MECHANISM OF ELAV DEPENDENT NEURAL SPECIFIC SPLICING

ELAV基因(胚胎致死，异常视觉系统)是 对神经系统发育至关重要。它有RNA结合基序， 并且是神经组织特有的。这种蛋白质的作用是交替地 将靶子拼接到组织特定的异构体。保守序列 分化的果蝇物种之间的关系可能会给出一个线索 调节序列存在于选择性剪接中。本报记者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 依赖内含子靶点，神经胶质细胞。她将定义以下配置项序列 受调节的内含子，由ELAV蛋白使用 生化方法，然后使用遗传方法来鉴定 对其他蛋白质进行诱变和筛选。 生化方法将包括直接的凝胶迁移率 结合内含子序列缺失分析的移位分析 在人工剪接实验中的选择性剪接所需的 模仿NRG。在这项实验中，我们将鉴定可能的蛋白质。这个 鉴定的方法是使用LacZ报告基因的体内剪接 建造。 建议的第二部分是一种筛查方法，它应该 在选择性剪接中衍生突变。一方面，使用记者来 在活体选择中，另一种在EMS后使用相当复杂的屏幕 诱变。

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.