Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases.

Epitranscriptomic RNA modifications can regulate fundamental biological processes, but we lack approaches to map modification sites and probe writer enzymes. Here we present a chemoproteomic strategy to characterize RNA 5-methylcytidine (m 5 C) dioxygenase enzymes in their native context based upon metabolic labeling and activity-based crosslinking with 5-ethynylcytidine (5-EC). We profile m 5 C dioxygenases in human cells including ALKBH1 and TET2 and show that ALKBH1 is the major hm 5 C- and f 5 C-forming enzyme in RNA. Further, we map ALKBH1 modification sites transcriptome-wide using 5-EC-iCLIP and ARP-based sequencing to identify ALKBH1-dependent m 5 C oxidation in a variety of tRNAs and mRNAs and analyze ALKBH1 substrate specificity in vitro. We also apply targeted pyridine borane-mediated sequencing to measure f 5 C sites on select tRNA. Finally, we show that f 5 C at the wobble position of tRNA-Leu-CAA plays a role in decoding Leu codons under stress. Our work provides powerful chemical approaches for studying RNA m 5 C dioxygenases and mapping oxidative m 5 C modifications and reveals the existence of novel epitranscriptomic pathways for regulating RNA function.