Deployment of non-canonical splicing in tunicate genomes is mediated by divergent U2AF function and changing m6A modification in U1 and U6 snRNA.

Spliceosomal small nuclear RNA (snRNA) U1 and the U2AF heterodimer play critical functions by recognizing the highly conserved GT and AG dinucleotides, respectively, located at the start and at the end of introns. Here, we explore how changing these components contributed to maintaining splicing function in genomes where 95% of introns escape the GT/AG rule. By gaining access to new tunicate genomes, we could reveal that the emergence of non-canonical introns in the Fritillaria borealis lineage coincides with the duplication of U2AF subunits. Our findings indicate that paralogs U2AF1α and U2AF2α have preserved conserved functions, while divergent paralogs U2AF1β and U2AF2β form novel heterodimers that recognize introns with non-canonical 3' ends. The conserved m6A present on U6 snRNA has been considerably reduced in F. borealis, but its U1 snRNA retains a stable 5'-terminal m6A, which is typically suppressed in humans and other chordates. We propose that this unique m6A pattern stabilizes the binding of snRNA to non-canonical 5' splice sites. Although the core components of the spliceosome remain preserved, functional changes implemented through gene duplication and post-transcriptional modifications can significantly broaden the range of target splice sites.