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

ABSTRACT: This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Homo sapiens

PROVIDER: GSE218307 | GEO | 2023/07/06

REPOSITORIES: GEO

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

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

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

2023-07-07 | GSE218305 | 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: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: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

A high throughput in vivo protein-DNA mapping approach reveals principles of dynamic gene regulation in mammals

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

2012-09-11 | GSE36104 | GEO

Network theory reveals principles of spliceosome structure and dynamics.

Project description:Cryoelectron microscopy has revolutionized spliceosome structural biology, and structures representing much of the splicing process have been determined. Comparison of these structures is challenging due to extreme dynamics of the splicing machinery and the thousands of changing interactions during splicing. We have used network theory to analyze splicing factor interactions by constructing structure-based networks from protein-protein, protein-RNA, and RNA-RNA interactions found in eight different spliceosome structures. Our analyses reveal that connectivity dynamics result in step-specific impacts of factors on network topology. The spliceosome's connectivity is focused on the active site, in part due to contributions from nonglobular proteins. Many essential factors exhibit large shifts in centralities during splicing. Others show transiently high betweenness centralities at certain stages, thereby suggesting mechanisms for regulating splicing by briefly bridging otherwise poorly connected network nodes. These observations provide insights into organizing principles of the spliceosome and provide frameworks for comparative analysis of other macromolecular machines.

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High-throughput mutagenesis reveals unique structural features of human ADAR1.

Project description:Adenosine Deaminases that act on RNA (ADARs) are enzymes that catalyze adenosine to inosine conversion in dsRNA, a common form of RNA editing. Mutations in the human ADAR1 gene are known to cause disease and recent studies have identified ADAR1 as a potential therapeutic target for a subset of cancers. However, efforts to define the mechanistic effects for disease associated ADAR1 mutations and the rational design of ADAR1 inhibitors are limited by a lack of structural information. Here, we describe the combination of high throughput mutagenesis screening studies, biochemical characterization and Rosetta-based structure modeling to identify unique features of ADAR1. Importantly, these studies reveal a previously unknown zinc-binding site on the surface of the ADAR1 deaminase domain which is important for ADAR1 editing activity. Furthermore, we present structural models that explain known properties of this enzyme and make predictions about the role of specific residues in a surface loop unique to ADAR1.

| S-EPMC7550611 | biostudies-literature

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