RNA/PROTEIN INTERACTIONS IN PREMRNA SPLICING REGULATION

性别决定是几乎所有后生动物在发育过程中都会遇到的基本决定。果蝇的性别决定涉及一系列可供选择的剪接决定，也是剪接调控的最好例子。剪接是从前体信使RNA中去除非编码序列(内含子)的过程。在高等真核生物中，结构性剪接和选择性剪接是许多重要细胞过程中基因调控的重要方面。大约15%与人类疾病有关的突变会影响RNA剪接信号，包括细胞转化、杜氏肌营养不良和肿瘤转移。我们的目标是了解RNA结合蛋白如何识别目标RNA，并调控结构性和选择性的前mRNA剪接。果蝇的性致死蛋白(SXL)在雄性和雌性细胞命运之间起着关键的二元开关作用。在过去，我们通过拮抗已知的剪接因子U2AF65来定义SXL调节选择性剪接的机制。专一性是生物调控的一个基本主题。U2AF65和SXL为剪接调控背景下的特定RNA-蛋白质相互作用提供了优秀的模型。例如，虽然通用剪接因子U2AF65识别各种各样的多嘧啶-链/3‘剪接位点，但高度特异的剪接抑制因子SXL识别特定的序列。虽然这两种蛋白质都含有核糖核蛋白共识基序，但它们具有不同的RNA结合特异性。然而，目前还不清楚这些看似相似的蛋白质是如何实现独特的RNA结合特异性的。为了确定U2AF65和SXL的RNA结合特异性的结构基础，我们将结合生化、分子和遗传方法扩展我们对RNA和蛋白质的分析。我们的发现也将直接适用于这个最大家族中可能调节RNA生物发生的不同方面的其他成员。此外，SXL还控制着许多女性特有的功能。然而，一些受SXL调控的相关基因仍有待鉴定。为了识别这些靶点，我们将使用最近开发的分子方法的组合-基因组SELEX和消减杂交/差异显示。这些方法应该是对基因分析的补充。

Sex determination is a fundamental decision that essentially all metazoans encounter during their development. Sex determination in Drosophila melanogaster involves a hierarchy of alternative splicing decisions, and is also the best understood example of splicing regulation. Splicing is a process by which non-coding sequences (introns) are removed from the precursor messenger RNA. In higher eukaryotes, constitutive and alternative splicing are important aspects of gene regulation in many important cellular processes. Approximately 15 percent of the mutations that have been linked to human diseases affect RNA splicing signals, including cellular transformation, Duchenne muscular dystrophy, and tumor metastasis. Our goal is to understand how RNA-binding proteins recognize target RNAs and regulate constitutive and alternative pre-mRNA splicing. The Drosophila protein Sex-lethal (SXL) acts as a key binary switch between the male and female cell fates. In the past, we defined the mechanism by which SXL regulates alternative splicing by antagonizing the known splicing factor U2AF65. Specificity is an underlying theme in biological regulation. U2AF65 and SXL offer excellent models for specific RNA-protein interactions in the context of splicing regulation. For example, while the general splicing factor U2AF65 recognizes a wide variety of polypyrimidine-tract/3' splice sites, the highly specific splicing repressor SXL recognizes a specific sequence. Although both proteins contain a ribonucleoprotein-consensus motif, they have distinct RNA-binding specificity. However, it is not understood how these seemingly similar proteins achieve unique RNA-binding specificities. To define the structural basis for the RNA-binding specificities of U2AF65 and SXL, we will extend our analysis of the RNA and the proteins by using a combination of biochemical, molecular, and genetic approaches. Our findings will also be directly applicable to other members of this largest family that likely regulate different aspects of RNA biogenesis. In addition, SXL controls many female-specific functions. However, some of the relevant genes that are regulated by SXL remain to be identified. To identify these targets, we will use a combination of recently developed molecular approaches - genomic SELEX and subtractive hybridization/differential display. These approaches should complement genetic analysis.