Molecular Genetic Analysis of RNA Editing in Drosophila

果蝇RNA编辑的分子遗传学分析

• 批准号: 9728737
• 负责人: Robert Reenan
• 金额: $ 27万
• 依托单位: University of Connecticut Health Center
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9728737&HistoricalAwards=false
• 关键词: Molecular; Genetic; Analysis; RNA; Editing

Reenan 9728737 Adenosine to inosine (A to I) RNA editing has emerged as a novel mechanism for regulating protein activity through alterations in a protein's primary amino-acid sequence. Mammalian RNA editases, such as DRADA and RED1, have been identified and shown to modify mRNA by hydrolytic deamination of certain adenosine residues to inosine. This reaction occurs via a double-stranded (ds) RNA intermediate. The goal of this research is to examine the process of A to I RNA editing in a model genetic system, Drosophila melanogaster. Preliminary results from this laboratory have identified RNA editing sites in transcripts of the para locus, which encodes a voltage-gated sodium channel. In addition, a candidate editase gene has been cloned from Drosophila, DRED-1 (Drosophila RNA editase-1). Experiments will be performed to address the nature of para RNA editing sites through in vivo and in vitro experiments using purified DRED-1 protein and synthetic para RNA substrates. Site-directed mutagenesis will be used to identify cis-acting regulatory sequences necessary for efficient RNA editing. In order to address whether the DRED-1 protein is essential for viability, site directed transposon mutagenesis of the DRED-1 locus will be performed. These experiments, in addition to determining the effect of DRED-1 knockouts, will address the necessity of DRED-1 activity for editing of para. Another approach, targeted tissue specific expression of a dominant negative DRED-1 protein, will address the effects of negating DRED-1 activity in selected tissues. Lastly, the investigator will utilize immunolocalization of the DRED-1 protein to Drosophila salivary gland polytene chromosomes to identify new targets for the process of RNA editing. The process whereby information encoded by genes is used to direct the synthesis of proteins is crucial to the proper development and function of all organisms. This research project will examine an important phenomenon called RNA editing, in which the infor mation in messenger RNAs, which are copies from genes, is changed or edited, resulting in a change in the final protein product. The changes in protein structure resulting from messenger RNA editing, can have a profound influence on a protein's function. This research will utilize a very powerful experimental system, the fruit fly Drosophila melanogaster, to dissect the role of RNA editing in the function of a complex organism, examining effects ranging from development to adult behavior.

Reenan 9728737 腺苷到肌苷（A到I）RNA编辑已经成为一种通过改变蛋白质一级氨基酸序列来调节蛋白质活性的新机制。 哺乳动物RNA编辑酶，如DRDA和RED 1，已被鉴定并显示通过将某些腺苷残基水解脱氨为肌苷来修饰mRNA。 该反应通过双链（ds）RNA中间体发生。 本研究的目的是研究在一个模式遗传系统，果蝇（Drosophila melanogaster）中A到I RNA编辑的过程。 该实验室的初步结果已经确定了编码电压门控钠通道的帕拉基因座转录本中的RNA编辑位点。 此外，已经从果蝇中克隆了一个候选编辑酶基因，DRED-1（果蝇RNA编辑酶-1）。 将通过使用纯化的DRED-1蛋白和合成的帕拉RNA底物的体内和体外实验进行实验，以解决帕拉RNA编辑位点的性质。 定点诱变将用于鉴定有效RNA编辑所必需的顺式作用调控序列。 为了确定DRED-1蛋白是否是生存力所必需的，将进行DRED-1基因座的定点转座子诱变。 这些实验，除了确定DRED-1基因敲除的影响，将解决DRED-1活性编辑的段落的必要性。另一种方法，显性负DRED-1蛋白的靶向组织特异性表达，将解决在选定的组织中否定DRED-1活性的影响。 最后，研究人员将利用DRED-1蛋白对果蝇唾液腺多线染色体的免疫定位来鉴定RNA编辑过程的新靶点。 基因编码的信息被用来指导蛋白质合成的过程对所有生物体的正常发育和功能至关重要。 该研究项目将研究一种称为RNA编辑的重要现象，其中信使RNA中的信息（来自基因的拷贝）被改变或编辑，导致最终蛋白质产物的变化。 由信使RNA编辑引起的蛋白质结构的变化可以对蛋白质的功能产生深远的影响。 这项研究将利用一个非常强大的实验系统，果蝇，来剖析RNA编辑在复杂生物体功能中的作用，研究从发育到成年行为的影响。