Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases

Abstract

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 (m5C) dioxygenase enzymes in their native context based upon metabolic labeling and activity-based crosslinking with 5-ethynylcytidine (5-EC). We profile m5C dioxygenases in human cells including ALKBH1 and TET2 and show that ALKBH1 is the major hm5C- and f5C-forming enzyme in RNA. Further, we map ALKBH1 modification sites transcriptome-wide using 5-EC-iCLIP and ARP-based sequencing to identify ALKBH1-dependent m5C 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 f5C sites on select tRNA. Finally, we show that f5C 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 m5C dioxygenases and mapping oxidative m5C modifications and reveals the existence of novel epitranscriptomic pathways for regulating RNA function.

The lncRNA epigenetics: The significance of m6A and m5C lncRNA modifications in cancer

“…ALKBH1 was reported, in 2022, to determine minor modification sites also in lncRNAs. However, it was reported that the main function of ALKBH1 is exerted on tRNAs ( 47 ).…”

Bioorthogonal Phosphines: Then and Now

“…Ketenes are high-energy motifs that serve as useful electrophilic traps in biologically relevant environments. [62,84] The ability to access them via bioorthogonal reagents provides a blueprint for a variety "chemically triggered" processes. In one area, we have been leveraging selective ketene formation for biomolecule crosslinking.…”

Activity‐Based Protein Profiling of Oxidases and Reductases

“…The group of Kleiner applied this principle for RNA‐mediated activity‐based protein profiling by feeding HEK293T cells with 5‐ethynylcytidine, which gets incorporated into cellular RNA [69] . Protein‐RNA enrichment and quantitative mass spectrometry proteomics showed that the Fe(II)‐α‐KG‐dependent dioxygenase ALKBH1 is the major m5C‐oxidizing enzyme in HEK293T cells, where it is largely responsible for the production of hm 5 C and f 5 C of within RNA.…”