MOLECULAR MECHANISM OF RNA EDITING ADENOSINE DEAMINASE

RNA编辑是一个用于描述RNA中核苷酸的结构改变、插入或删除的术语。如果这种修饰发生在信使RNA （mRNA）中，它可能导致翻译的蛋白质序列与基因DNA序列预测的蛋白质序列不同。由于发现了作用于真核细胞细胞核中编码的mRNA的RNA编辑酶，不能再假设高等生物的基因序列完全描述蛋白质序列。因此，RNA编辑在蛋白质表达过程中发生的信息传递的基本过程中起着关键作用。此外，从编辑信息中翻译的人类蛋白质(例如；谷氨酸和血清素受体)与许多神经退行性、精神和行为障碍（如中风、癫痫、帕金森病和精神分裂症）有关。人类病原体也会利用RNA编辑来发挥自己的优势。丁型肝炎病毒是一种亚病毒人类病原体，在同时感染乙型肝炎期间增加严重肝脏疾病的机会，需要在停止密码子处编辑RNA，以从同一信息产生两种蛋白质。这两种蛋白质都是丁型肝炎病毒适当复制和包装所必需的。在许多情况下，能够对特定RNA进行修饰的酶已经被鉴定和克隆。然而，我们对编辑反应中基本步骤的分子基础的理解，例如如何识别底物，令人惊讶地有限。本研究的目的是确定RNA编辑腺苷脱氨酶（ADAR-2）的机制特性。这将通过合成非天然RNA底物类似物以及使用酶动力学，凝胶迁移实验和荧光测量相结合的详细分析来完成。在我们的工作之前，没有实验系统可以对RNA编辑机制进行详细、系统的分析。这些研究的结果将扩展我们对RNA编辑重要过程的基本理解，并可能最终导致控制细胞或病毒RNA及其编码蛋白质的结构的能力。此外，我们的目标是开发可以协同使用的试剂，以阐明ADAR/RNA复合物沿反应坐标不同点的结构。

RNA editing is a term used to describe the structural alteration, insertion or deletion of nucleotides in RNA. If the modification occurs in messenger RNA (mRNA), it can result in the translation of a protein sequence different from that predicted by the DNA sequence of the gene. Since the discovery of RNA editing enzymes that act on the mRNA encoded in the nuclei of eukaryotic cells, the gene sequences of higher organisms can no longer be assumed to fully describe the protein sequences. Thus, RNA editing plays a pivotal role in the basic process of information transfer that takes place during protein expression. Moreover, human proteins translated from editing messages (erg. glutamate and serotonin receptors) have been implicated in a number of neurodegenerative, psychiatric and behavior disorders such as stroke, epilepsy, Parkinson's disease and schizophrenia. Human pathogens also use RNA editing to their advantage. Hepatitis delta virus, a subviral human pathogen that increases the chance of severe liver disease during concurrent infection with hepatitis B, requires RNA editing at a stop codon to produce two proteins from the same message. Both proteins are required for proper replication and packaging of hepatitis delta virus. In many cases, an enzyme capable of a particular RNA modification has been identified and cloned. However, our understanding of the molecular basis for the fundamental steps in the editing reactions, such as how the substrate is recognized, is surprisingly limited. The goal of this research is to define mechanistic properties of an RNA editing adenosine deaminase (ADAR-2). This will be accomplished through the synthesis of nonnatural RNA substrate analogs and their detailed analysis using a combination of enzyme kinetics, gel mobility shift experiments and fluorescence measurements. Prior to our work, no experimental system existed for a detailed, systematic analysis of RNA editing mechanisms. The results of these studies will extend our basic understanding of the important process of RNA editing and may ultimately lead to the ability to control the structure of cellular or viral RNAs and the proteins they encode. Furthermore, we aim to develop reagents that can be used in collaboration to elucidate the structure of the ADAR/RNA complex at distinct points along the reaction coordinate.