Functional splicing network reveals extensive regulatory plasticity of the core Spliceosomal machinery
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE56605 | GEO | 2015/12/31
SECONDARY ACCESSION(S): PRJNA244067
REPOSITORIES: GEO
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Project description:Pre-mRNA splicing relies on the still poorly understood dynamic interplay between more than 150 protein components of the Spliceosome, and the steps at which splicing can be regulated remain largely unknown. Here we systematically analyze the effect of knocking down the components of the splicing machinery on alternative splicing events relevant for cell proliferation and apoptosis and use this information to reconstruct a network of functional interactions. The network accurately captures well-established physical and functional associations and identifies new, revealing remarkable regulatory potential of core spliceosomal components, related to the order and duration of their recruitment during Spliceosome assembly. In contrast with standard models of regulation at early events of splice site recognition, factors involved in catalytic activation of the Spliceosome display regulatory properties. The network also sheds light on the antagonism between hnRNP C and U2AF and on targets of anti-tumor drugs, and can be widely used to identify mechanisms of splicing regulation. RNA from 3 biological replicates of 72 hours knockdowns of human IK or SMU1 and a control set were used. Changes between the control and knockdowns were measured based on using a splice-junction array (Affymetrix HJAY).
2015-12-31 | E-GEOD-56605 | biostudies-arrayexpress
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:Mutations affecting spliceosomal proteins are frequently found in hematological malignancies, including myelodysplastic syndromes and acute myeloid leukemia. DDX41/Abstrakt is a metazoan-specific spliceosomal DEAD-box RNA helicase found to be recurrently mutated in inherited myelodysplastic syndromes and in relapsing cases of acute myeloid leukemia. The genetic properties and genomic impacts of disease-causing missense mutations in DDX41 and other spliceosomal proteins have been uncertain. Here we conduct a comprehensive molecular genetic analysis of the C. elegans DDX41 ortholog, SACY-1. Our results reveal general essential functions for SACY-1 in both the germline and the soma, as well as specific functions affecting germline sex determination and cell cycle control. Certain sacy-1/DDX41 mutations, including the R525H human oncogenic variant, confer antimorphic activity, suggesting that they compromise the function of the spliceosome. Consistent with these findings, sacy-1 exhibits synthetic lethal interactions with several spliceosomal components, and biochemical analyses suggest that SACY-1 is a component of the C. elegans spliceosome. We used the auxin-inducible degradation system to analyze the impact of SACY-1 on the transcriptome using RNA sequencing. SACY-1 depletion impacts the transcriptome through splicing-independent and splicing-dependent mechanisms. The observed transcriptome changes suggest that disruption of spliceosomal function induces a stress response. Altered 3’ splice site usage represents the predominant splicing defect observed upon SACY-1 depletion, consistent with a role for SACY-1 as a second-step splicing factor. Missplicing events appear more prevalent in the soma than the germline, suggesting that surveillance mechanisms protect the germline from aberrant splicing.
2020-01-25 | GSE144003 | GEO
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: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.
2019-08-03 | E-MTAB-8182 | biostudies-arrayexpress
Project description:Sequencing-based profiling of ribose methylations is a new approach that allows for experiments adrressing dynamic changes on a large scale. Here, we apply such a method to spliceosomal snRNAs present in human whole cell RNA. Analysis of solid tissue samples confirmed all previously known sites and demonstrated close to full methylation at almost all sites. Methylation changes were revealed in biological experimental settings, using T cell activation as an example, and in the T cell leukemia model, Jurkat cells. Such changes could impact the dynamics of snRNA interactions during the spliceosome cycle and affect mRNA splicing efficiency and splicing patterns.
2017-10-20 | GSE104947 | GEO
Project description:Gene expression requires tight coordination of the molecular machineries that mediate transcription and splicing. While the interplay between transcription kinetics and spliceosome fidelity has been investigated before, less is known about mechanisms regulating the assembly of the spliceosomal machinery in response to transcription changes. Here we report an association of the Smn complex, which mediates spliceosomal snRNP biogenesis, with the 7SK complex involved in transcriptional regulation. We found that Smn interacts with the 7SK core components Larp7 and Mepce and specifically associates with 7SK subcomplexes containing hnRNP R. The association between Smn and 7SK complexes is enhanced upon transcriptional inhibition leading to reduced production of snRNPs. Taken together, our findings reveal a functional association of Smn and 7SK complexes that is governed by global changes in transcription. Thus, in addition to its canonical nuclear role in transcriptional regulation, 7SK has cytosolic functions in fine-tuning spliceosome production according to transcriptional demand.
