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

<i>N</i> ⁶-methyladenosine (m⁶A), a major modification of messenger RNAs (mRNAs), plays critical roles in RNA metabolism and function. In addition to the internal m⁶A, <i>N</i> ⁶, 2'-<i>O</i>-dimethyladenosine (m⁶Am) 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 <i>N</i> ⁶-methylation of m⁶Am. The crystal structure of CAPAM in complex with substrates revealed the molecular basis of cap-specific m⁶A formation. A transcriptome-wide analysis revealed that <i>N</i> ⁶-methylation of m⁶Am promotes the translation of capped mRNAs. Thus, a cap-specific m⁶A writer promotes translation of mRNAs starting from m⁶Am.