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. 2021 Jan 15;12:389. doi: 10.1038/s41467-020-20576-4

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — a 70% confluent CD₃-methionine labeled cells were incubated with fully labeled media for 2 h before the LD₅₀ dose of methyl methanesulfonate (MMS, yellow shaded area) was added. Control samples were treated the same, substituting the MMS stock solution with PBS. After 1 h the stress (or control) media was replaced by fresh labeled media. After set time points, cells were harvested and tRNA^Phe_GAA was purified and subjected to LC-MS/MS analysis. b Unlabeled modifications were referenced to unlabeled canonicals to calculate the amount of modifications arising from direct methylation damage by MMS. Red numbers at time point 0 give the percentage of damaged nucleoside referenced to the naturally occurring amount of the respective modification (amount of original modification before experiment initiation). c Labeled modifications were referenced to labeled canonicals to calculate the amount of modification in new tRNA transcripts. The numbers at time point 6 give the percentage of modification amount in the control sample referenced to the naturally occurring amount of the respective modification. All experiments are from n = 3 biol. replicates. Symbols reflect the mean and error bars reflect standard deviation. P-values from student t-test (equal distribution, two-sided): *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Where reasonable, exact P-values are given as grey numbers in the figure.