Detection of internal N7-methylguanosine (m7G) RNA modifications by mutational profiling sequencing

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.N7-甲基鸟苷（m7G）是广泛存在于所有生命域RNA内部位点的修饰形式，且与人类疾病密切相关，但迄今为止，m7G修饰的定位仅在有限的RNA分子中完成。本研究报道了m7G突变谱测序（m7G Mutational Profiling sequencing, m7G-MaP-seq）技术，可实现m7G修饰的高通量检测。该方法通过温和还原将m7G修饰位点转化为无碱基位点，经逆转录记录为互补DNA（cDNA）突变，随后通过测序比对，识别还原处理样本相较于对照样本突变率显著升高的位点，从而完成m7G修饰的定位。我们证实，m7G-MaP-seq可高效检测核糖体RNA（rRNA）中的m7G修饰，包括拟南芥中此前未被表征的核糖体RNA修饰位点。此外，我们在酿酒酵母、人类及拟南芥的转运RNA（tRNA）中鉴定出m7G修饰，并证实该修饰发生于tRNA剪接之前。我们未在微RNA（miRNA）、核仁小RNA（snoRNA）及小RNA（sRNA）等其他小RNA中发现内部m7G修饰的相关证据。同样，对大肠杆菌（E. coli）或酿酒酵母的信使RNA（mRNA）开展高覆盖度m7G-MaP-seq分析，也未检测到任何内部m7G修饰。