The bioorganic chemistry of RNA editing by ADARs

描述（由申请人提供）：RNA编辑阿达尔酶将人mRNA中的腺苷转化为肌苷，从而改变这些信息的编码特性。因此，ADARs在人类蛋白质表达过程中的信息传递的基本过程中起着关键作用。此外，从编辑的信息翻译的蛋白质涉及许多神经变性、精神和行为障碍，如中风、癫痫、帕金森病、精神分裂症和发作性共济失调。事实上，编码ADAR的基因的缺失导致模式生物中的显著行为缺陷。然而，我们对编辑反应中基本步骤的分子基础的理解是有限的。例如，为什么底物mRNA中的某些腺苷被选择性地脱氨基仍然是一个重要且具有挑战性的问题。此外，关于阿达尔编辑活动的调节信息很少。此外，目前还不存在控制阿达尔活性的药理学方法。在R01项目的竞争性更新中，我们将通过应用合成化学和生物化学技术来解决这些问题。这些研究的结果将扩展我们对RNA编辑过程的基本理解，并将导致新的控制方法。本论文的主要目的是：1）明确ADAR 2结构特征在RNA定点编辑中的重要性。这将使用酵母中的新功能筛选来实现，以将阿达尔结构的变化与RNA编辑效率的变化联系起来。此外，将确定ADAR 2的鉴定的功能结构域（dsRBM和脱氨酶结构域）的作用。这将通过使用羟基自由基探测、核苷酸类似物干扰、最近邻交联和X射线晶体学分析捕获的复合物来实现。2)我们将定义ADAR2的RNA编辑活性的调节途径，包括通过蛋白激酶，通过肌醇六磷酸的结合，以及通过开发结合ADAR2或其RNA底物的人工调节剂。3)我们将产生新的核苷类似物和修饰的RNA，以探测阿达尔反应的不同方面。这些类似物的设计由ADAR 2催化结构域的可用晶体结构和底物类似物反应性数据指导。

DESCRIPTION (provided by applicant): The RNA editing ADAR enzymes convert adenosines to inosines in human mRNAs, changing the coding properties of these messages. Thus, ADARs play a pivotal role in the basic process of information transfer during human protein expression. Moreover, proteins translated from edited messages have been implicated in a number of neurodegenerative, psychiatric and behavioral disorders such as stroke, epilepsy, Parkinson's disease, schizophrenia and episodic ataxia. Indeed, deletion of genes encoding ADARs leads to significant behavioral defects in model organisms. However, our understanding of the molecular basis for the fundamental steps in the editing reaction is limited. For instance, why certain adenosines in a substrate mRNA are selectively deaminated continues to be an important and challenging question. In addition, little information is available on regulation of ADAR editing activity. Furthermore, pharmacological methods for controlling ADAR activity do not currently exist. In this competitive renewal of an R01 project, we will address these issues through the application of synthetic chemistry coupled with techniques from biological chemistry. The results of these studies will extend our basic understanding of the process of RNA editing and will lead to new methods for its control. The research proposed here has the following specific aims: 1) We will define the importance of ADAR2 structural features in site-selective RNA editing. This will be accomplished using a novel functional screen in yeast to link changes in ADAR structure to changes in RNA editing efficiency. In addition, the role of ADAR2's identified functional domains (dsRBMs and deaminase domain) will be determined. This will be accomplished through the analysis of trapped complexes using hydroxyl radical probing, nucleotide analog interference, nearest neighbor crosslinking and x-ray crystallography. 2) We will define pathways for regulation of ADAR2's RNA editing activity including by protein kinases, by the binding of inositol hexaphosphate and through the development of artificial regulators that either bind ADAR2 or its RNA substrate. 3) We will generate new nucleoside analogs and modified RNAs to probe different aspects of the ADAR reaction. The design of these analogs is guided by the available crystal structure of the ADAR2 catalytic domain and substrate analog reactivity data.