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Surpassing limits of static RNA modification analysis with dynamic NAIL-MS

Author:
Reichle, Valentin F., Kaiser, Steffen, Heiss, Matthias, Hagelskamp, Felix, Borland, Kayla, Kellner, Stefanie
Source:
Methods 2019 v.156 pp. 91-101
ISSN:
1046-2023
Subject:
Escherichia coli, adenine, cell culture, cytidine, demethylation, genetic background, humans, isotope labeling, liquid chromatography, methyl methanesulfonate, methylation, tandem mass spectrometry, transfer RNA
Abstract:
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.
Agid:
6230137