Abstract
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 (s4U) 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 (ms4U) and confirm its presence and additional formation under stress in Escherichia coli. We used the synthetic ms4U 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.
Highlights
4-methylthiouridine and confirm its presence and additional formation under stress in Escherichia coli
Thiolation of uridine (4-thiouridine, s4U) is commonly found at position 8 of most tRNAs. s4U is a target of ultraviolet light;[2]
S4U-hypomodified tRNAs were found to be targeted by the Ribonucleic acid (RNA) degradosome; this leads to a reduced abundance of a subset of bacterial tRNAs.[4]
Summary
4-methylthiouridine (ms4U) and confirm its presence and additional formation under stress in Escherichia coli. Despite its important function in bacterial tRNA and its broad use as a metabolic label for RNA sequencing, little is known about its chemical reactivity inside cells. Another sulfur decorated tRNA modification, 2-thiocytidine (s2C) (blue in Figure 1) has been recently found to be endogenously methylated[8] and efficiently repaired, potentially through its function as a modulator of translation.[9] A direct methylation of s2C through electrophiles such as S-adenosylmethionine, methyl methanesulfonate (MMS) or antibiotics (streptozotocin) was observed.
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