RNA-Seq analysis of Yeast following temporal knockdown of core spliceosome components
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE121594 | GEO | 2018/10/23
REPOSITORIES: GEO
Similar Datasets
Project description:RNA-Seq analysis of Yeast following temporal knockdown of core spliceosome components
| PRJNA497849 | ENA
Project description:Appropriate expression of most eukaryotic genes requires the removal of introns from their pre-messenger RNAs (pre-mRNAs), a process catalyzed by the spliceosome. In higher eukaryotes a large family of auxiliary factors known as SR proteins can improve the splicing efficiency of transcripts containing suboptimal splice sites by interacting with distinct sequences present in those pre-mRNAs. The yeast Saccharomyces cerevisiae lacks functional equivalents of most of these factors; thus, it has been unclear whether the spliceosome could effectively distinguish among transcripts. To address this question, we have used a microarray-based approach to examine the effects of mutations in 18 highly conserved core components of the spliceosomal machinery. The kinetic profiles reveal clear differences in the splicing defects of particular pre-mRNA substrates. Most notably, the behaviors of ribosomal protein gene transcripts are generally distinct from other intron-containing transcripts in response to several spliceosomal mutations. However, dramatically different behaviors can be seen for some pairs of transcripts encoding ribosomal protein gene paralogs, suggesting that the spliceosome can readily distinguish between otherwise highly similar pre-mRNAs. The ability of the spliceosome to distinguish among its different substrates may therefore offer an important opportunity for yeast to regulate gene expression in a transcript-dependent fashion. Given the high level of conservation of core spliceosomal components across eukaryotes, we expect that these results will significantly impact our understanding of how regulated splicing is controlled in higher eukaryotes as well. Keywords: time course, splicing mutant, splicing-specific microarray
2007-04-04 | GSE7419 | GEO
Project description:Alternative splicing (AS) can produce multiple transcripts with different exon-intron structures from a single pre-mRNA. Pre-mRNA splicing is catalyzed by a dynamic macromolecular ribonucleoprotein (RNP) complex termed the spliceosome. The spliceosome consists of several small nuclear ribonucleoproteins (snRNPs) that bind uridine-rich small nuclear RNA (snRNA). In U1, U2, U4 and U5 snRNPs, snRNA interacts with the conserved Smith antigen (Sm) proteins via a bipartite Sm sequence motif. In eukaryotes, seven Sm proteins (B, D1/2/3, E, F and G) form a heptameric ring-shaped complex surrounding the snRNA. Here, we performed a tandem affinity purification using SMEB as a bait in Arabidopsis cell suspension cultures. At least 45 known/hypothesized and potential novel spliceosome components were identified in Arabidopsis.
2023-03-03 | PXD016607 | Pride
Project description:c-MYC (MYC) overexpression or hyperactivation is one of the most common drivers of human cancer. Despite intensive study, the MYC oncogene remains recalcitrant to therapeutic inhibition. Like other classic oncogenes, hyperactivation of MYC leads to collateral stresses onto cancer cells, suggesting that tumors harbor unique vulnerabilities arising from oncogenic activation of MYC. Herein, we discover the spliceosome as a new target of oncogenic stress in MYC-driven cancers. We identify BUD31 as a MYC-synthetic lethal gene, and demonstrate that BUD31 is a splicing factor required for the assembly and catalytic activity of the spliceosome. Core spliceosomal factors (SF3B1, U2AF1, and others) associate with BUD31 and are also required to tolerate oncogenic MYC. Notably, MYC hyperactivation induces an increase in total pre-mRNA synthesis, suggesting an increased burden on the core spliceosome to process pre-mRNA. In contrast to normal