Conserved and divergent functions of N6-methyladenosine RNA methyltransferase METTL4 during evolution.

This study aims to investigate the enzymatic properties and evolutionary conservation of METTL4, a novel internal m6A(m) methyltransferase involved in U2 snRNA modification during spliceosome assembly. Our research demonstrates that METTL4 is conserved during evolution and can efficiently catalyze N6-methylation into position A30 of U2 snRNA in both human and fruitfly. Unlike the Cap-m6Am modification mediated by PCIF1, METTL4 catalyzes N6-methylation in the late stage of U2 RNP biogenesis. We also discovered that the absence of 2'-O-methylation, directed by mgU2-30 snoRNA, determines the final modification status of U2 A30 site in fruitfly and worm. Moreover, we performed comprehensive analyses of splicing changes in METTL4 knockout models across various species, which demonstrated that METTL4 depletion affects splicing of short exons in higher organisms, including human and mouse. These findings provide new insights into the molecular mechanisms of U2 snRNA modification during spliceosome assembly and its role in mRNA splicing.