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 (ms²C) 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. ms²C is only found in 2-thiocytidine (s²C) containing tRNAs, namely tRNA^Arg_CCG, tRNA^Arg_ICG, tRNA^Arg_UCU and tRNA^Ser_GCU at low abundances. ms²C 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 s²C containing tRNA can be methylated to carry ms²C. With a pulse-chase NAIL-MS experiment, the repair mechanism by AlkB dependent sulfur demethylation is demonstrated in vivo. Overall, we describe ms²C 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.