m(7)G-quant-seq: Quantitative Detection of RNA Internal N(7)-Methylguanosine

[Image: see text] Methods for the precise detection and quantification of RNA modifications are critical to uncover functional roles of diverse RNA modifications. The internal m(7)G 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 m(7)G-quant-seq, a quantitative method that accurately detects internal m(7)G sites in human cytoplasmic tRNAs at single-base resolution. The efficient chemical reduction and mild depurination can almost completely convert internal m(7)G 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 m(7)G modification fraction at each methylated site. The calibration curves of all relevant motif contexts allow us to more quantitatively determine the m(7)G methylation level. We detected internal m(7)G sites in 22 human cytoplasmic tRNAs from HeLa and HEK293T cells and successfully estimated the corresponding m(7)G methylation stoichiometry. m(7)G-quant-seq could be applied to monitor the tRNA m(7)G methylation level change in diverse biological processes.