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

Pseudouridines (Ψs) are highly enriched in conserved, functionally critical regions of spliceosomal snRNAs, particularly within the U2 branch site recognition region (BSRR), which contains six Ψs 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, Ψ42 and Ψ44 generally enhanced splicing, whereas Ψ38 alone, and in some contexts together with Ψ35, 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 Ψs 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 Ψs 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.