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. Overall design: [dataset1] 20 samples (Sample 1-20): "Treated" samples for m7G-quant-seq to study the variation patterns at HeLa 18S rRNA m7G1639, under five RTs and different dNTP/dATP ratios. [dataset2] 6 samples (Sample 21-26): Calibration curves for estimating internal m7G methylation fractions, under HIV RT and 1mM dNTP. [dataset3] 8 samples (Sample 27-34): Duplicates for m7G-quant-seq "Treated" and "Input" samples to reveal tRNA m7G46 methylation fractions in HeLa and HEK293T cells, under HIV RT and 1mM dNTP.