Recently, studies about RNA modification dynamics in human RNAs are among the most controversially discussed. As a main reason, we identified the unavailability of a technique which allows the investigation of the temporal processing of RNA transcripts. Here, we present nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) for efficient, monoisotopic stable isotope labeling in both RNA and DNA in standard cell culture. We design pulse chase experiments and study the temporal placement of modified nucleosides in tRNA^Phe and 18S rRNA. In existing RNAs, we observe a time-dependent constant loss of modified nucleosides which is masked by post-transcriptional methylation mechanisms and thus undetectable without NAIL-MS. During alkylation stress, NAIL-MS reveals an adaptation of tRNA modifications in new transcripts but not existing ones. Overall, we present a fast and reliable stable isotope labeling strategy which allows in-depth study of RNA modification dynamics in human cell culture.

最近，关于人类 RNA 中 RNA 修饰动力学的研究是最具争议性的研究之一。主要原因是，我们发现无法使用一种技术来研究 RNA 转录本的时间处理。在这里，我们提出了核酸同位素标记偶联质谱 (NAIL-MS)，可在标准细胞培养物中的 RNA 和 DNA 中进行高效、单同位素稳定同位素标记。我们设计了脉冲追踪实验并研究了修饰核苷在 tRNA ^Phe和 18S rRNA 中的时间放置。在现有的 RNA 中，我们观察到修饰核苷的时间依赖性持续丢失，这种丢失被转录后甲基化机制掩盖，因此如果不使用 NAIL-MS 就无法检测到。在烷基化应激期间，NAIL-MS 揭示了新转录本中 tRNA 修饰的适应，但现有转录本中没有。总的来说，我们提出了一种快速可靠的稳定同位素标记策略，可以深入研究人类细胞培养物中的 RNA 修饰动力学。