Identification of the m6Am methyltransferase PCIF1 reveals the location and functions of m6Am in the transcriptome

mRNAs are regulated by nucleotide modifications that influence their cellular fate. Two of the most abundant modified nucleotides are N6-methyladenosine (m6A), found within mRNAs, and N6,2-O-dimethyladenosine (m6Am), which is found at the first-transcribed nucleotide. A long-standing challenge has been distinguishing these similar modifications in transcriptome-wide mapping studies. Here we identify and biochemically characterize, PCIF1, the methyltransferase that generates m6Am. We find that PCIF1 binds and is dependent on the m7G cap. By depleting PCIF1, we definitively identified m6Am sites and generated transcriptome-wide maps that are selective for m6Am and m6A. We find that m6A and m6Am misannotations largely arise from mRNA isoforms with alternate transcription-start sites. These isoforms contain m6Am that appear to map to \"internal\" sites, increasing the likelihood of misannotation. Using the new m6Am annotations, we find that depleting m6Am does not affect mRNA translation but reduces the stability of a subset of m6Am-annotated mRNAs. The discovery of PCIF1 and our accurate mapping technique will facilitate future studies to characterize m6Ams function.