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Transcriptome changes in spliceostatin A treated cells

ABSTRACT: Pre-mRNA in eukaryotes is subjected to post-transcriptional modifications, such as capping, polyadenylation and splicing. It is well known that transcription and post-transcriptional modifications are coupled and that this coupling stimulates post-transcriptional modifications, however, the effects of post-transcriptional modifications on transcription are not fully understood. Here, we report that inhibition of U2 snRNP by a splicing inhibitor, spliceostatin A (SSA), and antisense morpholino oligonucleotide to U2 snRNA caused 3’ end down regulation in a gene-specific manner. We also show that U2 snRNP inhibition resulted in the dephosphorylation of second serine residues (Ser2) within heptad repeats of the C-terminal domain (CTD) of Pol II, which is important for transcription elongation, and this dephosphorylation was correlated with splicing inactivation. Interestingly, removal of SSA from the culture media caused restoration of the Ser2 phosphorylation and expression of the 3’ end, suggesting that transcription elongation resumed. These findings suggest that a novel checkpoint mechanism prevents pre-mRNA accumulation and the production of aberrant proteins translated from pre-mRNA upon splicing deficiencies through the dephosphorylation of CTD Ser2.

ORGANISM(S): Homo sapiens

PROVIDER: GSE45379 | GEO | 2013/03/28

SECONDARY ACCESSION(S): PRJNA193597

REPOSITORIES: GEO

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Transcriptome changes in spliceostatin A treated cells

Project description:Pre-mRNA in eukaryotes is subjected to post-transcriptional modifications, such as capping, polyadenylation and splicing. It is well known that transcription and post-transcriptional modifications are coupled and that this coupling stimulates post-transcriptional modifications, however, the effects of post-transcriptional modifications on transcription are not fully understood. Here, we report that inhibition of U2 snRNP by a splicing inhibitor, spliceostatin A (SSA), and antisense morpholino oligonucleotide to U2 snRNA caused 3’ end down regulation in a gene-specific manner. We also show that U2 snRNP inhibition resulted in the dephosphorylation of second serine residues (Ser2) within heptad repeats of the C-terminal domain (CTD) of Pol II, which is important for transcription elongation, and this dephosphorylation was correlated with splicing inactivation. Interestingly, removal of SSA from the culture media caused restoration of the Ser2 phosphorylation and expression of the 3’ end, suggesting that transcription elongation resumed. These findings suggest that a novel checkpoint mechanism prevents pre-mRNA accumulation and the production of aberrant proteins translated from pre-mRNA upon splicing deficiencies through the dephosphorylation of CTD Ser2. Treated HeLa cells with methanol or spliceosotain A for 3 hours followed by labelling of nascent transcripts with EU for 1 hour. The EU labelled nascent RNA was purified and analyzed on human exon 1.0ST array. Three biological replicates were used for each treatment.

2013-03-28 | E-GEOD-45379 | biostudies-arrayexpress

Transcriptome changes in splicing inhibited cells

Project description:In eukaryotes, U1 small nuclear ribonucleoprotein (snRNP) forms spliceosomes in equal stoichiometry with U2, U4, U5 and U6 snRNPs; however, its abundance in human far exceeds that of the other snRNPs. Here we used antisense morpholino oligonucleotide to U1 snRNA to achieve functional U1 snRNP knockdown in HeLa cells, and identified accumulated unspliced pre-mRNAs by genomic tiling microarrays. In addition to inhibiting splicing, U1 snRNP knockdown caused premature cleavage and polyadenylation in numerous pre-mRNAs at cryptic polyadenylation signals, frequently in introns near (<5 kilobases) the start of the transcript. This did not occur when splicing was inhibited with U2 snRNA antisense morpholino oligonucleotide or the U2-snRNP-inactivating drug spliceostatin A unless U1 antisense morpholino oligonucleotide was also included. We further show that U1 snRNA–pre-mRNA base pairing was required to suppress premature cleavage and polyadenylation from nearby cryptic polyadenylation signals located in introns. These findings reveal a critical splicing-independent function for U1 snRNP in protecting the transcriptome, which we propose explains its overabundance.

2010-09-29 | GSE24179 | GEO

A genome-wide analysis indicates that yeast pre-mRNA splicing is predominantly post-transcriptional

Project description:Recent ChIP experiments indicate that spliceosome assembly and splicing can occur cotranscriptionally in S. cerevisiae. However, only a few genes have been examined, and all have long second exons. To extend these studies, we analyzed intron-containing genes with different second exon lengths, by ChIP as well as by whole-genome tiling arrays (ChIP-CHIP). The data indicate that U1 snRNP recruitment is independent of exon length. Recursive splicing constructs, which uncouple U1 recruitment from transcription, suggest that cotranscriptional U1 recruitment contributes to optimal splicing efficiency. In contrast, U2 snRNP recruitment as well as cotranscriptional splicing is deficient on short second exon-genes. We estimate that approximately 90% of endogenous yeast splicing is post-transcriptional, consistent with an analysis of post-transcriptional snRNP-associated pre-mRNA. Keywords: ChIP-CHIP

2007-01-22 | GSE6417 | GEO

The histone variant H2A.Z promotes efficient co-transcriptional splicing in S. cerevisiae

