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

N ⁶-methyladenosine (m⁶A), a major modification of messenger RNAs (mRNAs), plays critical roles in RNA metabolism and function. In addition to the internal m⁶A, N ⁶, 2'-O-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 N 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 N ⁶-methylation of m⁶Am promotes the translation of capped mRNAs. Thus, a cap-specific m⁶A writer promotes translation of mRNAs starting from m⁶Am.