A topologically conserved unstructured region helps positioning the evolutionarily conserved Prp40 WW domains to promote non-canonical intron splicing

Newly transcribed introns are immediately identified by early splicing factors that recognize the intron 5’ splice site (5’SS) and branch site (BS). In the budding yeast, these critical splice-site sequences are generally constrained, whereas degeneracy is the rule in higher eukaryotes. Yet, ∼40% of the yeast introns do diverge, to a certain degree, from the canonical sequences. Exactly how these non-canonical introns are recognized and spliced remains unknown. Here we show that the conserved Prp40 WW domains promote non-canonical intron splicing by enhancing stable U1 snRNP and BBP recruitments. AlphaFold predicts a topologically conserved unstructured region between Prp40 WW and FF domains. Alignment of the AlphaFold Prp40 structure with published U1 snRNP structure positions WW domains adjacent to 5’SS and Luc7, which is known to be critical for 5’SS recognition. Indeed, deletion of this unstructured region negatively impacts on splicing of the non-canonical 5’SS introns. Taken together, our results suggest that the conserved WW domains may have evolved to deal with the highly degenerate 5’SS and BS sequences in higher eukaryotes, so as to accommodate increased splicing complexity.