Project description:Methylation of N7-methylguanosine (m7G) is found at mRNA caps and at defined internal positions within abundant tRNAs and rRNAs. However, its detection within low abundance mRNAs and microRNAs (miRNAs) has been hampered by lack of sensitive detection strategies. Here, we adapt a chemical reactivity assay to detect internal m7G in RNA from eukaryotic cells. Using this approach, alongside a confirmational RNA immunoprecipitation assay, we identify m7G within miRNAs inhibiting cell migration, and show that METTL1 mediates this m7G methylation. Using Let-7 as an example we demonstrate that METTL1 activity is necessary for correct processing, is required for Let-7 dependent gene regulation and consequently has a negative effect on cell migration. These results identify m7G modification as a new pathway for the regulation of miRNAs.
Project description:Methylation of N7-methylguanosine (m7G) is found at mRNA caps and at defined internal positions within abundant tRNAs and rRNAs. However, its detection within low abundance mRNAs and microRNAs (miRNAs) has been hampered by lack of sensitive detection strategies. Here, we adapt a chemical reactivity assay to detect internal m7G in RNA from eukaryotic cells. Using this approach, alongside a confirmational RNA immunoprecipitation assay, we identify m7G within miRNAs inhibiting cell migration, and show that METTL1 mediates this m7G methylation. Using Let-7 as an example we demonstrate that METTL1 activity is necessary for correct processing, is required for Let-7 dependent gene regulation and consequently has a negative effect on cell migration. These results identify m7G modification as a new pathway for the regulation of miRNAs.
Project description:7-methylguanosine (m7G) is present at mRNA caps and at defined internal positions within tRNAs and rRNAs. However, its detection within low-abundance mRNAs and microRNAs (miRNAs) has been hampered by a lack of sensitive detection strategies. Here, we adapt a chemical reactivity assay to detect internal m7G in miRNAs. Using this technique (Borohydride Reduction sequencing [BoRed-seq]) alongside RNA immunoprecipitation, we identify m7G within a subset of miRNAs that inhibit cell migration. We show that the METTL1 methyltransferase mediates m7G methylation within miRNAs and that this enzyme regulates cell migration via its catalytic activity. Using refined mass spectrometry methods, we map m7G to a single guanosine within the let-7e-5p miRNA. We show that METTL1-mediated methylation augments let-7 miRNA processing by disrupting an inhibitory secondary structure within the primary miRNA transcript (pri-miRNA). These results identify METTL1-dependent N7-methylation of guanosine as a new RNA modification pathway that regulates miRNA structure, biogenesis, and cell migration.
Project description:Methylation of N7-methylguanosine (m7G) is found at mRNA caps and at defined internal positions within abundant tRNAs and rRNAs. However, its detection within low abundance mRNAs and microRNAs (miRNAs) has been hampered by lack of sensitive detection strategies. Here, we adapt a chemical reactivity assay to detect internal m7G in RNA from eukaryotic cells. Using this approach, alongside a confirmational RNA immunoprecipitation assay, we identify m7G within miRNAs inhibiting cell migration, and show that METTL1 mediates this m7G methylation. Using Let-7 as an example we demonstrate that METTL1 activity is necessary for correct processing, is required for Let-7 dependent gene regulation and consequently has a negative effect on cell migration. These results identify m7G modification as a new pathway for the regulation of miRNAs.
