Molecular and Genetic Analysis of Sex Determination in Drosophila

这项工作的长期目标是了解在多细胞生物体中调节选择性RNA剪接的分子机制。剪接如何被调控的问题尤其重要，因为选择性剪接已被证明是调控基因表达的一种常见手段。本研究以果蝇性别决定基因SXL的性别特异性剪接调控为模型系统。在这个奖项中，重点是sans-fille(Snf)基因在促进女性特有的SXL Pre-mRNA剪接中的作用。遗传学研究已经证实，非性别特异的SNF基因产物与雌性特异的SXL RNA结合蛋白一起作用，直接剪接SXL前-mRNA。值得注意的是，克隆的SNF基因证实它是两个已被广泛研究的人类剪接体蛋白ULA和U2B“的同源物。与ULA一样，SNF是U1 SnRNP蛋白。与U2B”一样，SNF是U2 SnRNP蛋白。这提供了第一个在调节RNA剪接中发挥作用的SnRNP蛋白的例子。具体目标是：1.确定SNF在U2 SnRNP中的存在是否对SXL剪接调控既是必要的，也是充分的。我们已经证实，SNF5MER等位基因编码一种突变蛋白，该蛋白优先整合在U1 SnRNP中，在体内不能在U2 SnRNP中检测到。我们对这一突变表型的描述与以下假设一致，即SNF的SXL剪接调控作用要求它是U2 SnRNP的组成部分。在这里，我们建议：a)继续我们对Snf5MER等位基因的生化特征，以确定其在SXL剪接调控中所必需的蛋白质的哪些性质。b)通过进行定向SNF突变，验证SNF在U2 SnRNP中的存在足以进行SXL剪接调控的假设，这些突变被预测为优先结合到U2 SnRNP中，并被排除在U1 SnRNP之外。如果U2SnRNP的掺入充足，那么我们预计这些新的突变型SNF等位基因在SXL剪接调控中不会有缺陷。2.确定SXL剪接调控所需的SNF蛋白结构域。我们已经证实，C-末端的RRM虽然对SNF+功能不是必需的，但可以补偿N-末端的突变。然而，初步研究表明，仅有N端RRM是不足以发挥SNF+功能的。在这里，我们建议：通过使表达最小N末端序列的转基因动物足以满足SNF的重要功能及其性别决定功能来识别蛋白质的关键功能域。作为这一特征的一部分，我们将需要产生识别蛋白质N端的抗体，并使用这些抗体来确定新的SNF突变蛋白是否被整合到U1和/或U2SnRNPs中。3.鉴定和分析参与SXL剪接自动调节的其他基因产物。为了继续阐明调控SXL剪接的机制，我们建议鉴定与SNF一起调控SXL剪接的其他关键基因产物。为此：a)我们使用了剂量敏感的修饰剂分析来调查已知的剪接因子，并确定U2AF35和HEL(UAP56同源物)可能参与SXL剪接调控。在哺乳动物细胞中，这两种基因产物都是U2SnRNP募集到Pre-mRNA所必需的，这表明遗传相互作用是由于SXL剪接的直接影响。在这里，我们建议通过确定这些蛋白中的一个或两个是否作为包含SXL的复合体的成员直接或间接与SNF相互作用来检验这一假设。B)我们正在使用酵母双杂交系统来鉴定与SNF相互作用的蛋白质。我们将通过生化分析和遗传相互作用测试来确认这些相互作用在体内的相关性。本奖项中描述的利用果蝇遗传学的力量从本奖项的实验中获得的研究将为了解这种保守的SnRNP蛋白在体内的功能提供关键信息。信息战线。首先，我们将增加我们对通用剪接因子(SNF)和组织特异性剪接因子(SXL)如何一起控制RNA剪接的理解。其次，我们将有助于我们全面了解剪接体的不同蛋白质成分如何有助于剪接的准确性。鉴于RNA剪接机制各组成部分的进化保守性，我们从研究中获得的信息将直接适用于哺乳动物系统，并将是我们对受调控的前mRNA剪接的理解的重大进步。

The long term goal of this work is to understand the molecular mechanisms that regulate alternative RNA splicing in a multicellular organism. The question of how splicing is regulated is particularly important because alternative splicing has proven to be a common means of regulating gene expression. Here the sex-specific splicing regulation of the Drosophila sex determination gene Sex-lethal (Sxl) is used as a model system. In this award, the focus is on the role of the sans-fille (snf) gene in promoting female-specific splicing of the Sxl pre-mRNA. Genetic studies have established that the non-sex specific snf gene product functions with the female-specific SXL RNA binding protein to direct splicing of the Sxl pre-mRNA. Remarkably, cloning of the snf gene identified it as the homolog of two well studied human spliceosomal proteins, UlA and U2B". Like UlA, SNF is a Ul snRNP protein. And, like U2B", SNF is a U2 snRNP protein.s. This provides the first example of an snRNP protein that plays a role in regulating RNA splicing. The specific aims are: 1. Determine if SNF's presence in the U2 snRNP is both necessary & sufficient for Sxl splicing regulation. We have established that the snf5MER allele, encodes a mutant protein that preferentially incorporates in the Ul snRNP and is not detected in the U2 snRNP in vivo. Our characterization of this mutant phenotype is consistent with the hypothesis that SNF's role Sxl splicing regulation requires that it be a component of the U2 snRNP. Here we propose to: a) Continue our biochemical characterization of the snf5MER allele