PCIF1 catalyzes m6Am mRNA methylation to regulate gene expression
mRNA modifications play an important role in regulating gene expression. One of the most abundant mRNA modifications is N6,2-O-dimethyladenosine (m6Am). Here, we demonstrate that m6Am is an evolutionarily conserved mRNA modification mediated by the Phosphorylated CTD Interacting Factor 1 (PCIF1), which catalyzes m6A methylation on 2-O-methylated adenine located at the 5 ends of mRNAs. Furthermore, PCIF1 catalyzes only 5 m6Am methylation of capped mRNAs, but not internal m6A methylation in vitro and in vivo. Our global mRNA methylation analysis revealed that there is no crosstalk between m6Am and m6A mRNA methylation events, suggesting that m6Am is functionally distinct from m6A. Importantly, our data indicate that m6Am negatively impacts translation of methylated mRNAs by antagonizing cap binding protein eIF4E. Together, we identify the first and only human mRNA m6Am methyltransferase and demonstrate a novel mechanism of gene expression regulation through PCIF1-mediated m6Am mRNA methylation in eukaryotes.\n\nHighlightsO_LIPCIF1 is an evolutionarily conserved mRNA m6Am methyltransferase\nC_LIO_LILoss of PCIF1 leads to a complete loss of m6Am, whereas m6A level and distribution are not affected\nC_LIO_LIPCIF1 mediated m6Am does not affect RNA Pol II transcription or mRNA stability\nC_LIO_LIm6Am-Exo-Seq is a robust methodology that enables global m6Am mapping\nC_LIO_LIm6Am suppresses cap dependent translation\nC_LI