@article{d89bd92fbc944790b6be906928502930,
title = "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.",
author = "Arguello, {A. Emilia} and Ang Li and Xuemeng Sun and Eggert, {Tanner W.} and Elisabeth Mairhofer and Kleiner, {Ralph E.}",
note = "Funding Information: The authors thank Saw Kyin and Henry Shwe at the Princeton University Mass Spectrometry and Proteomic Facility for performing proteomic analysis, and Christina DeCoste at the Princeton University Flow Cytometry Resource Facility for assistance with FACS analysis. R.E.K. acknowledges support from a National Science Foundation CAREER award (MCB-1942565), the National Institute of Health (R01 GM132189), the Sidney Kimmel Foundation and the Alfred P. Sloan Foundation. A.E.A. acknowledges the Edward C. Taylor 3rd Year Graduate Fellowship in Chemistry and an Eli Lilly-Edward C. Taylor Fellowship in Chemistry. T.W.E. acknowledges the Edward C. Taylor 3rd Year Graduate Fellowship in Chemistry. A.L. was supported by the Princeton Catalysis Initiative. All authors thank Princeton University for financial support. Funding Information: The authors thank Saw Kyin and Henry Shwe at the Princeton University Mass Spectrometry and Proteomic Facility for performing proteomic analysis, and Christina DeCoste at the Princeton University Flow Cytometry Resource Facility for assistance with FACS analysis. R.E.K. acknowledges support from a National Science Foundation CAREER award (MCB-1942565), the National Institute of Health (R01 GM132189), the Sidney Kimmel Foundation and the Alfred P. Sloan Foundation. A.E.A. acknowledges the Edward C. Taylor 3rd Year Graduate Fellowship in Chemistry and an Eli Lilly-Edward C. Taylor Fellowship in Chemistry. T.W.E. acknowledges the Edward C. Taylor 3rd Year Graduate Fellowship in Chemistry. A.L. was supported by the Princeton Catalysis Initiative. All authors thank Princeton University for financial support. Publisher Copyright: {\textcopyright} 2022, The Author(s).",
year = "2022",
month = dec,
doi = "10.1038/s41467-022-31876-2",
language = "English (US)",
volume = "13",
journal = "Nature Communications",
issn = "2041-1723",
publisher = "Nature Publishing Group",
number = "1",
}