Project description:In eukaryotes, a dynamic ribonucleic protein machine known as the spliceosome catalyzes the removal of introns from pre-messenger RNA (pre-mRNA). Recent studies show the process of RNA-synthesis and RNA-processing to be spatio-temporally coordinated, indicating that RNA splicing takes place in the context of chromatin. H2A.Z is a highly conserved histone variant of the canonical histone H2A. In S. cerevisiae, H2A.Z is deposited into chromatin by the SWR1-complex, is found near the 5’ ends of protein-coding genes, and has been implicated in transcription regulation. Here we show that splicing of intron-containing genes in cells lacking H2A.Z is impaired, particularly under suboptimal splicing conditions. Cells lacking H2A.Z are especially dependent on a functional U2 snRNP, as H2A.Z shows extensive genetic interactions with U2 snRNP associated proteins, and RNA-seq reveals introns with non-consensus branch points are particularly sensitive to H2A.Z loss. Consistently, H2A.Z promotes efficient spliceosomal rearrangements involving the U2 snRNP, as H2A.Z loss results in persistent U2 snRNP association and decreased recruitment of downstream snRNPs to nascent RNA. H2A.Z impairs transcription elongation, suggesting that spliceosome rearrangements are tied to H2A.Z’s role in elongation. Depletion of disassembly factor Prp43 suppresses H2A.Z-mediated splice defects, indicating that, in the absence of H2A.Z, stalled spliceosomes are disassembled and unspliced RNAs are released. Together these data demonstrate that H2A.Z is required for efficient pre-mRNA splicing and indicate a role for H2A.Z in coordinating the kinetics of transcription elongation and splicing.

2017-05-18 | GSE97416 | GEO

CDK11 phosphorylates SF3B1 and is essential for constitutive splicing (ChIP-seq)

Project description:Pre-mRNA splicing is a highly regulated process catalyzing intron excision by spliceosome. Spliceosome activation is a major control step requiring dramatic protein and RNA rearrangements leading to a catalytically active complex. Prior research has linked hyperphosphorylation of SF3B1, a subunit of U2 snRNP, with spliceosome activation and catalytically active spliceosome, rendering a relevant kinase a key player for pre-mRNA splicing. Here we use OTS964, the first potent inhibitor of cyclin-dependent kinase 11 (CDK11), to show rapid and selective dephosphorylation of SF3B1 on threonines required for spliceosome activation. CDK11 associates with SF3B1 and its inhibition causes massive intron retention, block in precatalytic spliceosome complex B to activated spliceosome complex Bact transition and accumulation of non-functional spliceosomes on pre-mRNA and chromatin. These studies reveal crucial regulatory role of CDK11 in human pre-mRNA splicing and define the compound OTS964 as a quality chemical biology probe for CDK11.

2022-07-09 | GSE185812 | GEO

CDK11 phosphorylates SF3B1 and is essential for constitutive splicing (iCLIP)

2022-07-09 | GSE185811 | GEO

CDK11 phosphorylates SF3B1 and is essential for constitutive splicing (4sU-seq)

2022-07-09 | GSE185810 | GEO

CDK11 phosphorylates SF3B1 and is essential for constitutive splicing (RNA-seq)

2022-07-09 | GSE185809 | GEO

Phospho-site mutants of the RNA Polymerase II C-terminal domain alter subtelomeric gene expression and chromatin modification state in fission yeast

Project description:Eukaryotic gene expression requires that RNA Polymerase II (RNAP II) gain access to DNA in the context of chromatin. The C-terminal domain (CTD) of RNAP II recruits chromatin modifying enzymes to promoters, allowing for transcription initiation or repression. Specific CTD phosphorylation marks facilitate recruitment of chromatin modifiers, transcriptional regulators, and RNA processing factors during the transcription cycle. However, the readable code for recruiting such factors is still not fully defined and how CTD modifications affect related families of genes or regional gene expression is not well understood. Here we examine the effects of manipulating the Y1S2P3T4S5P6S7 heptapeptide repeat of the CTD of RNAP II in Schizosaccharomyces pombe by substituting non-phosphorylatable alanines for Ser2 and/or Ser7 and the phosphomimetic glutamic acid for Ser7. Global gene expression analyses were conducted using splicing-sensitive microarrays and validated via RT-qPCR. The CTD mutations did not affect pre-mRNA splicing or snRNA levels. Rather, the data revealed upregulation of subtelomeric genes and alteration of the repressive histone H3 lysine 9 methylation (HeK9me) landscape. The data further indicate that H3K9me and expression status are not fully correlated, suggestive of CTD-dependent subtelomeric repression mechansims that act independently of H3K9me levels. Splicing sensitive S. pombe microarrays (Agilent-027365) were used to compare the splicing and expression profile of four mutant strains relative to WT control with 3-5 biological replicates and dye flipped samples

2016-08-10 | E-GEOD-83426 | biostudies-arrayexpress

Different effects of pre-mRNA splicing inhibitors on RNA polymerase II transcription

Project description:The production of a mature mRNA in eukaryotes requires both transcription by RNA polymerase (pol) II and co-transcriptional processes, including mRNA capping, splicing, and cleavage and polyadenylation. The pol II complex serves as a hub to coordinate transcription and co-transcriptional processes, especially via the carboxyl terminal domain (CTD) of the large subunit of pol II that is composed in human of 52 repeats of the heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 and that can be modified by several post-translational modifications. To understand better the effects of pre-mRNA splicing on pol II transcription, we investigated the transcriptional effects of two small molecule inhibitors of splicing: Madrasin and Isoginkgetin. We found that while Madrasin can quickly inhibit pre-mRNA splicing, it is not the case of Isoginkgetin, which affects transcription before any visible effect on pre-mRNA splicing. Interestingly, we found that these two small molecules promote a global transcriptional readthrough, including intronless protein-coding genes and histone genes, which is not the case of the splicing inhibitors targeting the splicing factor SF3B1.

2024-08-20 | GSE221279 | GEO

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