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Abstract
Summary7-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.
Highlights
Post-synthesis covalent modification of biological molecules is a key aspect of intracellular signaling, and it is critically important in many biological processes
This reaction is the basis of direct RNA sequencing (RNA-seq) by the Maxam and Gilbert method (Peattie, 1979) and has been used for mapping m7G residues in highly abundant rRNAs and tRNAs at single nucleotide resolution (Zueva et al, 1985)
We show that m7G promotes the processing of their precursor RNAs and that METTL1-dependent methylation is required to suppress cellular migration
Summary
Post-synthesis covalent modification of biological molecules is a key aspect of intracellular signaling, and it is critically important in many biological processes. Many RNA modifications have been identified by mass spectrometry (MS), and complex epitranscriptomes of tRNA and rRNA have been thoroughly studied This represents a mere snapshot of a much bigger picture, with the clear majority of modifications remaining uncharacterized. A few very recent analyses have used anti-modification antibodies (e.g., against N1-methyladenosine [Dominissini et al, 2016], N6-methyladenosine [Dominissini et al, 2012], and 5-hydroxymethylcytosine [Delatte et al, 2016]) or chemical reactivity of the modification (for pseudouridine [Carlile et al, 2014; Schwartz et al, 2014], m5C [Schaefer, 2015], and 20-O-methylation [Dai et al, 2017]) Their results clearly suggest that many of the modifications identified on rRNA and tRNA are present on other RNA classes. The development of epitranscriptomic methodologies (e.g., new antibody and chemical methods coupled to nextgeneration sequencing [NGS]) represents a bottleneck in deciphering the function of new RNA modifications
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