The protein U5-52K is required for T cell homeostasis by maintaining splicing efficiency
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE208003 | GEO | 2023/07/31
REPOSITORIES: GEO
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Project description:Pre-mRNA splicing catalyzed by the spliceosome represents a critical step in the regulation of gene expression contributing to transcriptome and proteome diversity. The spliceosome consists of five small nuclear ribonucleoprotein particles (snRNPs) biogenesis of which remains only partially understood. Here we define the evolutionarily conserved protein Ecdysoneless (Ecd) as a critical regulator of U5 snRNP assembly and Prp8 stability. Combining Drosophila genetics with proteomic approaches we demonstrate Ecd requirement for the maintenance of adult healthspan and lifespan and identify the Sm ring protein SmD3 as a novel interaction partner of Ecd. We show that the predominant task of Ecd is to deliver Prp8 to the Sm protein ring within emerging U5 snRNPs. Ecd deficiency leads to reduced Prp8 protein levels and compromised U5 snRNP biogenesis, causing loss of splicing fidelity and transcriptome integrity. Based on our findings, we propose that Ecd acts as a chaperone that delivers Prp8 to the forming U5 snRNP allowing completion of the cytoplasmic part of the U5 snRNP biogenesis necessary to meet the demand of cells for functional spliceosomes.
2021-04-21 | GSE149056 | GEO
Project description:U5 snRNP is a complex particle essential for RNA splicing. U5 snRNPs undergo an intricate biogenesis that ensures that only a fully mature particle assembles into a splicing competent U4/U6•U5 tri-snRNP and enters the splicing reaction. During splicing, U5 snRNP is substantially rearranged and leaves as a U5/PRPF19 post-splicing particle, which requires re-generation before a next round of splicing. Here, we show that a previously uncharacterized protein TSSC4 is a new component of U5 snRNP that promotes tri-snRNP formation. We provide evidence that TSSC4 associates with U5 snRNP chaperones, U5 snRNP and the U5/PRPF19 particle. Specifically, TSSC4 interacts with U5-specific proteins PRPF8, EFTUD2 and SNRNP200. We also identified TSSC4 domains critical for the interaction with U5 snRNP and the PRPF19 complex, as well as for TSSC4 function in tri-snRNP assembly. TSSC4 emerges as a specific chaperone that acts in U5 snRNP de novo biogenesis as well as post-splicing recycling.
2021-06-11 | PXD024929 | Pride
Project description:The goal of this study was to compare the transcriptional profile (RNA-seq) of Dengue virus 2 and mock infected cells at 24 and 36 hours post infection. Dengue virus NS5 protein plays multiple functions in the cytoplasm of infected cells, enabling viral RNA replication and counteracting host antiviral responses. Here, we demonstrate a novel function of NS5 in the nucleus where it interferes with cellular splicing. Using global proteomic analysis of infected cells together with functional studies, we found that NS5 binds spliceosome complexes and modulates endogenous splicing. In particular, we show that NS5 alone, or in the context of viral infection, interacts with core components of the U5 snRNP particle, CD2BP2 and DDX23, alters the inclusion/exclusion ratio of alternative splicing events, and changes mRNA isoform abundance of known antiviral factors. Interestingly, a genome wide transcriptome analysis, using recently developed bioinformatics tools, revealed an increase of intron retention upon dengue virus infection, and viral replication was improved by silencing specific U5 components. Different mechanistic studies indicate that binding of NS5 to the spliceosome reduces the efficiency of pre-mRNA processing, independently of NS5 enzymatic activities. We propose that NS5 binding to U5 snRNP proteins hijacks the splicing machinery resulting in a less restrictive environment for viral replication. A549 cells where infected with Dengue virus 2 or mock and after 24 and 36 hours post infection mRNA was purified. Then the transcriptional profile of these cells was analyzed using RNA-seq.
