m⁷G-quant-seq: Quantitative Detection of RNA Internal N⁷-Methylguanosine

Methods for the precise detection and quantification of RNA modifications are critical to uncover functional roles of diverse RNA modifications. The internal m⁷G 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 m⁷G-quant-seq, a quantitative method that accurately detects internal m⁷G sites in human cytoplasmic tRNAs at single-base resolution. The efficient chemical reduction and mild depurination can almost completely convert internal m⁷G 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 m⁷G modification fraction at each methylated site. The calibration curves of all relevant motif contexts allow us to more quantitatively determine the m⁷G methylation level. We detected internal m⁷G sites in 22 human cytoplasmic tRNAs from HeLa and HEK293T cells and successfully estimated the corresponding m⁷G methylation stoichiometry. m⁷G-quant-seq could be applied to monitor the tRNA m⁷G methylation level change in diverse biological processes.