Methylation of guanosine on position N7 (m7G) on internal RNA positions have been found in all domains of life and have been implicated in human disease, but m7G modifications has so far only been mapped in a limited number of RNA molecules. Here, we present m7G Mutational Profiling sequencing (m7G-MaP-seq), which allows high throughput detection of m7G modifications. In our method, m7G modified positions are converted to abasic sites by mild reduction, recorded as cDNA mutations through reverse transcription, sequenced and subsequently detected by identification of positions with increased mutation rates in the reduced sample compared to the control. We show that m7G-MaP-seq efficiently detects m7G modifications in rRNA, including a previously uncharacterised rRNA modification in Arabidopsis thaliana. Furthermore, we identify m7G tRNA modifications in budding yeast, human and arabidopsis tRNA and show that m7G modification occurs before tRNA splicing. We do not find any evidence for internal m7G modifications being present in other small RNA, such as miRNA, snoRNA and sRNA. Likewise, high coverage m7G-MaP-seq analysis of mRNA from E. coli or yeast cells failed to identify any internal m7G modifications.
Overall design
RNA from E. coli (strain BW25113 and RsmG KO), Saccharomyces cerevisiae (BY4741), HeLa, Arabidopsis Thaliana Columbia-0 . The RNA was treated with NaBH4 or mock-treated, then reverse transcribed.
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