Caenorhabditis elegans
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA369783 | ENA |
REPOSITORIES: ENA
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Project description:Pre-mRNA splicing requires assembly of spliceosome that consists of hundreds of factors forming various dynamic complexes with or without small nuclear RNAs (snRNAs). Systematic identification of the splicing factors remains a significant challenge especially in vivo. In our genetic screening for the factors required for division asynchrony during Caenorhabditis elegans embryogenesis, we identified a highly conserved but uncharacterized essential protein, GAD-1, that is necessary for setting cell cycle length of intestine progenitors, a function that is shared with many other factors involved in transcription, pre-mRNA splicing or polyadenylation, suggesting its potential role in mRNA biogenesis. Co-immunoprecipitation followed by mass spectrometry reveals that GAD-1 mainly interacts with non-snRNP type of splicing complex called NineTeen Complex (NTC). Consistent with this, RNA-seq analysis demonstrates pervasive defects in pre-mRNA splicing in gad-1 mutants. Transgenic reporter assay shows its ubiquitous and nuclear expression across developmental stages. Immunostaining of the C-terminal domain of RNA polymerase II demonstrates a GAD-1’s role in transcription elongation. In agreement with this, depletion of GAD-1 and its interacting partners inhibits expression of both ubiquitous and tissue-specific genes, supporting that both GAD-1 and many of its interacting proteins are novel components of NTC or its associated spliceosome. Taken together, we identify GAD-1 and its multiple interacting partners as novel components of spliceosome in vivo through which they regulate pre-mRNA splicing and transcription elongation.
2017-12-31 | GSE94463 | GEO
Project description:The spliceosome is a dynamic macromolecular machine that catalyzes the removal of introns from pre-mRNA to make mature message. Schizosaccharomyces pombe Cwf10 (homolog Saccharomyces cerevisiae Snu114 and of Human U5-116K), an integral member of the U5 snRNP, is a GTPase that shares sequence homology with the eukaryotic translation elongation factor EF2. Cwf10 is required for pre-mRNA splicing; however, its mechanism(s) of action is still not understood. Cwf10/Snu114 family members contain a conserved N-terminal extension (NTE) that lacks homology with EF2 and has been predicted to be an intrinsically unfolded domain. Using S. pombe as a model system, we show that the NTE is not essential, but cells lacking this domain are defective in pre-mRNA splicing at all temperatures. Genetic interactions between cwf10-M-NM-^TNTE and other pre-mRNA splicing mutants are consistent with a role for the NTE in spliceosome activation. Characterization of Cwf10-NTE by various biophysical techniques shows the NTE contains both regions of structure and disorder in solution. The first twenty-three highly-conserved amino acids of the NTE are essential for its role in splicing, but are not sufficient to restore pre-mRNA splicing to wild-type levels in cwf10-M-bM-^HM-^FNTE cells. When the NTE is overexpressed in the cwf10-M-NM-^TNTE background, it can complement the truncated Cwf10 protein in trans, and it also immunoprecipitates a complex similar in composition to the late-stage U5.U2/U6 spliceosome. These data show that the structurally flexible NTE is capable of making specific contacts within the spliceosome that may facilitate Cwf10M-bM-^@M-^Ys overall role facilitating spliceosome rearrangements. Interrogation of the S. pombe transcriptome using poly-A enriched RNA sequencing (Illumina HiSeq 2500) in wild type and cwf10-M-NM-^TNTE cultures. A total of 4 samples were analysed: two biological repeats of wild-type strain and two biological repeats of cwf10-M-NM-^TNTE
2013-09-25 | E-GEOD-47573 | biostudies-arrayexpress
Project description:There is good evidence for functional interactions between splicing and transcription in eukaryotes, but how and why these processes are coupled remain unknown. Prp5 is an RNA-stimulated ATPase required for pre-spliceosome formation in yeast. We demonstrate through in vivo RNA labelling that, in addition to a splicing defect, the prp5-1 mutation causes a defect in the transcription of intron-containing genes. We present chromatin immunoprecipitation evidence for a transcriptional elongation defect in which RNA polymerase that is phosphorylated at serine 5 of the largest subunit’s heptad repeat accumulates over introns, and that this defect requires the U2 snRNP-associated Cus2p. A similar accumulation of polymerase was observed when pre-spliceosome formation was blocked by a mutation in U2 snRNA. These results indicate the existence of a transcriptional elongation checkpoint that is associated with pre-spliceosome formation during co-transcriptional spliceosome assembly. We propose a role for Cus2p as a potential checkpoint factor in transcription. Examining the Pol II profile in MT strain and WT strain
2014-02-19 | E-GEOD-53647 | biostudies-arrayexpress
Project description:The spliceosome is a dynamic macromolecular machine that catalyzes the removal of introns from pre-mRNA to make mature message. Schizosaccharomyces pombe Cwf10 (homolog Saccharomyces cerevisiae Snu114 and of Human U5-116K), an integral member of the U5 snRNP, is a GTPase that shares sequence homology with the eukaryotic translation elongation factor EF2. Cwf10 is required for pre-mRNA splicing; however, its mechanism(s) of action is still not understood. Cwf10/Snu114 family members contain a conserved N-terminal extension (NTE) that lacks homology with EF2 and has been predicted to be an intrinsically unfolded domain. Using S. pombe as a model system, we show that the NTE is not essential, but cells lacking this domain are defective in pre-mRNA splicing at all temperatures. Genetic interactions between cwf10-ΔNTE and other pre-mRNA splicing mutants are consistent with a role for the NTE in spliceosome activation. Characterization of Cwf10-NTE by various biophysical techniques shows the NTE contains both regions of structure and disorder in solution. The first twenty-three highly-conserved amino acids of the NTE are essential for its role in splicing, but are not sufficient to restore pre-mRNA splicing to wild-type levels in cwf10-∆NTE cells. When the NTE is overexpressed in the cwf10-ΔNTE background, it can complement the truncated Cwf10 protein in trans, and it also immunoprecipitates a complex similar in composition to the late-stage U5.U2/U6 spliceosome. These data show that the structurally flexible NTE is capable of making specific contacts within the spliceosome that may facilitate Cwf10’s overall role facilitating spliceosome rearrangements.
2013-09-25 | GSE47573 | GEO
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
Project description:There is good evidence for functional interactions between splicing and transcription in eukaryotes, but how and why these processes are coupled remain unknown. Prp5 is an RNA-stimulated ATPase required for pre-spliceosome formation in yeast. We demonstrate through in vivo RNA labelling that, in addition to a splicing defect, the prp5-1 mutation causes a defect in the transcription of intron-containing genes. We present chromatin immunoprecipitation evidence for a transcriptional elongation defect in which RNA polymerase that is phosphorylated at serine 5 of the largest subunit’s heptad repeat accumulates over introns, and that this defect requires the U2 snRNP-associated Cus2p. A similar accumulation of polymerase was observed when pre-spliceosome formation was blocked by a mutation in U2 snRNA. These results indicate the existence of a transcriptional elongation checkpoint that is associated with pre-spliceosome formation during co-transcriptional spliceosome assembly. We propose a role for Cus2p as a potential checkpoint factor in transcription.
2014-02-19 | GSE53647 | GEO
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
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 | GSE185811 | GEO
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 | GSE185810 | GEO
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 | GSE185809 | GEO