The Role and Therapeutic Potential of Splicing Factor SF3B6 in Hepatocellular Carcinoma
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE318429 | GEO | 2026/02/11
REPOSITORIES: GEO
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Project description:The SF3B complex, a multiprotein component of the U2 snRNP of the spliceosome, mediates branch point selection and facilitates spliceosome assembly and activation. Several splicing modulators bind the SF3B1 and PHF5A subunits of the SF3B complex and globally inhibit splicing. Engineered mutant variants of SF3B1 and PHF5A conferred tolerance to splicing inhibitors with only minor effects on gene expression and AS.
2021-04-11 | GSE153234 | GEO
Project description:A major impediment to studying the human spliceosome in vivo has been the inability to program point mutations in endogenous genes on a large scale. CRISPR-Cas9 technology now provides such opportunities. Due to its scalability and ability to introduce point mutations, we chose CRISPR-Cas9 base editing for a forward genetic screen of the spliceosome in an eHAP haploid human cell line background. We maintained eHAP FNLS cells as haploid so that we could subsequently assign genotype-phenotype relationships. We designed a single guide RNA (sgRNA) library targeting a hand-curated list of 153 human spliceosomal proteins which engage at various steps of the splicing cycle. After mutagenesis, we interrogated the spliceosome using the potent inhibitor pladienolide B (PB), which targets U2 snRNP by binding to a pocket between the SF3B1 and PHF5A subunits of the SF3b complex, preventing stabilization of the U2-branchpoint RNA duplex. Validation and genomic sequencing revealed resistance mutations in SF3B1 and PHF5A in residues adjacent to the compound binding pocket. We mapped hypersensitive mutants to U2 snRNP components, but also to factors that act as late as the second chemical step, after SF3b has dissociated. Strikingly, we obtained resistance mutants in SUGP1, a spliceosomal G-patch protein of unknown function that lacks orthologs in yeast and is also a newly proposed tumor suppressor whose loss underpins the splicing changes induced by cancer-associated SF3B1 mutations.
2023-07-07 | GSE218306 | GEO
Project description:A major impediment to studying the human spliceosome in vivo has been the inability to program point mutations in endogenous genes on a large scale. CRISPR-Cas9 technology now provides such opportunities. Due to its scalability and ability to introduce point mutations, we chose CRISPR-Cas9 base editing for a forward genetic screen of the spliceosome in an eHAP haploid human cell line background. We maintained eHAP FNLS cells as haploid so that we could subsequently assign genotype-phenotype relationships. We designed a single guide RNA (sgRNA) library targeting a hand-curated list of 153 human spliceosomal proteins which engage at various steps of the splicing cycle. After mutagenesis, we interrogated the spliceosome using the potent inhibitor pladienolide B (PB), which targets U2 snRNP by binding to a pocket between the SF3B1 and PHF5A subunits of the SF3b complex, preventing stabilization of the U2-branchpoint RNA duplex. Validation and genomic sequencing revealed resistance mutations in SF3B1 and PHF5A in residues adjacent to the compound binding pocket. We mapped hypersensitive mutants to U2 snRNP components, but also to factors that act as late as the second chemical step, after SF3b has dissociated. Strikingly, we obtained resistance mutants in SUGP1, a spliceosomal G-patch protein of unknown function that lacks orthologs in yeast and is also a newly proposed tumor suppressor whose loss underpins the splicing changes induced by cancer-associated SF3B1 mutations.
2023-07-07 | GSE218305 | GEO
Project description:A major impediment to studying the human spliceosome in vivo has been the inability to program point mutations in endogenous genes on a large scale. CRISPR-Cas9 technology now provides such opportunities. Due to its scalability and ability to introduce point mutations, we chose CRISPR-Cas9 base editing for a forward genetic screen of the spliceosome in an eHAP haploid human cell line background. We maintained eHAP FNLS cells as haploid so that we could subsequently assign genotype-phenotype relationships. We designed a single guide RNA (sgRNA) library targeting a hand-curated list of 153 human spliceosomal proteins which engage at various steps of the splicing cycle. After mutagenesis, we interrogated the spliceosome using the potent inhibitor pladienolide B (PB), which targets U2 snRNP by binding to a pocket between the SF3B1 and PHF5A subunits of the SF3b complex, preventing stabilization of the U2-branchpoint RNA duplex. Validation and genomic sequencing revealed resistance mutations in SF3B1 and PHF5A in residues adjacent to the compound binding pocket. We mapped hypersensitive mutants to U2 snRNP components, but also to factors that act as late as the second chemical step, after SF3b has dissociated. Strikingly, we obtained resistance mutants in SUGP1, a spliceosomal G-patch protein of unknown function that lacks orthologs in yeast and is also a newly proposed tumor suppressor whose loss underpins the splicing changes induced by cancer-associated SF3B1 mutations.
2023-07-07 | GSE218304 | GEO
Project description:Transcription by RNA polymerase II (Pol II) is coupled to pre-mRNA splicing, but the underlying mechanisms remain poorly understood. Co-transcriptional splicing requires assembly of a functional spliceosome on nascent pre-mRNA, but whether and how this influences Pol II transcription remains unclear. Here we show that inhibition of pre-mRNA branch site recognition by the spliceosome component U2 snRNP leads to a widespread and strong decrease in new RNA synthesis in human cells. Multiomics analysis reveals that U2 snRNP inhibition increases the duration of Pol II pausing in the promoter-proximal region, impairs recruitment of the pause release factor P-TEFb, and reduces Pol II elongation velocity in the beginning of genes. Our results indicate that efficient release of paused Pol II into active transcription elongation requires formation of functional spliceosomes, and that eukaryotic mRNA biogenesis relies on positive feedback from the splicing machinery to the transcription machinery.