2016-08-09 | E-GEOD-84285 | 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-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:The goal of this study was to compare the transcriptional profile (RNA-seq) of Dengue virus 2 and mock infected cells at 24 and 36 hours post infection. Dengue virus NS5 protein plays multiple functions in the cytoplasm of infected cells, enabling viral RNA replication and counteracting host antiviral responses. Here, we demonstrate a novel function of NS5 in the nucleus where it interferes with cellular splicing. Using global proteomic analysis of infected cells together with functional studies, we found that NS5 binds spliceosome complexes and modulates endogenous splicing. In particular, we show that NS5 alone, or in the context of viral infection, interacts with core components of the U5 snRNP particle, CD2BP2 and DDX23, alters the inclusion/exclusion ratio of alternative splicing events, and changes mRNA isoform abundance of known antiviral factors. Interestingly, a genome wide transcriptome analysis, using recently developed bioinformatics tools, revealed an increase of intron retention upon dengue virus infection, and viral replication was improved by silencing specific U5 components. Different mechanistic studies indicate that binding of NS5 to the spliceosome reduces the efficiency of pre-mRNA processing, independently of NS5 enzymatic activities. We propose that NS5 binding to U5 snRNP proteins hijacks the splicing machinery resulting in a less restrictive environment for viral replication.
2016-08-09 | GSE84285 | GEO
Project description:DEAD-box proteins, a family of RNA-dependent ATPases, promote the numerous conformational rearrangements required for spliceosome assembly, activation, and disassembly. Previous work showed that a cold-sensitive substitution in DEAD-box protein Prp28 prevents the switch from U1 to U6 snRNA pairing with the 5’ splice site. Little is known about how Prp28 is regulated, although U5 snRNP protein Prp8 is a potential coordinator of Prp28 and other spliceosomal ATPases. We conducted a targeted selection in Prp8 for cold-insensitive suppressors of prp28-1, then used splicing specific microarrays to assess suppression of the prp28-1 splicing defect. Splicing specific microarrays were used to assess suppression of the prp28-1 splicing defect by a prp8 allele (prp8-tes) that suppresses prp28-1 cold-sensitivity
2014-01-01 | E-GEOD-42754 | biostudies-arrayexpress
Project description:The spliceosome is a large ribonucleoprotein complex responsible for pre-mRNA splicing and genome stability maintenance. Disruption of the spliceosome activity may lead to developmental disorders and tumorigenesis. However, the physiological role that the spliceosome plays in B cell development and function is still poorly defined. Here, we demonstrate that ubiquitin-specific peptidase 39 (Usp39), a spliceosome component of the U4/U6.U5 tri-snRNP complex, is essential for B cell development. Ablation of Usp39 in B cell lineage blocks pre-pro-B to pro-B cell transition in the bone marrow, leading to a profound reduction of mature B cells in the periphery. We show that Usp39 specifically regulates immunoglobulin gene rearrangement in a spliceosome-dependent manner, which involves modulating chromatin interactions at the Igh locus. Moreover, our results indicate that Usp39-deletion reduces the pre-malignant B cells in Eμ-Myc transgenic mice and significantly improves their survival.
2022-02-08 | GSE173597 | 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-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 this study, we analyzed the pre-mRNA splicing status in wild-type Col, atprmt5 mutants, and two atprmt5 suppressors (m90 and s215). RNA-seq analyses revealed that m90 and s215 can partially rescue the pre-mRNA splicing defects in atprmt5 mutants. Further study showed that m90 and s215 were identified in the highly conserved pre-mRNA splicing factor 8 (AtPrp8) and demonstrated that Prp19C/NTC complex failed to be assembled into U5 snRNP to form activated spliceosome in atprmt5 mutants, while restored in the suppressor. Our findings uncover a key molecular mechanism for AtPRMT5 in the regulation of pre-mRNA splicing.
2017-05-19 | GSE62611 | GEO
Project description:DEAD-box proteins, a family of RNA-dependent ATPases, promote the numerous conformational rearrangements required for spliceosome assembly, activation, and disassembly. Previous work showed that a cold-sensitive substitution in DEAD-box protein Prp28 prevents the switch from U1 to U6 snRNA pairing with the 5’ splice site. Little is known about how Prp28 is regulated, although U5 snRNP protein Prp8 is a potential coordinator of Prp28 and other spliceosomal ATPases. We conducted a targeted selection in Prp8 for cold-insensitive suppressors of prp28-1, then used splicing specific microarrays to assess suppression of the prp28-1 splicing defect.
2014-01-01 | GSE42754 | GEO