The bioorganic chemistry of RNA editing by ADARs

描述（由申请人提供）：RNA编辑ADAR酶将人类mrna中的腺苷转化为肌苷，改变这些信息的编码特性。因此，ADARs在人类蛋白表达过程中信息传递的基本过程中起着举足轻重的作用。此外，从编辑信息中翻译的蛋白质与许多神经退行性疾病、精神疾病和行为疾病（如中风、癫痫、帕金森病、精神分裂症和发作性共济失调）有关。事实上，编码ADARs的基因缺失会导致模式生物显著的行为缺陷。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去氨化仍然是一个重要而具有挑战性的问题。此外，关于ADAR编辑活动的监管信息很少。此外，控制ADAR活性的药理学方法目前还不存在。在这个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编辑过程的基本认识，并将为其控制带来新的方法。本文提出的研究有以下具体目的：1)我们将定义ADAR2结构特征在位点选择性RNA编辑中的重要性。这将通过酵母中一种新的功能筛选来实现，将ADAR结构的变化与RNA编辑效率的变化联系起来。此外，还将确定ADAR2已确定的功能域（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射线晶体学分析被捕获的配合物来完成。2)我们将定义ADAR2 RNA编辑活性的调控途径，包括通过蛋白激酶，通过与六磷酸肌醇的结合，以及通过开发结合ADAR2或其RNA底物的人工调节剂。3)我们将生成新的核苷类似物和修饰rna来探索ADAR反应的不同方面。这些类似物的设计是由ADAR2催化域的晶体结构和底物类似物的反应性数据指导的。

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.