N⁶-methyladenosine (m⁶A), an abundant modification in eukaryotic mRNAs and long-noncoding RNAs, has been recognized as a major epitranscriptome mark that plays critical roles in RNA metabolism and function. In addition to the internal m⁶A, N⁶, 2′-O-dimethyladenosine (m⁶Am) is present at the transcription start site of capped mRNAs in vertebrates. Previous studies reported that an eraser protein, FTO, demethylates N⁶-methyl group of m⁶Am and destabilizes a subset of mRNAs, suggesting a possible function of m⁶Am in stabilizing A-starting capped mRNAs. However, the biogenesis and functional role of m⁶Am remain elusive.

To reveal the functional and physiological roles of m⁶Am, it is necessary to identify a writer protein for N⁶-methylation of m⁶Am. We first established a highly sensitive method to analyze 5′-terminal chemical structures of the capped mRNAs using mass spectrometry (RNA-MS), and then measured m⁶Am methylation status accurately. We employed RNA-MS to identify the writer gene by a reverse genetic approach. We chose several candidates of uncharacterized methyltransferases (MTases) that are conserved in vertebrates, but not in yeast, which does not have m⁶Am. Each of the candidates was knocked out in human cells. If the target gene is disrupted, RNA-MS can detect the absence of m⁶Am in mRNAs prepared from the knockout cells.

RNA-MS analysis revealed that m⁶Am modification in human mRNAs is more abundant (92%) than previously estimated. We identified human PCIF1 as cap-specific adenosine-N⁶-MTase (CAPAM) responsible for N⁶-methylation of m⁶Am. Indeed, m⁶Am disappeared completely and converted to Am modification in mRNAs prepared from the CAPAM knockout (KO) cells. The CAPAM KO cells were viable, but sensitive to oxidative stress, implying the physiological importance of m⁶Am. We showed that CAPAM catalyzes N⁶-methylation of m⁶Am in the capped mRNAs in an S-adenosylmethionine (SAM)–dependent manner. A series of biochemical studies revealed that CAPAM specifically recognizes the 7-methylguanosine (m⁷G) cap structure and preferentially N⁶-methylates m⁷GpppAm rather than m⁷GpppA, indicating the importance of the 2′-O-methyl group of the target site for efficient m⁶Am formation. CAPAM has a N-terminal WW domain that specifically interacts with the Ser⁵-phosphorylated C-terminal domain (CTD) of RNA polymerase II (RNAPII), suggesting that the CAPAM is recruited to the early elongation complex of RNAPII and introduces m⁶Am in a nascent mRNA chain cotranscriptionally. We also solved the crystal structure of CAPAM complexed with the cap analog and SAM analog. The core region of CAPAM is composed of MTase and helical domains. The m⁷G cap is bound to a pocket formed by these two domains. The SAM analog is recognized by an active site with the characteristic NPPF motif in the MTase domain. This structure reveals the molecular basis of cap-specific m⁶A formation. RNA-sequencing analysis of the CAPAM KO cells revealed that loss of m⁶Am does not affect transcriptome alteration. This result does not support the proposed function of m⁶Am in stabilizing A-starting capped mRNAs. Instead, ribosome profiling of the CAPAM KO cells showed that N⁶-methylation of m⁶Am promotes the translation of capped mRNAs.

We identified PCIF1/CAPAM as a cap-specific m⁶A writer for vertebrate mRNAs. Structural analysis revealed the molecular basis of cap-specific m⁶A formation by CAPAM. The ribosome profiling experiment revealed that CAPAM-mediated m⁶Am formation promotes …