The Bioorganic Chemistry of RNA editing by ADARs

ADAR 编辑 RNA 的生物有机化学

• 批准号: 8635357
• 负责人: PETER A. BEAL
• 金额: $ 27.25万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8635357
• 关键词: ADAR1; Address; Adenosine; Aging; Amino Acids; Amyotrophic Lateral Sclerosis; Antisense Oligonucleotides; Behavior; Biochemistry; Biological Process; Chemistry; Codon Nucleotides; Complex; Coupled; Crystallization; Cyclic Peptides; DNA Repair; DNA Repair Enzymes; DRADA2b protein; Deaminase; Deamination; Enzymes; Epigenetic Process; Epilepsy; G-Protein-Coupled Receptors; Gated Ion Channel; Gene Expression; Genetic Techniques; Guanosine; Human; Inosine; Knowledge; Lead; Length; Ligands; Link; Malignant Neoplasms; Maps; Mental Depression; Messenger RNA; Methods; Modification; Molecular Biology Techniques; Nervous System Physiology; Nervous system structure; Peptoids; Prader-Willi Syndrome; Process; Proteins; RNA; RNA Binding; RNA Editing; Reaction; Regulation; Reporting; Research; Research Design; Role; Saccharomyces cerevisiae; Site; Structure; Structure-Activity Relationship; Synthesis Chemistry; Testing; Therapeutic; Time; Tissues; Transcript; Translations; Vertebral column; base; follow-up; genetic selection; human disease; inhibitor/antagonist; mRNA Precursor; mutant; novel; nucleoside analog; protein function; small molecule; tool; voltage

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.

描述(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，这种修改可能会导致密码子含义的变化(重新编码)。在人类的mRNA中，至少有50个不同的A到I位点会导致重新编码。重新编码在神经系统中很常见，其靶标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对于正常运作的神经系统是必要的，而且众所周知，它可以调节后生动物的行为。然而，人们对神经系统以外的目标重新编码的影响知之甚少。在几种人类疾病中观察到A到I编辑的扰动，包括肌萎缩侧索硬化症(ALS)、遗传性对称性色素异常症(DSH)、Prader-Willi综合征(PWS)、癫痫、抑郁症和癌症。最近的一项研究还表明，ADAR在控制衰老方面也有一定的作用。尽管这种形式的表观遗传学具有重要意义，但我们对A到I编辑的机制和调节的理解是不足的。例如，由于缺乏对ADAR-RNA复合体的详细表征，ADAR底物中特定腺苷的选择性仍然难以完全解释。此外，目前还不存在控制RNA编辑的药理学方法，限制了可能探索其生物学功能的研究类型。在这次R01项目的竞争性更新中，这些知识差距将通过应用合成化学以及分子生物学和生物化学的技术来解决。这些研究结果将扩大我们对RNA编辑过程及其对蛋白质功能影响的基本理解，并为其控制提供新的方法。将开发选择性抑制RNA编辑的方法。为此目的，将研究以RNA编辑底物为靶标的骨架修饰的反义寡核苷酸和螺旋丝状肽。此外，我们还将定义控制编辑选择性的因素和ADAR2反应的抑制机制。功能筛选将用于确定在控制两个ADAR2-RNA相互作用域之间的连接物结构中的长度和序列的编辑位点选择性方面的重要性。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将被用于绘制ADAR2-RNA相互作用的图谱。一种新的遗传选择将识别ADAR2的环肽抑制剂，对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，我们还将定义ADAR1编辑DNA修复酶NEIL1的信使核糖核酸的基础。这些工作将确定ADAR1反应的结构/活性关系，并扩大我们对具有神经系统以外功能的靶标重新编码的理解。最后，该项目将生产用于ADAR-RNA络合物结晶试验的新分子。