NAIL-MS reveals the repair of 2-methylthiocytidine by AlkB in E. coli

RNAs contain post-transcriptional modifications, which fulfill a variety of functions in translation, secondary structure stabilization and cellular stress survival. Here, 2-methylthiocytidine (ms(2)C) is identified in tRNA of E. coli and P. aeruginosa using NAIL-MS (nucleic acid isotope labeling coupled mass spectrometry) in combination with genetic screening experiments. ms(2)C is only found in 2-thiocytidine (s(2)C) containing tRNAs, namely tRNA(Arg)(CCG), tRNA(Arg)(ICG), tRNA(Arg)(UCU) and tRNA(Ser)(GCU) at low abundances. ms(2)C is not formed by commonly known tRNA methyltransferases. Instead, we observe its formation in vitro and in vivo during exposure to methylating agents. More than half of the s(2)C containing tRNA can be methylated to carry ms(2)C. With a pulse-chase NAIL-MS experiment, the repair mechanism by AlkB dependent sulfur demethylation is demonstrated in vivo. Overall, we describe ms(2)C as a bacterial tRNA modification and damage product. Its repair by AlkB and other pathways is demonstrated in vivo by our powerful NAIL-MS approach.