The Bioorganic Chemistry of RNA editing by ADARs

描述（由申请人提供）：RNA编辑ADAR酶将RNA中的腺苷转化为肌苷。由于肌苷在翻译过程中被解码为鸟苷，这种修饰会导致密码子意义的改变（重新编码）。人类mrna中至少有50个不同的A到I位点导致重新编码。重编码在神经系统中很常见，其靶点包括配体门控离子通道、电压门控离子通道和g蛋白偶联受体。事实上，adar对于神经系统的正常运作是必要的，并且在后生动物中调节行为。然而，对于在神经系统之外发挥作用的靶标的重新编码的效果知之甚少。在包括肌萎缩性侧索硬化症（ALS）、遗传性对称性异常症（DSH）、Prader-Willi综合征（PWS）、癫痫、抑郁症和癌症在内的几种人类疾病中，已经观察到A到I编辑的扰动。最近的一项研究也暗示了ADARs在控制衰老方面的作用。尽管这种形式的表观遗传学具有重要意义，但我们对A to I编辑的机制和调控的理解仍然不足。例如，由于缺乏对ADAR- rna复合物的详细表征，ADAR底物中特定腺苷的选择性仍然难以完全解释。此外，控制RNA编辑的药理学方法目前还不存在，限制了可能探索其生物学功能的研究类型。在这个R01项目的竞争性更新中，这些知识缺口将通过合成化学与分子生物学和生物化学技术的应用来解决。这些研究结果将扩展我们对RNA编辑过程及其对蛋白质功能影响的基本认识，并为其控制带来新的方法。将开发RNA编辑的位点选择性抑制方法。针对RNA编辑底物的主链修饰反义寡核苷酸和螺旋穿线肽将为此目的进行研究。此外，我们将定义控制编辑选择性的因素和抑制ADAR2反应的机制。功能筛选将用于定义两个ADAR2-RNA相互作用域之间的连接器结构中长度和序列在控制编辑位点选择性中的重要性。此外，含有非天然氨基酸的ADAR2突变体和含有核苷类似物的RNA将用于绘制ADAR2-RNA相互作用的图谱。一种新的基因选择将确定ADAR2的环肽抑制剂，这些抑制剂的后续研究将确定ADAR2反应中易受小分子控制的点。此外，我们将定义ADAR1编辑DNA修复酶NEIL1 mRNA的基础。这些努力将定义ADAR1反应的结构/活性关系，并扩展我们对神经系统外功能靶标重新编码的理解。最后，本项目将产生用于ADAR-RNA复合物结晶试验的新分子。

DESCRIPTION (provided by applicant): The RNA editing ADAR enzymes convert adenosines to inosines in RNA. Since inosine is decoded as guanosine during translation, this modification can lead to changes in the meaning of codons (recoding). There are at least 50 different A to I sites in human mRNAs that cause recoding. Recoding is common in the nervous system with targets including ligand-gated ion channels, voltage-gated ion channels and G-protein coupled receptors. Indeed, ADARs are necessary for a properly functioning nervous system and are known to regulate behavior in metazoans. However, little is known about the effect of recoding for targets with roles outside the nervous system. Perturbations in A to I editing have been observed in several human diseases including amyotrophic lateral sclerosis (ALS), dyschromatosis symmetrica hereditaria (DSH), Prader-Willi syndrome (PWS), epilepsy, depression and cancer. A recent study also implicates ADARs in the control of aging. Despite the significance of this form of epigenetics, our understanding of the mechanism and regulation of A to I editing is deficient. For instance, the selectivity for specific adenosines within ADAR substrates remains difficult to fully explain due to a lack of detailed characterization for ADAR-RNA complexes. Furthermore, pharmacological methods for controlling RNA editing do not currently exist limiting the types of studies possible to probe its biological function. In this competitive renewal of an R01 project, these knowledge gaps will be addressed through the application of synthetic chemistry coupled with techniques from molecular biology and biochemistry. The results of these studies will extend our basic understanding of the process of RNA editing and its effects on protein function as well as lead to new methods for its control. Methods for site-selective inhibition of RNA editing will be developed. Backbone modified antisense oligonucleotides and helix- threading peptoids that target RNA editing substrates will be investigated for this purpose. In addition, we will define factors controlling editing selectivity and mechanisms of inhibition for the ADAR2 reaction. A functional screen will be used to define the importance in controlling editing site selectivity of the length and sequence in a linker structure between two ADAR2-RNA interaction domains. In addition, ADAR2 mutants containing unnatural amino acids and RNA containing nucleoside analogs will be used to map ADAR2-RNA interactions. A novel genetic selection will identify cyclic peptide inhibitors of ADAR2 and follow-up studies with these inhibitors will identify points in the ADAR2 reaction susceptible to small molecule control. In addition, we will define the basis for ADAR1 editing of the mRNA for the DNA repair enzyme NEIL1. These efforts will define structure/activity relationships for the ADAR1 reaction and extend our understanding of the recoding of targets with functions outside the nervous system. Finally, this project will produce new molecules for crystallization trials of ADAR-RNA complexes. PUBLIC HEALTH RELEVANCE: RNA editing catalyzed by the ADAR enzymes is a form of epigenetic control of gene expression that is perturbed in a variety of human diseases including amyotrophic lateral sclerosis (ALS), dyschromatosis symmetrica hereditaria (DSH), Prader-Willi syndrome (PWS), epilepsy, depression and cancer. The proposed studies will extend our basic understanding of the process of RNA editing as well as lead to new methods for its control.