Abstract 2289 Understanding the dynamic design of the human spliceosome

Most of our understanding of how the spliceosome operates in vivo comes from studies of mutants in yeasts, but human splicing displays major differences. However, applying genetics to understand the spliceosomal basis for these differences is challenging. To identify informative alleles, we implemented CRISPR-Cas9 base editing in haploid human cells (eHAPs), producing a library that generates point mutations in thousands of individual residues in over 150 spliceosomal proteins. We tested this approach by performing a screen to identify mutants resistant or hypersensitive to pladienolide B (plaB), an inhibitor of SF3b in the U2 snRNP. Hypersensitive mutants occurred in SF3a and SF3b subunits and other A complex components but also in factors thought to act only later in splicing. Resistant mutations showed anticipated sequence changes in SF3b subunits near the plaB binding pocket. Viable resistance substitutions map not only to the pladienolide B binding site but also to factors not involved in drug binding. Amongst these, two resistance mutations map 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. More broadly, we propose that the ATPases associated with the human spliceosome that are not found in S. cerevisiae function in kinetic proofreading mechanism, offering an explanation for complex and dynamic nature of the spliceosome. Supported by R01 GM71801.