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

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. [Display omitted] •Global mutagenesis of the human spliceosome using CRISPR-Cas9 base editing•Pladienolide B-hypersensitive and -resistant mutants identified•Resistance mutations in SUGP1 G-patch disrupt interaction with DHX15/hPrp43•SUGP1-DHX15/hPrp43 may mediate an early spliceosome proofreading step Beusch et al. use base editing to investigate the human spliceosome in haploid cells. Selection for resistance to the U2 snRNP inhibitor pladienolide B revealed mutations in the drug-binding factors and in the early spliceosomal protein SUGP1. Further studies reveal the spliceosome disassemblase DHX15/hPrp43 to be the ligand of SUGP1.