Methylation profiling by high throughput sequencing Other
Summary
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.
Overall design
[dataset 1] 12 samples. Duplicates for PAR-CLIP of METTL1 and WDR4 in HeLa cell line. Duplicates for PAR-CLIP of eIF4E, RNMT, METTL1 and WDR4 in HEK293T cell line. [dataset 2] 8 samples. Duplicates for Ribo-Seq samples, mRNA input and ribosome-bound RNA of siControl vs siMETTL1 in HeLa cell. [dataset 3] 46 samples. Duplicates for m7G-seq of small RNA (<200 nt) and mRNA samples, including three sections of RNA input, before_pulldown, and pulldown in HeLa, HepG2, and HEK293T cell lines. [dataset 4] 38 samples. Duplicates for m7G-MeRIP-seq (anti-m7G antibody, MBL) of cell mRNA samples, including two sections of RNA input and IP in HeLa, HepG2, HEK293T, MEF and mESC wild-type cell lines; also in HeLa cell siControl vs siMETTL1, and HepG2 cell shControl vs shMETTL1.
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