mRNA (2'-O-methyladenosine-N(6)-)-methyltransferase
mRNA (2'-O-methyladenosine-N6-)-methyltransferase activity
Identification of the m<sup>6</sup>Am Methyltransferase PCIF1 Reveals the Location and Functions of m<sup>6</sup>Am in the Transcriptome.
mRNAs are regulated by nucleotide modifications that influence their cellular fate. Two of the most abundant modified nucleotides are N<sup>6</sup>-methyladenosine (m<sup>6</sup>A), found within mRNAs, and N<sup>6</sup>,2'-O-dimethyladenosine (m<sup>6</sup>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<sup>6</sup>Am. We find that PCIF1 binds and is dependent on the m<sup>7</sup>G cap. By depleting PCIF1, we generated transcriptome-wide maps that distinguish m<sup>6</sup>Am and m<sup>6</sup>A. We find that m<sup>6</sup>A and m<sup>6</sup>Am misannotations arise from mRNA isoforms with alternative transcription start sites (TSSs). These isoforms contain m<sup>6</sup>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<sup>6</sup>Am-annotated mRNAs. The discovery of PCIF1 and our accurate mapping technique will facilitate future studies to characterize m<sup>6</sup>Am's function.