New tools for RNA modification dynamics analysis

转录后，对RNA进行加工和化学修饰。这些修饰包括例如甲基化和硫代化，其中一些用于翻译的微调。特别是在tRNA中，这种修饰状态会随着压力的变化而改变。细胞如何实现这种适应尚不清楚。本项目的目标是研究这些适应过程的机制。为此，我们希望使用我们独特的核酸同位素标记耦合质谱仪(钉子-MS)。利用这种方法，我们可以区分RNA的来源，并观察胁迫前存在的RNA和胁迫期间转录的新RNA的命运。因此，我们可以确定哪些RNA适应它们的修饰密度来克服压力。我们计划利用酵母细胞，通过全消化和核糖体RNA(RRNA)的核苷定量分析来研究tRNA和rRNA。此外，我们还计划建立一种基于寡核苷酸的钉钉方法。这种寡聚-指甲-MS技术将在蛋白质研究方面与SILAC蛋白质组学相媲美。我们主要想用这种方法来研究rRNA的修饰，以及在应激过程中，这种RNA中指定位置的修饰密度是如何变化的。

After transcription, RNA is processed and chemically modified. These modifications comprise e.g. methylations and thiolations and some of them serve in fine-tuning of translation. Especially in tRNA, the modification status changes in response to stress. How the cells achieve this adaptation remains elusive.The goal of this project is to study the mechanisms of these adaptation processes. For this, we want to use our unique nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS). With this method we can distinguish the origin of the RNA and observe the fate of the RNA present before the stress and the new RNA that is transcribed during the stress. Thus we can identify which RNAs adapt their modification density to overcome the stress. We plan to use yeast cells and study tRNA and ribosomal RNA (rRNA) by total digest and quantitative mass spectrometry of the nucleosides.In addition, we plan to establish an oligonucleotide based NAIL method. This Oligo-NAIL-MS technique will be comparable to SILAC proteomics in protein research. We want to use this method primarily to study modifications in rRNA and how the modification density changes at a defined location in this RNA during stress.