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Transcriptome Mapping of Internal N7-methylguanosine modification via m7G MeRIP-seq [m7g.2]

ABSTRACT: Previous study found the presence of internal N7-methylguanosine (m7G) within mammalian mRNA. We performed antibody-based m7G MeRIP-seq for transcriptome-wide mapping the internal m7G sites in U2OS cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE192845 | GEO | 2023/05/17

REPOSITORIES: GEO

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Transcriptome Mapping of Internal N7-methylguanosine modification via m7G MeRIP-seq [m7g.1]

Project description:Previous study found the presence of internal N7-methylguanosine (m7G) within mammalian mRNA. We performed antibody-based m7G MeRIP-seq for transcriptome-wide mapping the internal m7G sites in HepG2 cells.

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Identification of potential readers for eukaryotic internal mRNA N7-methylguanosine (m7G) modification

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Transcriptome Mapping of Internal N7-methylguanosine modification via m7G MeRIP-seq [m7g.1]

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Transcriptome Mapping of Internal N7-methylguanosine modification via m7G MeRIP-seq [m7g.2]

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Transcriptome Mapping of Internal N7-methylguanosine Methylome in Mammalian mRNA

Project description: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.

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Recognition of internal mRNA N7-methylguanosine by QKI shuttles transcripts into stress granules and regulates translation

Project description:Recent studies reported that N7-methylguanosine (m7G) modification exists in internal mRNAs; however, the “reader” protein for mRNA internal m7G modification is still unrevealed. Here, by performing m7G MeRIP-seq and RIP-seq, we identified quaking protein (QKI) as a novel internal m7G modification reader. Internal mRNA m7G modification acts as a key player in mRNA translation under stress condition. To explore the mechanism underlying the function of QKI in translation regulation, we employed RNA-seq and Ribo-seq, and demonstrated that QKI7 regulates the translation efficiency of a subset of internal m7G-modified transcripts under stress condition.

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m7G-quant-seq: Quantitative Detection of RNA Internal N7-Methylguanosine

Project description:Methods for the precise detection and quantification of RNA modifications are critical to uncover functional roles of diverse RNA modifications. The internal m7G modification in mammalian cytoplasmic tRNAs is known to affect tRNA function and impact embryonic stem cell self-renewal, tumorigenesis, cancer progression, and other cellular processes. Here, we introduce m7G-quant-seq, a quantitative method that accurately detects internal m7G sites in human cytoplasmic tRNAs at single-base resolution. The efficient chemical reduction and mild depurination can almost completely convert internal m7G sites into RNA abasic sites (AP sites). We demonstrate that RNA abasic sites induce a mixed variation pattern during reverse transcription, including G → A or C or T mutations as well as deletions. We calculated the total variation ratio to quantify the m7G modification fraction at each methylated site. The calibration curves of all relevant motif contexts allow us to more quantitatively determine the m7G methylation level. We detected internal m7G sites in 22 human cytoplasmic tRNAs from HeLa and HEK293T cells and successfully estimated the corresponding m7G methylation stoichiometry. m7G-quant-seq could be applied to monitor the tRNA m7G methylation level change in diverse biological processes.

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