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

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

PROVIDER: PRJNA903135 | ENA |

REPOSITORIES: ENA

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

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

| PRJNA903130 | ENA

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: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 [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 [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

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

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

Project description:Adenosine-to-inosine (A-to-I) RNA editing catalyzed by ADAR enzymes occurs in double-stranded RNAs. Despite a compelling need towards predictive understanding of natural and engineered editing events, how the RNA sequence and structure determine the editing efficiency and specificity (i.e., cis-regulation) is poorly understood. We apply a CRISPR/Cas9-mediated saturation mutagenesis approach to generate libraries of mutations near three natural editing substrates at their endogenous genomic loci. We use machine learning to integrate diverse RNA sequence and structure features to model editing levels measured by deep sequencing. We confirm known features and identify new features important for RNA editing. Training and testing XGBoost algorithm within the same substrate yield models that explain 68 to 86 percent of substrate-specific variation in editing levels. However, the models do not generalize across substrates, suggesting complex and context-dependent regulation patterns. Our integrative approach can be applied to larger scale experiments towards deciphering the RNA editing code.

2021-03-05 | GSE168234 | GEO

Spliceosome iCLIP

Project description:Spliceosome iCLIP

| PRJEB27592 | ENA