Deployment of non-canonical splicing in tunicate genomes is mediated by divergent U2AF function and re-patterning of snRNA m6A modification

Abstract

In eukaryotes, the critical function of the spliceosome is to remove introns from newly transcribed pre-messenger RNA to produce functional mRNA. Spliced segments normally begin with a GT and end with an AG, and precise intron removal relies on the recognition of terminal dinucleotides by the spliceosomal RNA (snRNA) and by the U2AF heterodimer. Here, we reveal how the modification of these molecules was instrumental for shifting spliceosome specificity, enforcing splicing accuracy in genomes where 95% of introns escape the GT/AG rule. We show that the emergence of non-canonical introns in the Fritillaria borealis lineage is associated with the duplication of each U2AF subunit. Subunit paralogues can be combined to form multiple U2AF complexes that involve novel protein-protein interaction rules. Binding assays on RNA and transcriptome analysis of human cells expressing U2AF subunits show that divergent paralogues can contribute to the recognition of non-canonical splice signals at the 3 end of introns. In transfected cells, levels of N6-methyladenosine (m6A) modification on snRNA U2 and U6 are impacted by the presence of F. borealis U2AF2 and by AHCYL1, a specific partner of the divergent subunit U2AF2{beta}. In striking contrast with other species, F. borealis has lost m6A on the U6 snRNA while U1 snRNA has gained a stable, 5-terminal m6A. We used structure prediction to show how these snRNA modifications could play decisive roles during the recognition of non-GT/AG introns. In conclusion, we argue that spliceosome function can be profoundly impacted by gene neofunctionalization and post-transcriptional modifications, without implying major changes to conserved genetic components.