RESUMEN
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.
Asunto(s)
Mapeo Cromosómico/métodos , Guanosina/análogos & derivados , Metiltransferasas/genética , ARN Mensajero/genética , ARN de Transferencia/genética , Transcriptoma , Animales , Secuencia de Bases , Línea Celular , Fibroblastos/citología , Fibroblastos/metabolismo , Guanosina/metabolismo , Células HEK293 , Células HeLa , Células Hep G2 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Metilación , Metiltransferasas/metabolismo , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Transcripción ReversaRESUMEN
m7G-seq detects internal 7-methylguanosine (m7G) sites within mRNAs and noncoding RNAs by misincorporation signatures. A chemical-assisted sequencing approach selectively converts internal m7G sites into abasic sites, triggering misincorporation at these sites in the presence of a specific reverse transcriptase. The further enrichment of m7G-induced abasic sites by biotin pull-down reveals hundreds of internal m7G sites in human mRNA. The misincorporation ratio before pull-down enrichment can be used for estimating the methylation fraction of some highly methylated m7G sites.