cells, partial inhibition of the spliceosome in MYC-hyperactivated cells leads to global intron retention, widespread defects in pre-mRNA maturation, and deregulation of many essential cell processes. Importantly, genetic or pharmacologic inhibition of the spliceosome in vivo impairs survival, tumorigenicity, and metastatic proclivity of MYC-dependent breast cancers. Collectively, these data suggest that oncogenic MYC confers a collateral stress on splicing and that components of the spliceosome may be therapeutic entry points for aggressive MYC-driven cancers. Examination of intron rentention in MYC-ER HMECs, in 4 conditions
2015-09-03 | E-GEOD-66182 | biostudies-arrayexpress
Project description:Very little is known about splicing and its regulation in germ cells, particularly during meiosis. This paper describes the role of a male germ cell-specific protein, Tudor containing protein 6 (TDRD6), in assembly of the spliceosome in spermatocytes. We show that in spermatocytes, TDRD6 interacts with the key protein methyl transferase of the splicing pathway PRMT5. PRMT5 methylates arginines in substrate proteins. In a methylation dependent manner, TDRD6 also associates with spliceosomal core protein SmB in the absence of RNA, thus before an RNP-type spliceosome has been assembled. In Tdrd6-/- primary spermatocytes, PRMT5's association with SmB and the arginine dimethylation of SmB are much reduced. Abrogation of arginine methylation impaired the assembly of spliceosomes and the presence of the spliceosomal RNA U5 is aberrantly increased. These deficiencies in spliceosome maturation correlated with decreased numbers of Cajal bodies and gems involved in later stages, i.e. nuclear snRNP maturation. To reveal functional consequences of these deficiencies, transcriptome analysis of primary spermatocytes showed high numbers of splicing defects such as aberrant usage of intron and exons as well as aberrant representation of splice junctions upon TDRD6 loss. This study reveals a novel function of TDRD6 in spliceosome maturation and mRNA splicing in spermatocytes.
2017-03-01 | GSE94791 | GEO
Project description:Purpose: The goals of this study were to determine whether the spliceosome interacts with non-intronic mRNAs Methods: RNAseq was performed on RNA that immunoprecipitated with the yeast SMD1 protein. Tandem-affinity-purified RNAs were extracted and RNAseq libraries were generated using the EpiCentre ScriptSeq kit (v1). We also performed RNAseq experiments on rRNA depleted total RNA extracted from an exosome mutant (rrp6M-NM-^T), a temperature-sensitive splicing mutant (prp40-1) and a parental strain (BY4741). The rRNA was depleted using the Invitrogen RiboMinus kit, according to manufactureres procedures. The depleted RNA was subsequently treated with Turbo DNAse I (Ambion) and RNAseq libraries were generated using the EpiCentre ScriptSeq kit (v1). Results: The SM RNAseq data identified a number of non-intronic mRNAs that appeard to be bound by the spliceosome. Among these was the BDF2 mRNA, which enocdes for a bromo-domain protein. BDF2 was highly enriched in both SM-IP datasets and was therefore analyzed in more detail. To determine if other non-intronic mRNAs could be regulated by the spliceosome, we analysed the transcriptome in the rrp6M-NM-^T, the prp40-1 and a parental strain. Bioinformatic analysis of these data sets revealed that roughly 1% of the non-intronic mRNAs in yeast could be targeted by the spliceosome. TopHat revealed cannonical splice junctions in roughly 30 non-intronic mRNAs, indicating that these messages are spliced. Conclusions: We demonstrate, for the first time, that the spliceosome can regulate expression of non-intronic mRNAs via one and/or two RNA cleavage events. We refer to this process as Spliceosome Mediated Decay (SMD). We report RNAseq data for two SM immunoprecipitation experiments and RNAseq datasets for the parental strain (BY4741), the prp40-1 mutant, and the rrp6M-NM-^T strain.
