GSE120454
m7G methylation by METTL1 regulates let-7 microRNAs [m7G-RIP-Seq Caco2]
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. Overall design: Identification of miRNAs harboring m7G modification. Total RNA from a human lung cancer cell line (A549 cells) was decapped, 5' end dephosphorylated, and treated with NaBH4 and low pH to generate abasic sites at positions harbouring m7G. These sites were exposed to a biotin-coupled aldehyde reactive probe (N-(aminooxyacetyl)-n'-(D-biotinoyl) hydrazine; ARP) that covalently binds to the abasic RNA sites. Modified RNAs were then pulled-down using streptavidin-beads, small RNA libraries were prepared and RNAs were identified by NGS. To confirm the validity of this approach and to provide an independent verification of m7G-methylated miRNAs, we performed a RIP-Seq experiment employing an antibody that recognizes m7G in RNA.