2021-01-26 | PXD021360 | Pride
Project description:Mutations in genes encoding splicing factors (which we refer to as spliceosomal genes) are commonly found in patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). These mutations recurrently affect specific amino acid residues, leading to perturbed normal splice site and exon recognition. Spliceosomal gene mutations are always heterozygous and rarely occur together with one another, suggesting that cells may tolerate only a partial deviation from normal splicing activity. To test this hypothesis, we engineered mice to express a mutated allele of serine/arginine-rich splicing factor 2 (Srsf2P95H) - which commonly occurs in individuals with MDS and AML - in an inducible, hemizygous manner in hematopoietic cells. These mice rapidly succumbed to fatal bone marrow failure, demonstrating that Srsf2-mutated cells depend on the wild-type Srsf2 allele for survival. In the context of leukemia, treatment with the spliceosome inhibitor E7107 resulted in substantial reductions in leukemic burden, specifically in isogenic mouse leukemias and patient-derived xenograft AMLs carrying spliceosomal mutations. Whereas E7107 treatment of mice resulted in widespread intron retention and cassette exon-skipping in leukemic cells regardless of Srsf2 genotype, the magnitude of splicing inhibition following E7107 treatment was greater in Srsf2-mutated than in Srsf2-wild-type leukemia, consistent with the differential effect of E7107 on survival. Collectively, these data provide genetic and pharmacologic evidence that leukemias with spliceosomal gene mutations are preferentially susceptible to additional splicing perturbations in vivo as compared to leukemias without such mutations. Modulation of spliceosome function may thus provide a new therapeutic avenue in genetically defined subsets of individuals with MDS or AML.
2016-03-31 | GSE74064 | GEO
Project description:Mutations in spliceosomal genes are commonly found in patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). These mutations occur at highly restricted amino acid residues and perturb normal splice site and exon recognition. Spliceosomal mutations are always heterozygous and rarely co-occur with one another, suggesting that cells may only tolerate a partial deviation from normal splicing activity. To test this hypothesis, we generated mice with inducible hemizygous expression of the commonly occurring SRSF2P95H mutation in the hematopoietic system. These mice rapidly developed lethal bone marrow failure upon activation of the Srsf2P95H mutation with concomitant deletion of the wildtype Srsf2 allele, demonstrating that Srsf2-mutant cells depend on the wildtype Srsf2 allele for survival. We next tested whether spliceosomal-mutant leukemias display greater sensitivity to pharmacologic splicing inhibition induced by the small molecule E7107. Treatment of isogenic murine leukemias as well as patient-derived xenograft (PDX) AMLs showed significant reductions in leukemic burden specifically in samples carrying spliceosomal mutations. Collectively, these data provide genetic and pharmacologic evidence that leukemias with spliceosomal mutations are preferentially susceptible to additional splicing perturbations in vivo compared with wildtype counterparts. Modulation of spliceosome function may provide a novel therapeutic avenue in genetically defined subsets of MDS/AML patients. We created an isogenic murine leukemia model by retroviral overexpression of the MLL-AF9 fusion oncogene in Vav-Cre Srsf2+/+ or Vav-Cre Srsf2P95H/+ BM cells followed by transplantation into lethally irradiated recipient mice. In order to determine the mechanistic origins of the Srsf2 mutant-selective effects of E7107, we analyzed transcriptional changes after five consecutive days of E7107 or vehicle treatment in vivo. GFP+ Cd11b+ cells were purified from the BM of recipient mice exactly three hours after the last dose of E7107 and were subjected to paired-end 2x50bp RNA-seq. For the knock-in/knock-out experiments, we generated inducible, hemizygous Srsf2P95H mice to study the effects of wildtype Srsf2 deletion with concomitant activation of the Srsf2P95H allele. Mx1-cre Srsf2fl/+ mice were crossed to Srsf2P95H/+ mice to generate Srsf2 wildtype (Mx1-cre Srsf2+/+), Srsf2 heterozygous knockout (Mx1-Cre Srsf2+/-), Srsf2 heterozygous P95H mutant (Mx1-Cre Srsf2P95H/+), and Srsf2 hemizygous P95H mutant (Mx1-Cre Srsf2P95H/-) mice, which were subjected to single-end 101bp RNA-seq.
2016-03-31 | E-GEOD-74064 | biostudies-arrayexpress
Project description:Cwc21 (complexed with CEF1 protein 21) is a 135 amino acid yeast protein that shares homology with the N-terminal domain of human SRm300, a large serine/arginine-repeat protein shown previously to associate with the splicing coactivator SRm160. Proteomic analysis of spliceosomal complexes has suggested a role for Cwc21 and SRm300 at the core of the spliceosome. However, specific functions for these proteins have remained elusive. In this report we employ quantitative genetic interaction mapping, mass-spectrometry of tandem affinity purified complexes and microarray profiling to investigate genetic, physical and functional interactions involving Cwc21. Combined data from these assays support multiple roles for Cwc21 in the formation and function of splicing complexes. Consistent with a role for Cwc21 at the core of the spliceosome, we observe strong genetic, physical and functional interactions with Isy1, a protein previously implicated in the first catalytic step of splicing and splicing fidelity. Together, the results suggest multiple functions for Cwc21/SRm300 in the splicing process, including an important role in the activation of splicing in association with Isy1.
2009-12-31 | GSE17395 | GEO