PhOxi-seq detects enzyme-dependent m2G in multiple RNA types

Abstract

ABSTRACTIn 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 optimised PhOxi-seq workflow, coupled to a novel bioinformatic pipeline, that is capable of detecting enzyme-dependent m2G sites throughout the transcriptome, including low abundant mRNAs. In this way, we generated the first database of high confidence sites of THUMPD3-dependent m2G in multiple RNA classes within a human cancer cell line and further identify non-THUMPD3 controlled sites throughout the transcriptome.