2021-01-31 | GSE148433 | 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:U12-type introns were originally recognized based on their highly conserved non-consensus AT-AC termini1,2, which are spliced by a separate minor spliceosome3,4. Padgett and Krainer groups later showed that terminal dinucleotides do not differentiate U12-type from U2-type introns, as there are U12-type introns with GT-AG termini and U2-type introns with AT-AC termini5,6. Rather, U12-type introns are recognized by their divergent and highly conserved 5’ splice site (5’ss) and branch point sequences, which both differ from the consensus sequences found in U2-type introns. To date, no functional differences have been ascribed to AT-AC or GT-AG subtypes of U12-type introns, nor have RNAseq analyses of minor spliceosome diseases reported any subtype specificity. Here, we describe a novel protein component of the minor spliceosome, encoded by the CENATAC locus, that is required for accurate splicing of AT-AC but not GT-AG type minor introns. CENATAC was initially identified in a subset of Mosaic Variegated Aneuploidy (MVA) patients with mutations in CENATAC, which lead to chromosome congression defects during mitosis. Earlier large-scale proteomic analyses tentatively classified CENATAC as a spliceosome component and phylogenetic analyses showed co-segregation of CENATAC with minor spliceosome components. Targeted depletion of CENATAC in HeLa cells, followed by RNAseq revealed global retention of AT-AC minor subtype introns with more than 60% showing statistically significant (up to 90%) intron retention (IR). Additionally, U12-type introns with 5’-AT, but divergent 3’-terminal dinucleotides also showed significant IR. We also detected cryptic U2-type splice site activation near affected AT-AC introns. In contrast, about 10% of GT-AG subtype introns responded to CENATAC depletion. Co-IP experiments revealed that CENATAC is not a U11/U12 di-snRNP component as expected for a specificity factor, but rather associates with the U4atac/U6atac.U5 tri-snRNP via interaction with PRPF3/4, suggesting a role for minor tri-snRNP in initial 5’ss recognition. CENATAC also interacts with TXNL4B, a paralog of TXNL4A in the major tri-snRNP. Finally, several genes encoding chromosome congression factors harbor U12 AT-AC-type introns that were highly retained in CENATAC depleted cells, potentially explaining the aneuploidy phenotype observed in MVA patients.
2021-04-19 | GSE143392 | GEO
Project description:The spliceosome is a dynamic RNA-protein complex that executes pre-mRNA splicing and is composed of five core small nuclear ribonucleoprotein particles (U1, U2, U4/5/6 snRNP) and >150 additional proteins specific for each snRNP. We report a circadian role for Pre-mRNA Processing factor 4 (PRP4), a conserved component of the spliceosomal U4/U6.U5 triple small nuclear ribonucleoprotein (tri-snRNP) complex. We broadly hypothesized that downregulation of prp4 led to the aberrant splicing of one or many of the core clock transcripts. To identify these splicing events in an unbiased way, we performed RNA-Sequencing (RNA-Seq) analysis. We reasoned that we could have a more targeted approach if we could zoom in on the overlapping splicing changes that would be driven by the knockdown of at least two different tri-snRNP components. Because the pan-neuronal knockdown of all tri-snRNP components tested in our study led to lethality, we decided to utilize an alternative broad driver. For that purpose, we selected a strong eye-specific Glass Multiple Promoter driver (GMR-Gal4). Because most of the signal from head lysates comes directly from the eye tissue and because the core splicing factors are ubiquitously expressed, GMR-specific downregulation of prp4 and prp8 promised to be a viable alternative to the pan-neuronal knockdown. We examined changes in both the total transcript levels and splicing events upon prp4 knockdown in the eye. The overall gene expression seemed to be dramatically influenced by prp4 downregulation (433 DOWN, 310 UP at FDR < 0.05). Despite the fact that PRP4 is a component of the core spliceosome that is required for constitutive exon splicing, we did not detect dramatic effects on global splicing. Only 45 genes exhibited differential alternate splicing upon prp4 downregulation at FDR < 0.05).
2018-11-30 | GSE115163 | GEO
Project description:We isolated snRNP complexes derived from precatalytic A or B-like spliceosomes solubilized from the chromatin pellet of lysed nuclei. These complexes contained U2 snRNP proteins and a portion of the U2 snRNA, bound with intronic branch sites prior to the first catalytic step of splicing. The complexes have similar composition, whether directly isolated from extracted material, or after selection from glycerol gradients. Here we describe sequencing libraries prepared from RNA derived from directly isolated (IP-seq), or gradient-purified complexes (RNP-seq). Libraries from deproteinized RNA were prepared after selective degradation of U2 snRNA, each in a triplicate. An additional library from RBM5-Flag RNP complex was prepared without such degradation. The majority of sequenced pre-mRNA fragments map to intronic branch sites.
2024-03-08 | GSE240596 | GEO
Project description:The branch site / 3' splice site recognition machinery is a hotspot for disease-associated mutations. A single amino acid substitution K700E in the U2 snRNP-specific protein SF3B1 is frequently associated with cancer and is particularly common among myelodysplastic syndromes. The goal of this study is to employ the U2 IP-seq strategy in examining the differences in branch site selection in cells expressing wild type SF3B1 vs those carrying the K700E mutation.
2025-01-28 | GSE280659 | GEO