RNA/PROTEIN INTERACTIONS IN PREMRNA SPLICING REGULATION

性别决定是一个基本的决定，基本上所有后生动物在他们的发展过程中遇到。 黑腹果蝇的性别决定涉及一系列选择性剪接决定，也是最好理解的剪接调控的例子。 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，组成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%的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结合蛋白如何识别靶RNA并调节组成性和选择性前mRNA剪接。 果蝇蛋白质Sex-lethal（SXL）是雄性和雌性细胞命运之间的关键二元开关。 在过去，我们定义了SXL通过拮抗已知的剪接因子U2 AF 65来调节选择性剪接的机制。特异性是生物调节的一个基本主题。U2 AF 65和SXL为剪接调控背景下的特定RNA-蛋白质相互作用提供了极好的模型。 例如，通用剪接因子U2 AF 65识别多种多嘧啶段/3'剪接位点，而高度特异性剪接阻遏物SXL识别特异性序列。虽然这两种蛋白质都含有核糖核蛋白共有基序，但它们具有不同的RNA结合特异性。 然而，目前尚不清楚这些看似相似的蛋白质如何实现独特的RNA结合特异性。 为了定义U2 AF 65和SXL的RNA结合特异性的结构基础，我们将通过使用生物化学、分子和遗传方法的组合来扩展对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的其他成员，这些成员可能调节RNA生物合成的不同方面。此外，SXL还控制着许多女性特有的功能。然而，SXL调控的一些相关基因仍有待鉴定。 为了确定这些目标，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。这些方法应补充遗传分析。

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.