Dynamic nature of RNA modifications and their influence in neurological diseases

RNA及其修饰在细胞应激反应中受到动态调节。因此，发现与RNA相互作用的基因突变是许多疾病的原因并不令人惊讶。我的研究重点是RNA修饰在神经元疾病中的作用，并冒险进入表观转录组学(也称为RNA表观遗传学)的新领域。为了回答RNA修饰的动态性质如何与神经元疾病相联系的问题，工作方案被细分为三个相互作用和相辅相成的目标。在目标1中，我们将建立已知与神经元疾病有关的RNA修饰酶(RNA编写者)的突变和击倒细胞系。我们将纯化主要的RNA，如tRNA，rRNA和mRNA，以及特定的RNA，如tRNA片段和miRNA，以评估它们的修饰谱。利用我们的新技术，核酸同位素标记与质谱联用(NAIL-MS)，我们将研究这些RNA的稳定性依赖于它们的修饰状态。利用指甲质谱仪，我们还将分析在应激暴露期间RNA编写器突变体和野生型细胞中RNA修饰的动态。这些研究将揭示表位转录应激反应和潜在的RNA去修饰酶(RNA擦除器)的机制。野生型和突变型细胞系的比较蛋白质组学将被用来分析不平衡RNA修饰的分子后果。在目标2中，我们将研究在这些神经元疾病中积累的小的非编码RNA(如tRNA片段)的相互作用组(RNA阅读器)。我们将鉴定它们的相互作用组，并将天然、修饰的RNA的相互作用组与合成的、未修饰的RNA的相互作用组进行比较。这种方法将回答以下问题：哪些下游过程受到RNA编写器突变的影响，这可能是导致发病的原因。目标2的另一个方面涉及tRNA片段的命运。这些RNA是RNA编写器和/或RNA擦除器的底物吗？此外，我们将研究修饰对RNA细胞分布的影响。目标3涵盖了目标1和2中观察到的研究，并将使我们能够更深入地了解表位转录组。它将揭示RNA擦除器的存在，并确定负责去甲基化/去修饰过程的候选酶。以类似的方式，我们将筛选新的RNA作者和已知的具有未知RNA底物的RNA作者。利用我们开发的工具和知识，我们将寻找在应激或突变细胞系中积累的未知小非编码RNA，并确定它们参与细胞同源稳定。拟议的研究将在神经性疾病患者中发现RNA编写者的突变与目前鲜为人知的发病机制有关。

RNA and its modifications are dynamically regulated in cellular stress response. Therefore it is not surprising to find mutations in RNA interacting genes as causes for many diseases. My research focuses on the role of RNA modifications in neuronal diseases and ventures into the new field of epitranscriptomics (also referred to as RNA epigenetics). To answer the question how the dynamic nature of RNA modifications is linked to neuronal diseases, the work programme is subdivided into three objectives which interact and complement one another. In objective 1 we will generate mutant and knock-down cell lines of RNA modifying enzymes (RNA writers) with a known connection to neuronal diseases. We will purify the major RNAs like tRNA, rRNA and mRNA and specific RNAs like tRNA fragments and miRNA to assess their modification profile. Using our new technique, nucleic acid isotope labeling coupled with mass spectrometry (NAIL-MS), we will study the stability of these RNAs in dependence of their modification status. With NAIL-MS we will also analyze the dynamics of RNA modifications in the RNA writer mutants and wildtype cells during stress-exposure. These studies will reveal the mechanism of epitranscriptomic stress-response and potential RNA demodification enzymes (RNA erasers). Comparative proteomics of wildtype and mutant cell lines will be used to analyze the molecular consequences of imbalanced RNA modification.In objective 2 we will study the interactome (RNA readers) of small non-coding RNAs like tRNA fragments which have been found to accumulate in these neuronal diseases. We will identify their interactome and compare the interactome of the native, modified RNAs with the interactome of synthetic, non-modified RNAs. This approach will answer the question which downstream processes are affected by the RNA writer mutations which might be causative to pathogenesis. Another aspect of objective 2 covers the fate of tRNA fragments. Are these RNAs substrate to RNA writers and/or RNA eraser? Furthermore we will study the influence of modification on the cellular distribution of RNAs.Objective 3 covers studies arising from the observations made in objective 1 and 2 and will allow a deeper insight into the epitranscriptome. It will reveal the existence of RNA erasers and identify enzyme candidates responsible for the demethylation/demodification process. In a similar fashion we will screen for novel RNA writers and known RNA writers with yet unknown RNA substrates. With our developed tools and knowledge, we will search for yet unkown small non-coding RNAs which accumulate during stress or in the mutant cell lines and determine their involvement in cell homeostasis.The proposed studies will link the mutations of RNA writers found in patients with neuronal diseases to the currently poorly understood pathogenesis.