PhOxi-seq Detects Enzyme-Dependent m²G in Multiple RNA Types

In recent years, RNA-modifying enzymes have gained significant attention due to their impact on critical RNA-based processes and, consequently, human pathology. However, identifying sites of modifications throughout the transcriptome remains challenging largely due to the lack of accurate and sensitive detection technologies. Recently, we described PhOxi-seq as a method capable of confirming known sites of m2G within abundant classes of RNA, namely, purified rRNA and purified tRNA. Here, we further explore the selectivity of PhOxi-seq and describe an optimized PhOxi-seq workflow, coupled to a novel bioinformatic pipeline, that is capable of detecting enzyme-dependent m2G sites throughout the transcriptome. In this way, we generated a database of potential THUMPD3-dependent m2G sites in multiple RNA classes within a human cancer cell line and further identify potential non-THUMPD3 controlled m2G sites. These potential sites should serve as the basis for further confirmation studies for m2G within the human transcriptome.近年来，RNA修饰酶因其对关键RNA调控过程以及人类病理状态的显著影响而受到广泛关注。然而，在全转录组范围内鉴定修饰位点仍颇具挑战，这在很大程度上源于缺乏精准且灵敏的检测技术。此前，我们报道了可用于确认丰度较高RNA类别中已知N2-甲基鸟苷（m2G）修饰位点的PhOxi-seq技术，该技术可针对纯化的核糖体RNA（rRNA）和转运RNA（tRNA）进行检测。本研究进一步探究了PhOxi-seq的选择性，并报道了一套优化后的PhOxi-seq实验流程，结合全新的生物信息学分析流程，可在全转录组范围内检测酶依赖型m2G修饰位点。据此，我们在人类癌细胞系的多种RNA类别中构建了依赖THUMPD3的潜在m2G修饰位点数据库，并进一步鉴定了非THUMPD3调控的潜在m2G修饰位点。这些潜在修饰位点可作为后续人类转录组中m2G修饰位点验证研究的基础。