Pseudouridines in the U2 Branch Site Recognition Region Differentially Contribute to Branch Site Recognition during Pre-mRNA Splicing

Pseudouridines ({Psi}s) are highly enriched in conserved, functionally critical regions of spliceosomal snRNAs, particularly within the U2 branch site recognition region (BSRR), which contains six {Psi}s in humans and three in S. cerevisiae. To investigate how U2 BSRR pseudouridylation influences branch site sequence (BSS) recognition, we developed a large-scale, high-throughput screening system in S. cerevisiae that allows a library of pre-mRNAs with randomized BSSs to be spliced in distinct U2 BSRR pseudouridylation backgrounds. Screening and next-generation sequencing (NGS) revealed that different U2 pseudouridylation backgrounds exhibit distinct recognition patterns and efficiencies for specific BSSs. Notably, {Psi}42 and {Psi}44 generally enhanced splicing, whereas {Psi}38 alone, and in some contexts together with {Psi}35, consistently impaired BSS recognition. The differential effects were validated using endogenous S. cerevisiae genes. In addition, splicing assays with engineered pre-mRNA constructs guided by the screening results demonstrated that BSRR {Psi}s directly influence BSS selection, supporting a model in which U2 pseudouridylation modulates BSS recognition and could contribute to alternative splicing in more complex eukaryotes. Finally, synthetic-lethality analyses with a Prp5 mutant, together with Prp5-U2-pre-mRNA binding assays, indicate that U2 BSRR {Psi}s are critical for Prp5 recruitment and, consequently, for proper U2-BSS interactions during spliceosome assembly. Collectively, these findings establish U2 pseudouridylation as a key determinant of branch site recognition and spliceosome function.