GENETIC DISSECTION OF PRE-MRNA EDITING IN DROSOPHILA

果蝇 mRNA 前编辑的基因解剖

• 批准号: 6798389
• 负责人: ROBERT A. REENAN
• 金额: $ 2.9万
• 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/6798389
• 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.

经由阿达尔酶（作用于RNA的腺苷脱氨酶）的RNA编辑已经成为改变由单个遗传基因座编码的蛋白质产物的新颖且重要的转录后机制。 已在亲缘关系较远的生物体和生物体内的多种组织中检测到阿达尔活性。 巧合的是，ADAR的mRNA靶点的数量仍然很小，并且主要限于哺乳动物神经系统的配体门控离子通道。 已经表征了有限数量的用于ADAR的充分表征的mRNA底物，并且已经证明了前mRNA中对dsRNA中间体的一般要求。 尽管如此，没有一致答案的中心问题仍然存在，例如：是什么决定了阿达尔的特异性？ RNA编辑位点是由什么组成的？我们能否预测它们的存在，而不是依赖于偶然发现？ 编辑特定RNA编辑位点对生物体的表型影响是什么？ RNA编辑在基因调控中的全球作用是什么？ 在这个建议中，这些主要领域是作为一个模型遗传系统使用果蝇。 首先，已知的RNA编辑位点在几个果蝇电压和配体门控离子通道，以及一个在酶将被用来阐明通过在体内转基因和体外方法的前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编辑的影响。 最后，最近分离的果蝇阿达尔敲除突变d阿达尔将在分子、遗传和行为水平上进行表征，以评估特异性RNA编辑的全球意义。