Transcriptome-wide Mapping of Internal N⁷-Methylguanosine Methylome in Mammalian mRNA

N⁷-methylguanosine (m⁷G) is a positively charged, essential modification at the 5' cap of eukaryotic mRNA, regulating mRNA export, translation, and splicing. m⁷G also occurs internally within tRNA and rRNA, but its existence and distribution within eukaryotic mRNA remain to be investigated. Here, we show the presence of internal m⁷G sites within mammalian mRNA. We then performed transcriptome-wide profiling of internal m⁷G methylome using m⁷G-MeRIP sequencing (MeRIP-seq). To map this modification at base resolution, we developed a chemical-assisted sequencing approach that selectively converts internal m⁷G sites into abasic sites, inducing misincorporation at these sites during reverse transcription. This base-resolution m⁷G-seq enabled transcriptome-wide mapping of m⁷G in human tRNA and mRNA, revealing distribution features of the internal m⁷G methylome in human cells. We also identified METTL1 as a methyltransferase that installs a subset of m⁷G within mRNA and showed that internal m⁷G methylation could affect mRNA translation.