GENETIC DISSECTION OF PRE-MRNA EDITING IN DROSOPHILA

通过ADAR酶（作用于RNA的腺苷脱氨酶）进行RNA编辑已经成为一种新的重要的转录后机制，可以改变由单个遗传位点编码的蛋白质产物。ADAR活性已在不同种类的生物体和生物体内的多种组织中被检测到。矛盾的是，ADARs的mRNA靶点数量仍然很少，并且主要局限于哺乳动物神经系统的配体门控离子通道。已经鉴定了有限数量的表征良好的ADARs mRNA底物，并且已经证明了pre-mRNA中对dsRNA中间体的一般要求。尽管如此，没有一致答案的核心问题仍然存在，例如；什么决定了ADAR的特异性？RNA编辑位点是由什么组成的？我们能否预测它们的存在，而不是依靠意外发现？特定RNA编辑位点的编辑对生物体的表型后果是什么？RNA编辑在基因调控中的全局作用是什么？在本提案中，这些主要领域是解决使用果蝇作为模型遗传系统。首先，我们将利用果蝇电压和配体门控离子通道中已知的RNA编辑位点以及酶中的RNA编辑位点，通过体内转基因和体外方法阐明mrna前编辑位点的顺式决定因素。其次，遗传和分子方法将确定敲除或减少已知对神经系统功能和行为有影响的离子通道基因中特定RNA编辑位点的RNA编辑的影响。最后，最近分离的果蝇ADAR敲除突变dADAR将在分子，遗传和行为水平上进行表征，以评估特定RNA编辑的全球意义。

RNA editing via the ADAR enzymes (adenosine deaminases that act on RNA) has emerged as a novel and significant post- transcriptional mechanism for altering protein products encoded by a single genetic locus. ADAR activity has been detected across phyletically distant organisms and within a variety of tissues within an organism. Paradoxically, the number of mRNA targets of ADARs remains small and limited primarily to ligand- gated ion channels of the mammalian nervous system. A limited number of well characterized mRNA substrates for ADARs have been characterized and a general requirement for a dsRNA intermediate in pre-mRNA has been proven. Still, central questions with no consistent answers remain such as; What determines ADAR specificity? What comprises an RNA editing site and can we predict their existence rather than relying on serendipity? What are the phenotypic consequences for the organism of editing of a particular RNA editing site? What is the global role of RNA editing in gene regulation? In this proposal, these major areas are addressed using Drosophila as a model genetic system. First, known RNA editing sites in several Drosophila voltage- and ligand-gated ion channels as well as one in an enzyme will be utilized to elucidate the cis-determinants of pre-mRNA editing sites via in vivo transgenic and in vitro methods. Second, a genetic and molecular approach will determine the effects of knocking out or reducing RNA editing of a specific RNA editing site in a ion-channel gene known to have effects on nervous system function and behavior. Lastly, a recently isolated knock- out mutation of the Drosophila ADAR, dADAR, will be characterized at the molecular, genetic and behavioral level to assess the global significance of specific RNA editing.