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 (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 inâ vivo. Our findings reveal the complex nature of bacterial RNA damage repair.
Subject(s)
Escherichia coli/metabolism , RNA, Bacterial/metabolism , RNA, Transfer/metabolism , Thiouridine/metabolism , Models, Molecular , Molecular Structure , RNA, Bacterial/chemistry , RNA, Transfer/chemistry , Thiouridine/chemical synthesis , Thiouridine/chemistryABSTRACT
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 tRNAArgCCG, tRNAArgICG, tRNAArgUCU and tRNASerGCU 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.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Mixed Function Oxygenases/metabolism , RNA Processing, Post-Transcriptional , RNA, Bacterial/metabolism , RNA, Transfer/metabolism , Cytidine/analogs & derivatives , Cytidine/metabolism , Demethylation , Escherichia coli/genetics , Gene Knockout Techniques , Isotope Labeling/methods , Mass Spectrometry/methods , Methylation , Nucleic Acids/metabolism , Pseudomonas aeruginosa/metabolism , tRNA Methyltransferases/metabolismABSTRACT
Ribonucleic acids (RNA) are extensively modified. These modifications are quantified by mass spectrometry (LC-MS/MS) to determine the abundance of a modification under certain conditions or in various genetic backgrounds. With LC-MS/MS the steady state of modifications is determined, and thus we only have a static view of the dynamics of RNA modifications. With nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) we overcome this limitation and get access to the dynamics of RNA modifications. We describe labeling techniques for E. coli, S. cerevisiae and human cell culture and the current instrumental limitations. We present the power of NAIL-MS but we also outline validation experiments, which are necessary for correct data interpretation. As an example, we apply NAIL-MS to study the demethylation of adenine and cytidine, which are methylated by the damaging agent methyl-methanesulfonate in E. coli. With NAIL-MS we exclude the concurrent processes for removal of RNA methylation, namely RNA degradation, turnover and dilution. We use our tool to study the speed and efficiency of 1-methyladenosine and 3-methylcytidine demethylation. We further outline current limitations of NAIL-MS but also potential future uses for e.g. relative quantification of tRNA isoacceptor abundances.
Subject(s)
Adenosine/analogs & derivatives , Cytidine/analogs & derivatives , Isotope Labeling/methods , Mass Spectrometry/methods , RNA Processing, Post-Transcriptional , RNA, Messenger/chemistry , RNA, Transfer/chemistry , Adenosine/chemistry , Adenosine/metabolism , Carbon Isotopes , Cytidine/chemistry , Cytidine/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , HEK293 Cells , Humans , Hydrolysis , Methyl Methanesulfonate/chemistry , Nitrogen Isotopes , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Transfer/genetics , RNA, Transfer/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , TranscriptomeABSTRACT
In all domains of life, the nucleobases of tRNA can be methylated. These methylations are introduced either by enzymes or by the reaction of methylating agents with the nucleophilic centers of the nucleobases. Herein, we present a systematic approach to identify the methylation sites within RNA in vitro and in vivo. For discrimination between enzymatic tRNA methylation and tRNA methylation damage in bacteria, we used nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS). With NAIL-MS, we clearly observed the formation of 7-methylguanosine, 3-methyluridine, and 6-methyladenosine during exposure of bacteria to the alkylating agent methyl methanesulfonate (MMS) in vivo. These damage products were not reported to form in tRNA in vivo, as they were masked by the enzymatically formed modified nucleosides in previous studies. In addition, we found formation of the known damage products 1-methyladenosine and 3-methylcytidine in vivo. With a dynamic NAIL-MS setup, we observed tRNA repair by demethylation of these two RNA modifications in vivo. Furthermore, we saw the potential repair of 6-methyladenosine but not 7-methylguanosine in bacterial tRNA.
Subject(s)
Escherichia coli/genetics , RNA, Transfer/chemistry , Deuterium , Isotope Labeling/methods , Mass Spectrometry/methods , Methylation , Nitrogen Isotopes , Ribonucleosides/chemistryABSTRACT
RNA in yeast, especially rRNA and tRNA are heavily modified to fulfill their function in protein translation. Using biosynthetic stable isotope labeled internal standards we quantified 12 modified nucleosides in tRNA from S. cerevisiae over 24 hours. We observed different quantities of modified nucleosides in dependence of the growth phase. To elucidate the underlying mechanism of the observed tRNA modification profile adaptation, it is necessary to distinguish the pre-existing tRNA pool and its modifications from newly-synthesized tRNAs. By combination of 2 differentially isotope labeled media we developed NAIL-MS, nucleic acid isotope labeling coupled mass spectrometry. During the yeast growth cycle we observe dilution of pre-existing tRNAs by newly-synthesized tRNAs by the growing number of cells. tRNA was found to be highly stable with only little degradation over the observed period. The method was further used to quantify the levels of modified nucleosides in the original and new tRNA pools. By addition of deuterium-labeled methionine, we could observe the incorporation of new methyl marks on pre-existing tRNAs. For 2'-O-methylcytidine (Cm) we observed a global increase in log phase. We identified extensive 2'-OH-cytidine methylation of the pre-existing tRNAs and the new tRNAs which masks an actual decrease of pre-existing Cm. In contrast, global 5-methylcytidine (m5C) levels decreased during growth due to a drop in m5C quantities in the original tRNA pool. The NAIL-MS data suggests different mechanisms for tRNA modification adaptation depending on the individual modification observed. With this new tool it is possible to follow the fate of methylated RNAs during growth and potentially compare the impact of different stress conditions on the epitranscriptome.
Subject(s)
Isotope Labeling , Mass Spectrometry , Nucleic Acids , RNA, Transfer/genetics , RNA, Transfer/metabolism , Methylation , Molecular Structure , Nucleic Acid Conformation , Nucleic Acids/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolismABSTRACT
We explore the chemical space of Pseudomonas quinolone signal analogs as privileged structures and report the discovery of a thioquinolone as a potent inhibitor of the important virulence factor elastase of the human pathogen Pseudomonas aeruginosa. We provide evidence that the derivative binds to the active site zinc of elastase and additionally acts as a fluorescent zinc sensor.
