Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases

Kleiner and co-workers profile RNA 5-methylcytidine (m(5)C) dioxygenase enzymes using an activity-based metabolic probing strategy. They reveal ALKBH1 as the major 5-formylcytidine (f(5)C) writer and characterize modification sites across mRNA and tRNA. 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.

Automated summary

Kleiner and his colleagues profile RNA 5-methylcytidine dioxygenase enzymes using an activity-based metabolic probing strategy, identifying ALKBH1 as the major writer of 5-formylcytidine. Their study characterizes modification sites across mRNA and tRNA, revealing the regulatory role of epitranscriptomic RNA modifications. They propose a chemoproteomic strategy to study RNA 5-methylcytidine dioxygenases in their native context and map oxidative m5C modifications. The research uncovers novel epitranscriptomic pathways for regulating RNA function.