Cap-specific terminal N ⁶ -methylation of RNA by an RNA polymerase II–associated methyltransferase

N ⁶ -methyladenosine (m ⁶ A), a major modification of messenger RNAs (mRNAs), plays critical roles in RNA metabolism and function. In addition to the internal m ⁶ A, N ⁶ , 2′-O-dimethyladenosine (m ⁶ Am) is present at the transcription start nucleotide of capped mRNAs in vertebrates. However, its biogenesis and functional role remain elusive. Using a reverse genetics approach, we identified PCIF1, a factor that interacts with the serine-5–phosphorylated carboxyl-terminal domain of RNA polymerase II, as a cap-specific adenosine methyltransferase (CAPAM) responsible for N ⁶ -methylation of m ⁶ Am. The crystal structure of CAPAM in complex with substrates revealed the molecular basis of cap-specific m ⁶ A formation. A transcriptome-wide analysis revealed that N ⁶ -methylation of m ⁶ Am promotes the translation of capped mRNAs. Thus, a cap-specific m ⁶ A writer promotes translation of mRNAs starting from m ⁶ Am.