2013-10-11 | E-GEOD-49966 | biostudies-arrayexpress
Project description:The studies of spliceosomal interactions are challenging due to their dynamic nature. Here we developed spliceosome iCLIP, which immunoprecipitates SmB along with snRNPs and auxiliary RNA binding proteins (RBPs) to simultaneously map the spliceosomal binding to human snRNAs and pre-mRNAs. This identified 9 distinct regions on pre-mRNAs, which overlap with position-dependent binding patterns of 15 RBPs. Using spliceosome iCLIP, we additionally identified >50,000 branchpoints (BPs) that have canonical features, unlike those identified by RNA-seq. The iCLIP BPs generally overlap with the computationally predicted BPs, and alternative BPs are associated with extended regions of structurally accessible RNA. We find that the position and strength of BPs defines the binding patterns of SF3 and U2AF complexes, whereas the RNA structure around BPs affects the sensitivity of exons to perturbation of these complexes. Our findings introduce spliceosome iCLIP as a new method for transcriptomic studies of BPs and splicing mechanisms.
2019-08-30 | E-MTAB-6950 | biostudies-arrayexpress
Project description:c-MYC (MYC) overexpression or hyperactivation is one of the most common drivers of human cancer. Despite intensive study, the MYC oncogene remains recalcitrant to therapeutic inhibition. Like other classic oncogenes, hyperactivation of MYC leads to collateral stresses onto cancer cells, suggesting that tumors harbor unique vulnerabilities arising from oncogenic activation of MYC. Herein, we discover the spliceosome as a new target of oncogenic stress in MYC-driven cancers. We identify BUD31 as a MYC-synthetic lethal gene, and demonstrate that BUD31 is a splicing factor required for the assembly and catalytic activity of the spliceosome. Core spliceosomal factors (SF3B1, U2AF1, and others) associate with BUD31 and are also required to tolerate oncogenic MYC. Notably, MYC hyperactivation induces an increase in total pre-mRNA synthesis, suggesting an increased burden on the core spliceosome to process pre-mRNA. In contrast to normal cells, partial inhibition of the spliceosome in MYC-hyperactivated cells leads to global intron retention, widespread defects in pre-mRNA maturation, and deregulation of many essential cell processes. Importantly, genetic or pharmacologic inhibition of the spliceosome in vivo impairs survival, tumorigenicity, and metastatic proclivity of MYC-dependent breast cancers. Collectively, these data suggest that oncogenic MYC confers a collateral stress on splicing and that components of the spliceosome may be therapeutic entry points for aggressive MYC-driven cancers.
2015-09-03 | GSE66182 | GEO
Project description:Purpose: The goals of this study were to determine whether the spliceosome interacts with non-intronic mRNAs Methods: RNAseq was performed on RNA that immunoprecipitated with the yeast SMD1 protein. Tandem-affinity-purified RNAs were extracted and RNAseq libraries were generated using the EpiCentre ScriptSeq kit (v1). We also performed RNAseq experiments on rRNA depleted total RNA extracted from an exosome mutant (rrp6Δ), a temperature-sensitive splicing mutant (prp40-1) and a parental strain (BY4741). The rRNA was depleted using the Invitrogen RiboMinus kit, according to manufactureres procedures. The depleted RNA was subsequently treated with Turbo DNAse I (Ambion) and RNAseq libraries were generated using the EpiCentre ScriptSeq kit (v1). Results: The SM RNAseq data identified a number of non-intronic mRNAs that appeard to be bound by the spliceosome. Among these was the BDF2 mRNA, which enocdes for a bromo-domain protein. BDF2 was highly enriched in both SM-IP datasets and was therefore analyzed in more detail. To determine if other non-intronic mRNAs could be regulated by the spliceosome, we analysed the transcriptome in the rrp6Δ, the prp40-1 and a parental strain. Bioinformatic analysis of these data sets revealed that roughly 1% of the non-intronic mRNAs in yeast could be targeted by the spliceosome. TopHat revealed cannonical splice junctions in roughly 30 non-intronic mRNAs, indicating that these messages are spliced. Conclusions: We demonstrate, for the first time, that the spliceosome can regulate expression of non-intronic mRNAs via one and/or two RNA cleavage events. We refer to this process as Spliceosome Mediated Decay (SMD).
2013-10-11 | GSE49966 | GEO