Genetic dissection of nonconventional introns reveals codominant noncanonical splicing code in <i>Euglena</i>.

Abstract

Pre-mRNA splicing is essential for eukaryotic gene expression and is achieved through the accurate recognition of exon-intron boundaries. Although nonconventional introns, which do not follow the conventional GT-AG splicing rule, have been identified in several species, these introns are typically rare in any given genome. Here, we demonstrate the widespread occurrence of nonconventional introns (71.8% of all introns) in the <i>Euglena agilis</i> genome and identify consensus motifs at these nonconventional exon-intron boundaries. We assessed the splicing efficiency of nonconventional introns and variants with point mutations via genomic knock-in within the second exon of <i>Glucan synthase-like 2</i> in <i>Euglena gracilis</i> and genetically defined the sequence signature (5'-N₃CDG-/-CH'GN_5-6|R_exon-3') required for their proper splicing. This signature is present in 61.2% of all nonconventional introns detected in the <i>E. agilis</i> genome. Accordingly, we present a noncanonical splicing code for <i>Euglena</i> introns, highlighting the global coexistence of dual splicing rules for conventional and nonconventional introns.