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

N-7-methylguanosine (m(7)G) is a positively charged, essential modification at the 5' cap of eukaryotic mRNA, regulating mRNA export, translation, and splicing. m(7)G 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 m(7)G sites within mammalian mRNA. We then performed transcriptome-wide profiling of internal m(7)G methylome using m(7)G-MeRIP sequencing (MeRIP-seq). To map this modification at base resolution, we developed a chemical-assisted sequencing approach that selectively converts internal m(7)G sites into abasic sites, inducing misincorporation at these sites during reverse transcription. This base-resolution m(7)G-seq enabled transcriptome-wide mapping of m(7)G in human tRNA and mRNA, revealing distribution features of the internal m(7)G methylome in human cells. We also identified METTL1 as a methyltransferase that installs a subset of m(7)G within mRNA and showed that internal m(7)G methylation could affect mRNA translation.