Molecular Genetic Analysis of RNA Editing in Drosophila

Adenosine to inosine (A to 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。哺乳动物的RNA编辑酶，如DRADA和RED1，已被鉴定并显示通过水解某些腺苷残基为肌苷来修饰mRNA。该反应通过双链RNA中间体发生。这项研究的目的是研究A到I RNA编辑的过程在一个模型遗传系统，果蝇黑腹。本实验室的初步结果已经确定了副位点转录本中的RNA编辑位点，其编码电压门控钠通道。此外，还从果蝇中克隆了一个候选编辑酶基因DRED-1 （Drosophila RNA编辑酶-1）。实验将通过使用纯化的DRED-1蛋白和合成的对RNA底物进行体内和体外实验来解决对RNA编辑位点的性质。定点诱变将用于鉴定有效RNA编辑所必需的顺式调控序列。为了确定DRED-1蛋白是否对生存至关重要，将对DRED-1基因座进行定点转座子诱变。这些实验除了确定DRED-1基因敲除的效果外，还将解决DRED-1活性对段落编辑的必要性。另一种方法是靶向组织特异性表达显性负DRED-1蛋白，将在选定的组织中解决负DRED-1活性的影响。最后，研究者将利用果蝇唾液腺多烯染色体上DRED-1蛋白的免疫定位来确定RNA编辑过程的新靶点。利用基因编码的信息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重要的现象，称为RNA编辑，其中信使RNA（基因拷贝）中的信息被改变或编辑，导致最终蛋白质产品的变化。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，果蝇黑腹果蝇，来解剖RNA编辑在复杂生物体功能中的作用，检查从发育到成年行为的影响。

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.