The conserved Prp40 WW domains facilitate non-canonical intron splicing via bridging 5'-splice-site and branch-site interaction

Precise regulation of molecular interactions is essential for the proper execution of cellular pathways. In pre-mRNA splicing, the spliceosome must accurately process introns containing both canonical and non-canonical splice-site sequences, the latter often requiring additional regulatory mechanisms. Here, we investigate how cells cope with introns carrying non-canonical splice signals, focusing on spliceosomal components that recognize the 5′ splice site (5′SS) and branch point sequence (BPS). Unexpectedly, we find that the N-terminal WW repeats of the U1 snRNP protein Prp40, previously considered non-essential, play a critical role in facilitating the recognition and splicing of introns with suboptimal 5′SS and BPS elements. We provide genetic and biochemical evidence supporting this function and propose a structural model consistent with predictions from AlphaFold3. Together, our findings reveal a previously underappreciated role for Prp40 in fine-tuning splice-site recognition and ensuring robust splicing of non-canonical introns.