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

被引：20

作者：

Beusch, Irene ^{[1
]}

Rao, Beiduo ^{[1
,4
]}

Studer, Michael K. ^{[1
,2
]}

Luhovska, Tetiana ^{[2
]}

Sukyte, Viktorija ^{[2
]}

Lei, Susan ^{[1
,5
]}

Oses-Prieto, Juan ^{[3
]}

SeGraves, Em ^{[1
]}

Burlingame, Alma ^{[1
,3
]}

Jonas, Stefanie ^{[1
,2
]}

Madhani, Hiten D. ^{[1
]}

机构：

[1] Univ Calif San Francisco, Dept Biochem & Biophys, San Francisco, CA 94143 USA

[2] Swiss Fed Inst Technol, Inst Mol Biol & Biophys, Dept Biol, Zurich, Switzerland

[3] Univ Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA USA

[4] Calico Life Sci LLC, South San Francisco, CA USA

[5] Univ Calif Davis, Davis, CA USA

来源：

MOLECULAR CELL | 2023年 / 83卷 / 14期

基金：

瑞士国家科学基金会; 芬兰科学院;

关键词：

SPLICING FIDELITY; STRUCTURAL BASIS; LARIAT-INTRON; BOX ATPASES; RNA; PRP43; ACTIVATION; COMPONENTS; MUTATIONS; PROOFREAD;

D O I：

10.1016/j.molcel.2023.06.003

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：2578 / +

页数：27

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