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Targeted high throughput mutagenesis of the human spliceosome reveals its in vivo operating principles

ABSTRACT: Targeted high throughput mutagenesis of the human spliceosome reveals its in vivo operating principles

PROVIDER: PRJNA903130 | ENA |

REPOSITORIES: ENA

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Targeted high throughput mutagenesis of the human spliceosome reveals its in vivo operating principles [screen]

Project description:Targeted high throughput mutagenesis of the human spliceosome reveals its in vivo operating principles [screen]

| PRJNA903134 | ENA

Targeted high throughput mutagenesis of the human spliceosome reveals its in vivo operating principles [editing]

Project description:Targeted high throughput mutagenesis of the human spliceosome reveals its in vivo operating principles [editing]

| PRJNA903135 | ENA

Targeted high throughput mutagenesis of the human spliceosome reveals its in vivo operating principles [RNA-Seq]

Project description:Targeted high throughput mutagenesis of the human spliceosome reveals its in vivo operating principles [RNA-Seq]

| PRJNA903133 | ENA

Targeted high throughput mutagenesis of the human spliceosome reveals its in vivo operating principles [editing]

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

Targeted high throughput mutagenesis of the human spliceosome reveals its in vivo operating principles [screen]

2023-07-07 | GSE218306 | GEO

Targeted high throughput mutagenesis of the human spliceosome reveals its in vivo operating principles [RNA-Seq]

2023-07-07 | GSE218304 | GEO

Targeted high throughput mutagenesis of the human spliceosome reveals its in vivo operating principles

Project description:This SuperSeries is composed of the SubSeries listed below.

2023-07-06 | GSE218307 | GEO

Spliceosome iCLIP

Project description:Spliceosome iCLIP

| PRJEB27592 | ENA

Learning cis-regulatory principles of ADAR-based RNA editing from CRISPR-mediated mutagenesis

Project description:Learning cis-regulatory principles of ADAR-based RNA editing from CRISPR-mediated mutagenesis

| PRJNA706647 | ENA

Spliceosome iCLIP

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

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