Transcriptome carries a wide range of different chemical modifications. Among these modifications, RNA methylation is the most abundant, exerting important functions in multiple biological processes in eukaryotes. Especially, the recent discoveries of N6-methyladenosine (m6A) in transcriptome with reversible dynamics and regulatory roles have drawn considerable attentions from world-wide scientists. While N6-methyladenosine (m6A) is widely observed to regulate gene expression in mammals, our lab has also reasoned that positively-charged mRNA modifications such as N1-methyladenosine (m1A) could tune RNA secondary structures or protein-RNA interactions, which could impact key biological functions. N7-methylguanosine (m7G) is the positively-charged, well-known and essential modification at the 5’ cap of eukaryotic messenger RNA (mRNA), regulating mRNA export, translation, and splicing. m7G also occurs internally within transfer RNA (tRNA) and ribosomal RNA (rRNA), but whether it exists within eukaryotic mRNA remains to be investigated. Here, we show the presence of internal m7G sites within mammalian mRNA via mass spectrometry quantification; then illustrate the transcriptome-wide profile of internal m7G methylome with m7G-MeRIP-seq. To map this modification at base resolution, we developed a chemical-assisted sequencing approach that selectively convert internal m7G sites into abasic sites, which finally leads to misincorporation at these m7G sites during reverse transcription. With accurately detecting internal m7G methylation in tRNA and mRNA, the high resolution of this misincorporation-based method enables us to reveal key features of 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 may promote mRNA translation. Collectively, our approaches reveal internal m7G methylome in mammalian mRNA and suggest potential functional roles of m7G-mediated epitranscriptomic regulation.



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