The Biology and Biochemistry of ADAR RNA Editing Enzymes

描述(由申请人提供)： 项目摘要/摘要ADAR是一种RNA编辑酶，可将细胞和病毒双链RNA(DsRNA)中的腺苷转化为肌苷。ADAR的一个功能是靶向mRNA中的密码子，以便从单个基因的信息中获得多种蛋白质异构体。密码子编辑在许多神经重要的mRNAs中被发现，编辑的异常水平与神经系统疾病有关。要确定ADAR如何对人类健康、有时对疾病作出贡献，最根本的是完全了解它们的催化机制。有人建议通过实验来确定人类ADAR2催化结构域中的氨基酸，这些氨基酸控制其催化效率并导致精确的腺苷靶向。筛查已经确定了影响这些过程的突变，并将使用各种生化分析来表征这些突变。尽管密码子很重要，但用密码子编辑的情况很少，而且在非编码的RNA序列中含有更多的肌苷。然而，肌苷在非编码序列中的功能尚不清楚。由于ADAR针对任何dsRNA序列，一种可能性是它们影响dsRNA介导的基因沉默途径。将进行高通量测序，将野生型线虫的小RNA与缺乏ADAR的菌株的小RNA进行比较。重点将放在从dsRNA前体加工的小RNA上，即microRNAs和内源siRNAs。改变的小RNA水平及其编辑位点将被列表，后者将与测序错误区分开来，因为它们在缺乏ADAR编辑的动物中不存在。ADAR对小RNA的影响将与利用微阵列分析预测的mRNA水平变化相关。虽然缺乏ADAR的线虫是可行的，但它们有趋化缺陷，预计它们还有其他尚未被认识到的微妙缺陷。在生物信息学研究的观察结果得到验证后，将对分子缺陷所暗示的表型进行测试。

DESCRIPTION (provided by applicant): Project Summary/Abstract ADARs are RNA editing enzymes that convert adenosine to inosine in cellular and viral double- stranded RNA (dsRNA). One function of ADARs is to target codons in mRNA to allow multiple protein isoforms from the information in a single gene. Codon editing is found in many neuronally important mRNAs, and aberrant levels of editing have been linked to neurological disease. Fundamental to determining how ADARs contribute to human health, and sometimes disease, is a complete understanding of their catalytic mechanism. Experiments are proposed to determine the amino acids in the human ADAR2 catalytic domain that control its catalytic efficiency and lead to targeting of precise adenosines. A screen has identified mutations that affect these proceses, and these wil be characterized using a variety of biochemical assays. Despite its importance, editing in codons is rare, and there are far more inosines in noncoding RNA sequences. Yet, the function of inosines in noncoding sequences is unclear. Since ADARs target any dsRNA sequence, one posibility is that they affect dsRNA-mediated gene silencing pathways. High- throughput sequencing will be performed to compare the small RNAs of wildtype C. elegans with those in strains lacking ADARs. The focus will be on small RNAs that are processed from a dsRNA precursor, namely, microRNAs and endogenous siRNAs. Altered levels of small RNAs as well as their editing sites will be tabulated, and the latter will be distinguished from sequencing errors by their absence in animals lacking ADAR editing. Effects of ADARs on small RNAs will be correlated with predicted changes in mRNA levels using microarray analyses. While C. elegans lacking ADARs are viable, they have chemotaxis defects, and it is anticipated they have other subtle defects that have not been recognized. After validation of observations made with bioinformatics studies, phenotypes suggested by molecular defects will be tested.