Overlapping roles of spliceosomal components SF3B1 and PHF5A in rice splicing regulation
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ABSTRACT:
ORGANISM(S): Oryza sativa Japonica Group
PROVIDER: GSE153234 | GEO | 2021/04/11
REPOSITORIES: GEO
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Project description:Developing small-molecule splicing modulators represents a promising therapeutic approach for various diseases. Natural products such as pladienolide, herboxidiene, and spliceostatin have been identified as potent splicing modulators that target SF3B1 in the SF3b subcomplex. Using integrated chemogenomic, structural and biochemical approaches, we show that PHF5A, another core component of the SF3b complex, is also targeted by these compounds. Mutations in PHF5A-Y36, SF3B1-K1071, SF3B1-R1074, and SF3B1-V1078 confer resistance to these modulators, suggesting a common site of interaction. Whole-transcriptome RNA-seq analysis reveals that PHF5A-Y36C has minimal effect on basal splicing but alters the action of splicing modulators from inducing intron-retention to exon-skipping. Relative intron strength to splicing inhibition correlates with the differential in GC content between adjacent introns and exons, leading to this differential global splicing pattern. We determine the crystal structure of human PHF5A and find that Y36 is located on the surface in a region of high sequence conservation. Structural analysis of the cryo-EM spliceosome Bact complex shows that these mutations cluster in a well-defined pocket surrounding the branch point adenosine suggesting a possible competitive mode of action for these splicing modulators, which interact with the aromatic side-chain of PHF5A-Y36. Collectively, we propose that PHF5A-SF3B1 forms a central node for binding to these small-molecule splicing modulators, offering insights to modulate splicing.
2017-05-25 | GSE96917 | 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: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: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 | 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 | 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
Project description:SF3B1 is one of shared components of U2 and U12 snRNPs that are required for splicing of U2-type and U12-type introns, respectively. Interestingly, recurrent somatic mutations have been identified in the spliceosome components in human cancer. By using CLIP-seq for SF3B1, binding sites in human embryonic stem cells were determined. The most prominent binidng sites were at 3' splice sites on pre-mRNA. Interestingly, 5' and 3' ends of introns spliced out were enriched by CLIP.
2023-04-26 | GSE222867 | GEO
Project description:Much remains unknown concerning the mechanism by which the splicing machinery pinpoints short exons within intronic sequences and how splicing factors are directed to their pre-mRNA targets. Part of the explanation probably lies in differences in chromatin organization between exons and introns. Proteomic, co-immunoprecipitation, and sedimentation analyses described here indicated that SF3B1, an essential splicing component of the U2 snRNP complex, is strongly associated with nucleosomes. ChIP-seq and RNA-seq analyses revealed that SF3B1 is specifically bound to nucleosomes located at exonic positions. SF3B1 binding is enriched at nucleosomes positioned over short exons flanked by long introns that are also characterized by differential GC content between exons and introns. Disruption of SF3B1 binding to such nucleosomes affected the splicing of these exons similarly to inhibition of SF3B1 expression. Our findings suggest that the association of SF3B1 with nucleosomes is functionally important for splice site recognition and that SF3B1 conveys splicing-relevant information embedded in chromatin structure. MNase-seq on Input and SF3B1 pull-down, mRNA-seq on control and SF3B1 si-RNA treated cells as well as on TSA (Trichostatin A) treated and untreated cells.
2015-04-22 | E-GEOD-65644 | biostudies-arrayexpress