicing and splicing efficiency and faster elongation reduces co transcriptional splicing and splicing efficiency in budding yeast, suggesting that splicing is more efficient when co-transcriptional. Moreover, we demonstrate that altering RNA polymerase II elongation rate in either direction compromises splicing fidelity, and we reveal that splicing fidelity depends largely on intron length together with secondary structure and splice site score. These effects are notably stronger for the highly expressed ribosomal protein coding transcripts. We propose that transcription by RNA polymerase II is tuned to optimise the efficiency and accuracy of ribosomal protein gene expression, while allowing flexibility in splice site choice with the nonribosomal protein transcripts.
2017-11-25 | GSE99161 | GEO
Project description:Purpose: To assess the global impacts of RNaseH domain alleles of Prp8 on the in vivo splice site selection for native introns. Conclusions: Our study reveals functional links between distinct spliceosomal conformations and splicing fidelity.
2017-06-21 | GSE96891 | GEO
Project description:The functional consequences of alternative splicing on altering the transcription rate have been the subject of intensive study in mammalian cells but less is known about effects of splicing on changing the transcription rate in yeast. We present several lines of evidence showing that slow RNA polymerase II elongation increases both cotranscriptional splicing and splicing efficiency and that faster elongation reduces cotranscriptional splicing and splicing efficiency in budding yeast, suggesting that splicing is more efficient when cotranscriptional. Moreover, we demonstrate that altering the RNA polymerase II elongation rate in either direction compromises splicing fidelity, and we reveal that splicing fidelity depends largely on intron length together with secondary structure and splice site score. These effects are notably stronger for the highly expressed ribosomal protein coding transcripts. We propose that transcription by RNA polymerase II is tuned to optimize the efficiency and accuracy of ribosomal protein gene expression, while allowing flexibility in splice site choice with the nonribosomal protein transcripts.
| S-EPMC5793784 | biostudies-literature
Project description:Prp45 is a budding yeast NineTeen-Complex associated factor, which plays a role in pre-mRNA splicing. In addition to its documented involvement in the second step of splicing, here we show that it is important in the early stages of co-transcriptional spliceosome assembly as well. To determine the overall splicing efficiency of prp45 mutant cells and to reveal how the efficiency depends on the sequences which define introns (whether they are consensual or non-consensual), we performed RNA-seq analysis of prp45(1-169) and corresponding wild-type cells (two biological replicates each). Total RNA was isolated by combining phenol-chlorophorm extraction with MasterPure Yeast RNA Purification Kit (Epicentre). Ribodepletion, library preparation and sequencing were performed by BGI Genomics.
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Project description:RNA interference (RNAi) is a gene-silencing pathway that can play roles in viral defense, transposon silencing, heterochromatin formation, and post-transcriptional gene silencing. Although absent from Saccharomyces cerevisiae, RNAi is present in other budding-yeast species, including Naumovozyma castellii, which have an unusual Dicer and a conventional Argonaute that are both required for gene silencing. To identify other factors that act in the budding-yeast pathway, we performed an unbiased genetic selection. This selection identified Xrn1p, the cytoplasmic 5′-to-3′ exoribonuclease, as a cofactor of RNAi in budding yeast. Deletion of XRN1 impaired gene silencing in N. castellii, and this impaired silencing was attributable to multiple functions of Xrn1p, including affecting the composition of siRNA species in the cell, influencing the efficiency of siRNA loading into Argonaute, degradation of cleaved passenger strand, and degradation of sliced target RNA.
2020-05-20 | GSE143548 | 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.
2008-12-31 | GSE11491 | GEO
Project description:Vex-seq is a method developed to study the impact of genetic variants on pre-mRNA splicing efficiency
2018-04-17 | GSE113163 | 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