Identification of the m 6 Am Methyltransferase PCIF1 Reveals the Location and Functions of m 6 Am in the Transcriptome

mRNAs are regulated by nucleotide modifications that influence their cellular fate. Two of the most abundant modified nucleotides are N -methyladenosine (m A), found within mRNAs, and N ,2'-O-dimethyladenosine (m Am), which is found at the first transcribed nucleotide. Distinguishing these modifications in mapping studies has been difficult. Here, we identify and biochemically characterize PCIF1, the methyltransferase that generates m Am. We find that PCIF1 binds and is dependent on the m G cap. By depleting PCIF1, we generated transcriptome-wide maps that distinguish m Am and m A. We find that m A and m Am misannotations arise from mRNA isoforms with alternative transcription start sites (TSSs). These isoforms contain m Am that maps to "internal" sites, increasing the likelihood of misannotation. We find that depleting PCIF1 does not substantially affect mRNA translation but is associated with reduced stability of a subset of m Am-annotated mRNAs. The discovery of PCIF1 and our accurate mapping technique will facilitate future studies to characterize m Am's function.