to establish which properties of the protein are necessary for its function in Sxl splicing regulation.b) Test the hypothesis that SNF's presence in the U2 snRNP is sufficient for Sxl splicing regulation by making directed snf mutations that are predicted to preferentially incorporates into the U2 snRNP's and be left out of U1 snRNPs. If U2 snRNP incorporation is sufficient then we expect that these new mutant snf alleles will not be defective in Sxl splicing regulation. 2. Identify the protein domains of SNF necessary for Sxl splicing regulation. We have established that the C-terminal RRM, while not essential for snf+ function can compensate for mutations in the N-termin`us. Preliminary studies, however, suggest that the N-terminal RRM alone is not sufficient for snf+ function. Here we propose to: Identify critical functional domains of the protein by making transgenic animals that express the minimum N-terminal sequences sufficient for both snf's vital function and its sex determination function. As part of this characterization, we will need to generate antibodies that recognize the N-terminal end of the protein and use these antibodies to determine whether the new SNF mutant proteins are incorporated into Ul and/or U2 snRNPs. 3. Identify and analyze other gene products that function in Sxl splicing autoregulation. To continue to elucidate the mechanism by which Sxl splicing is regulated, we propose to identify other critical gene products that function with SNF in regulating Sxl splicing. To accomplish this: a) we have used a dosage-sensitive modifier assay to survey known splicing factors and we have identified U2AF35 and hel (the UAP56 homolog) as likely players in Sxl splicing regulation. In mammalian cells, both of these gene products are required for the recruitment of the U2 snRNP to the pre-mRNA, suggesting that the genetic interaction is due to a direct effect on Sxl splicing. Here we propose to test this hypothesis by determining if either or both these proteins interact with SNF, either directly or indirectly, as a member of the SXL containing complex. b) we are using the yeast two-hybrid system to identify proteins that interact with SNF. We will confirm the in vivo relevance of these interactions by both biochemical assays and genetic interaction tests.The studies described in this award exploit the power of Drosophila genetics to gained from the experiments in this award will provide critical information on two understand the function of this conserved snRNP protein in vivo. The information fronts. First, we will add to our understanding of how a general splicing factor (SNF) and a tissue-specific splicing factor (SXL) function together to control RNA splicing. Second, we will contribute to our overall understanding of how the different protein components of the spliceosome contribute to the accuracy of splicing. Given the evolutionary conservation of the components of the RNA splicing machinery, the information gained from our studies will be directy applicable to mammalian systems and will be a significant advance in our understanding of regulated pre-mRNA splicing.