METTL4 catalyzes m(6)Am methylation in U2 snRNA to regulate pre-mRNA splicing

N (6)-methylation of 2′-O-methyladenosine (Am) in RNA occurs in eukaryotic cells to generate N(6),2′-O-dimethyladenosine (m(6)Am). Identification of the methyltransferase responsible for m(6)Am catalysis has accelerated studies on the function of m(6)Am in RNA processing. While m(6)Am is generally found in the first transcribed nucleotide of mRNAs, the modification is also found internally within U2 snRNA. However, the writer required for catalyzing internal m(6)Am formation had remained elusive. By sequencing transcriptome-wide RNA methylation at single-base-resolution, we identified human METTL4 as the writer that directly methylates Am at U2 snRNA position 30 into m(6)Am. We found that METTL4 localizes to the nucleus and its conserved methyltransferase catalytic site is required for U2 snRNA methylation. By sequencing human cells with overexpressed Mettl4, we determined METTL4’s in vivo target RNA motif specificity. In the absence of Mettl4 in human cells, U2 snRNA lacks m(6)Am thereby affecting a subset of splicing events that exhibit specific features such as 3′ splice-site weakness and an increase in exon inclusion. These findings suggest that METTL4 methylation of U2 snRNA regulates splicing of specific pre-mRNA transcripts.