Synthesis and Metabolic Fate of 4-Methylthiouridine in Bacterial tRNA

Ribonucleic acid (RNA) is central to many life processes and, to fulfill its function, it has a substantial chemical variety in its building blocks. Enzymatic thiolation of uridine introduces 4-thiouridine (s⁴ U) into many bacterial transfer RNAs (tRNAs), which is used as a sensor for UV radiation. A similar modified nucleoside, 2-thiocytidine, was recently found to be sulfur-methylated especially in bacteria exposed to antibiotics and simple methylating reagents. Herein, we report the synthesis of 4-methylthiouridine (ms⁴ U) and confirm its presence and additional formation under stress in Escherichia coli. We used the synthetic ms⁴ U for isotope dilution mass spectrometry and compared its abundance to other reported tRNA damage products. In addition, we applied sophisticated stable-isotope pulse chase studies (NAIL-MS) and showed its AlkB-independent removal in vivo. Our findings reveal the complex nature of bacterial RNA damage repair.