m(6)A modification of U6 snRNA modulates usage of two major classes of pre-mRNA 5’ splice site

Alternative splicing of messenger RNAs is associated with the evolution of developmentally complex eukaryotes. Splicing is mediated by the spliceosome, and docking of the pre-mRNA 5’ splice site into the spliceosome active site depends upon pairing with the conserved ACAGA sequence of U6 snRNA. In some species, including humans, the central adenosine of the ACAGA box is modified by N(6) methylation, but the role of this m(6)A modification is poorly understood. Here, we show that m(6)A modified U6 snRNA determines the accuracy and efficiency of splicing. We reveal that the conserved methyltransferase, FIONA1, is required for Arabidopsis U6 snRNA m(6)A modification. Arabidopsis fio1 mutants show disrupted patterns of splicing that can be explained by the sequence composition of 5’ splice sites and cooperative roles for U5 and U6 snRNA in splice site selection. U6 snRNA m(6)A influences 3’ splice site usage. We generalise these findings to reveal two major classes of 5’ splice site in diverse eukaryotes, which display anti-correlated interaction potential with U5 snRNA loop 1 and the U6 snRNA ACAGA box. We conclude that U6 snRNA m(6)A modification contributes to the selection of degenerate 5’ splice sites crucial to alternative splicing.