Molecular Genetic Analysis of RNA Editing in Drosophila

Reenan 9728737腺苷到肌苷(A到I)的核糖核酸编辑已经成为一种通过改变蛋白质的主要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DRADA和Red1，已被发现并被证明通过将某些腺苷残基水解脱氨基为肌苷来修饰mRNA。该反应通过双链(DS)RNA中间体进行。这项研究的目标是在一个模型遗传系统--黑腹果蝇中检查A到I RNA编辑的过程。这个实验室的初步结果已经在编码电压门控钠通道的对位基因的转录本中发现了RNA编辑位点。此外，还从果蝇中克隆了一个候选编辑酶基因Dred-1(Drophila RNA编辑酶-1)。将进行实验，通过使用纯化的Dred-1蛋白和合成的parRNA底物进行体内和体外实验，以解决parRNA编辑位点的性质。定点突变将被用来识别有效的RNA编辑所必需的顺式作用调控序列。为了解决Dred-1蛋白是否对生存是必要的，将进行Dred-1基因座的定点转座子突变。这些实验除了确定Dred-1基因敲除的效果外，还将解决Dred-1活动对编辑段落的必要性。另一种方法是靶向组织特异性表达显性负Dred-1蛋白，将解决在选定组织中否定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确定RNA编辑过程的新靶点。由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。这项研究项目将研究一种名为RNA编辑的重要现象，在这种现象中，信使RNA中的信息被改变或编辑，从而导致最终蛋白质产品的变化。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--来剖析RNA编辑在复杂有机体功能中的作用，检查从发育到成年行为的各种影响。

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.