Targeted High-throughput Mutagenesis of the Human Spliceosome Reveals Its In vivo Operating Principles

Overview

Journal Mol Cell

Publisher Cell Press

Specialty Cell Biology

Date 2023 Jul 4

PMID 37402368

Authors

Irene Beusch

Beiduo Rao

Michael K Studer

Tetiana Luhovska

Viktorija Sukyte

Susan Lei

Juan Oses-Prieto

Em SeGraves

Alma Burlingame

Stefanie Jonas

Hiten D Madhani

Affiliations

Soon will be listed here.

Abstract

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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