Targeted high throughput mutagenesis of the human spliceosome reveals its in vivo operating principles
Beusch, I.; Rao, B.; Studer, M.; Luhovska, T.; Sukyte, V.; Lei, S.; Oses-Prieto, J. A.; SeGraves, E.; Burlingame, A.; Jonas, S.; Madhani, H. D.
Show 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 interrogated 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 (ATPase activator) 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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