GENETIC DISSECTION OF PRE-MRNA EDITING IN DROSOPHILA

果蝇 mRNA 前编辑的基因解剖

• 批准号: 6599081
• 负责人: ROBERT A. REENAN
• 金额: $ 3.38万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6599081
• 关键词: Drosophilidae; adenosine deaminase; arthropod genetics; behavior test; behavioral /social science research tag; chemical structure function; double stranded RNA; electrophysiology; gene expression; gene targeting; genetic promoter element; genetic regulation; genetically modified animals; growth /development; helicase; membrane channels; molecular cloning; molecular site; neuropsychology; nucleic acid sequence; posttranscriptional RNA processing; precursor mRNA; site directed mutagenesis; tissue /cell culture; voltage gated channel

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.

通过 ADAR 酶（作用于 RNA 的腺苷脱氨酶）进行的 RNA 编辑已成为一种新颖且重要的转录后机制，用于改变单个基因位点编码的蛋白质产物。 ADAR 活性已在不同种系的生物体以及生物体内的多种组织内检测到。 矛盾的是，ADAR 的 mRNA 靶标数量仍然很少，并且主要限于哺乳动物神经系统的配体门控离子通道。 ADAR 的有限数量的经过充分表征的 mRNA 底物已得到表征，并且前体 mRNA 中 dsRNA 中间体的一般要求已得到证实。 尽管如此，仍然没有一致答案的核心问题仍然存在，例如： ADAR 特异性由什么决定？ RNA 编辑位点由什么组成？我们能否预测它们的存在而不是依赖偶然性？ 特定 RNA 编辑位点的编辑会对生物体产生哪些表型后果？ RNA 编辑在基因调控中的全球作用是什么？ 在该提案中，使用果蝇作为模型遗传系统来解决这些主要领域。 首先，几种果蝇电压门控离子通道和配体门控离子通道以及酶中的已知RNA编辑位点将用于通过体内转基因和体外方法阐明前mRNA编辑位点的顺式决定簇。 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编辑的效果。 最后，最近分离出的果蝇 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。