Highlights d PCIF1 is the N6-adenosine methylase that produces m 6 Am in an m 7 G cap-dependent manner d PCIF1 depletion allows transcriptome-wide mapping of m 6 A and m 6 Am d m 6 Am mapping identifies alternative ''internal'' transcription start sites d m 6 Am increases stability of a subset of mRNAs and has no effect on translation

mRNAs are regulated by nucleotide modifications that influence their cellular fate. Two of the most abundant modified nucleotides are N 6 -methyladenosine (m 6 A), found within mRNAs, and N 6 ,2'-O-dimethyladenosine (m 6 Am), which is found at the first-transcribed nucleotide. A longstanding challenge has been distinguishing these similar modifications in transcriptome-wide mapping studies. Here we identify and biochemically characterize, PCIF1, the methyltransferase that generates m 6 Am. We find that PCIF1 binds and is dependent on the m 7 G cap. By depleting PCIF1, we definitively identified m 6 Am sites and generated transcriptomewide maps that are selective for m 6 Am and m 6 A. We find that m 6 A and m 6 Am misannotations largely arise from mRNA isoforms with alternate transcription-start sites. These isoforms contain m 6 Am that appear to map to "internal" sites, increasing the likelihood of misannotation. Using the new m 6 Am annotations, we find that depleting m 6 Am does not affect mRNA translation but reduces the stability of a subset of m 6 Am-annotated mRNAs. The discovery of PCIF1 and our accurate mapping technique will facilitate future studies to characterize m 6 Am's function.