METTL4 catalyzes m6Am methylation inU2 snRNAto regulate pre-mRNA splicing

AbstractN6-methylation of 2’-O-methyladenosine (Am) in RNA occurs in eukaryotic cells to generateN6,2’-O-dimethyladenosine (m6Am). Identification of the methyltransferase responsible for m6Am catalysis has accelerated studies on the function of m6Am in RNA processing. While m6Am is generally found in the first transcribed nucleotide of mRNAs, the modification is also found internally withinU2 snRNA. However, the writer required for catalyzing internal m6Am 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 atU2 snRNAposition 30 into m6Am. We found that METTL4 localizes to the nucleus and its conserved methyltransferase catalytic site is required forU2 snRNAmethylation. By sequencing human cells with overexpressedMettl4, we determined METTL4’sin vivotarget RNA motif specificity. In the absence ofMettl4in human cells,U2 snRNAlacks m6Am thereby affecting a subset of splicing events that exhibit specific features such as overall 3’ splice-site weakness with certain motif positions more affected than others. This study establishes that METTL4 methylation ofU2 snRNAregulates splicing of specific pre-mRNA transcripts.