a Department of Chemistry , Ludwig-Maximilians-Universität München , Munich , Germany.
RNA Biol. 2017 Sep 2;14(9):1260-1268. doi: 10.1080/15476286.2017.1325063. Epub 2017 May 10.
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 (mC) levels decreased during growth due to a drop in mC 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.
酵母中的 RNA，尤其是 rRNA 和 tRNA，经过大量修饰以满足其在蛋白质翻译中的功能。我们使用生物合成稳定同位素标记的内标，在 24 小时内定量测定了来自 S. cerevisiae 的 tRNA 中的 12 种修饰核苷。我们观察到不同的修饰核苷数量取决于生长阶段。为了阐明观察到的 tRNA 修饰谱适应的潜在机制，有必要区分预先存在的 tRNA 池及其修饰与新合成的 tRNA。通过组合两种不同同位素标记的培养基，我们开发了 NAIL-MS，即核酸同位素标记结合质谱。在酵母生长周期中，我们观察到随着细胞数量的增加，预先存在的 tRNA 被新合成的 tRNA稀释。tRNA 被发现具有高度稳定性，在观察期间只有少量降解。该方法进一步用于定量原始和新 tRNA 池中的修饰核苷水平。通过添加氘标记的蛋氨酸，我们可以观察到新的甲基标记在预先存在的 tRNA 上的掺入。对于 2'-O-甲基胞嘧啶（Cm），我们在对数期观察到整体增加。我们发现预先存在的 tRNA 和新 tRNA 发生了广泛的 2'-OH-胞嘧啶甲基化，掩盖了预先存在的 Cm 的实际减少。相比之下，由于原始 tRNA 池中的 mC 数量下降，全局 5-甲基胞嘧啶（mC）水平在生长过程中降低。NAIL-MS 数据表明，不同的修饰适应机制取决于观察到的个别修饰。有了这个新工具，就有可能在生长过程中跟踪甲基化 RNA 的命运，并有可能比较不同应激条件对表转录组的影响。
