MECHANISM OF ELAV DEPENDENT NEURAL SPECIFIC SPLICING

ELAV 依赖性神经特异性剪接机制

• 批准号: 2420650
• 负责人: JUAN QIU
• 金额: $ 2.54万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-14 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2420650
• 关键词: DNA footprinting; Drosophilidae; RNA binding protein; RNA splicing; developmental neurobiology; embryonic stem cell; gel mobility shift assay; gene expression; gene mutation; genetic regulatory element; glial fibrillary acidic protein; introns; lethal genes; protein isoforms; protein structure function; regulatory gene; reporter genes

The ELAV gene (embryonic lethal, abnormal visual system) is essential for nervous system development. It has RNA binding motifs, and is specific for nervous tissue. The protein acts to alternatively splice a target to a tissue specific isoform. Conserved sequences between diverged Drosophila species may give a hint as to where the regulatory sequences reside for alternative splicing. A reporter GFP will be used to detect the spliced RNA and quantitative the expression. The candidate will investigate the neural specific splicing of an ELAV dependent intron target, neuroglian. She will define cis sequences of the regulated intron that are identified by the ELAV protein using a biochemical approach and then use a genetic approach to identify other proteins by mutagenesis and screening. The biochemical approach will involve straight-forward gel mobility shift assays combined with a deletion analysis of the intron sequences required for alternative splicing in an artificial splicing assay, which mimics nrg. In this assay putative proteins will be identified. The method of identification is in vivo splicing using the LacZ reporter construct. The second part of the proposal is a screening method which should derive mutations in alternative splicing. One aspect uses a reporter for in vivo selection, the other uses a rather complicated screen after EMS induced mutations.

Elav基因（胚胎致死，异常视觉系统）是 神经系统发展至关重要。 它具有RNA结合基序， 并且是特定于神经组织的。 蛋白质作用为 将靶标剪接到组织特异性同工型。 保守序列 在分散的果蝇之间，可能会暗示 调节序列存在于替代剪接。 记者GFP 将用于检测剪接的RNA并定量表达。 候选人将研究ELAV的神经特异性剪接 依赖的内含子靶标，神经元。 她将定义顺式序列 使用A的Elav蛋白鉴定的调节内含子 生化方法，然后使用遗传方法来识别 通过诱变和筛选的其他蛋白质。 生化方法将涉及直线凝胶迁移率 移位测定与内含子序列的缺失分析相结合 在人造剪接测定中进行替代剪接所需的 模仿NRG。 在此测定中，将确定推定的蛋白质。 这 识别方法是使用LACZ Reporter的体内剪接 构造。 提案的第二部分是一种筛选方法，应 在替代剪接中得出突变。 一个方面使用记者 在体内选择，另一个在EMS之后使用相当复杂的屏幕 诱导的突变。