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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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			Action	DRS
	SRR22333000.fastq.gz	Fastqsanger.gz
	SRR22333001.fastq.gz	Fastqsanger.gz
	SRR22333002.fastq.gz	Fastqsanger.gz
	SRR22333003.fastq.gz	Fastqsanger.gz
	SRR22333004.fastq.gz	Fastqsanger.gz

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Project description:The spliceosome is a staggeringly complex machine, comprising, in humans, 5 snRNAs and >150 proteins. We scaled haploid CRISPR-Cas9 base editing to target the entire human spliceosome and investigated the mutants using the U2 snRNP/SF3b inhibitor, pladienolide B. Hypersensitive substitutions define functional sites in the U1/U2-containing A complex but also in components that act as late as the second chemical step after SF3b is dissociated. Viable resistance substitutions map not only to the pladienolide B-binding site but also to the G-patch domain of SUGP1, which lacks orthologs in yeast. We used these mutants and biochemical approaches to identify the spliceosomal disassemblase DHX15/hPrp43 as the ATPase ligand for SUGP1. These and other data support a model in which SUGP1 promotes splicing fidelity by triggering early spliceosome disassembly in response to kinetic blocks. Our approach provides a template for the analysis of essential cellular machines in humans.

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