GENERAL PRE-MRNA SPLICING FACTORS IN DROSOPHILA

这项建议的目标是了解这些机制 参与保守的精氨酸-丝氨酸的功能(R/S) 含有前mRNA剪接因子U2的SnRNP辅助结构域 果蝇体内的因子(U2AF)。这 该提案充分利用了果蝇作为一种 实验生物体，并允许多学科方法来 生物问题。使用遗传学、分子生物学的组合 生物学和生物化学，我们将通过以下方式阐明其机制 哪些R/S结构域在前信使核糖核酸剪接中起作用。R/S域 已在许多哺乳动物和果蝇中发现 调控剪接因子的体内实验分析 它们在生理水平上的功能还没有被执行。 我们已经分离出了果蝇U2AF的编码基因 同源物，表明重组dU2AF50具有所有的RNA HU2AF65的结合和剪接活性并表达了 DU2AF38蛋白在大肠杆菌中的表达。细胞遗传学和生殖系 在我们实验室进行的转化实验表明， 先前发现的致命性突变位于基因的14c1-2位 X染色体在一个未特化的互补组中称为 9-21对应于大的U2AF亚基dU2AF50。我们还有 结果表明，dU2AF38小亚基基因位于 第二条染色体，并发现了致命的dU2AF38突变。 细胞遗传学实验已将dU2AF38基因定位到 在第二条染色体上定位21B-C，我们现在已经分离到 DU2AF38小亚基基因的致死性突变 基因是必不可少的。这些观察结果提供了依据 一种必需的前信使核糖核酸突变形式的功能分析 转基因后生动物体内的剪接因子。已经在那里了 现有许多U2AF功能的生化检测方法，因此 任何体内表型的突变型U2AF衍生物都可以直接 与体外生化活动的改变有关。在.期间 这一资金期是为了实现这个目标 建议，我们将1)进行野生型和 体内突变型U2AF大小亚基衍生物；2) 确定体内和体内U2AF异二聚体相互作用结构域 体外培养；3)利用裂殖酵母及其序列信息 突变的PRP-2等位基因产生温度敏感的dU2AF50 突变体及其在果蝇中的功能检测；4)检测野生型 和突变的U2AF异源二聚体，用于剪接活性和剪接 5)核糖核酸的结构研究。 性致死蛋白与dU2AF50的结合域及其相互作用 DU2AF异源二聚体的结构域。很明显，许多人类 癌基因通过交替的Pre-mRNA进行差异表达 拼接。这些癌基因蛋白亚型可能具有不同的 在不同的组织中发挥作用。因为许多人类疾病， 如β-地中海贫血，是由前-信使核糖核酸缺陷引起的 剪接，替代剪接缺陷很可能会导致 某些体细胞类型的致癌转化。

The goal of this proposal is to understand the mechanisms involved in the function of the conserved arginine-serine (R/S) domain containing pre-mRNA splicing factor U2 snRNP auxiliary factor (U2AF) in the fruit fly, Drosophila melanogaster. This proposal capitalizes on the strengths of Drosophila as an experimental organism and allows a multidisciplinary approach to biological problems. Using a combination of genetics, molecular biology, and biochemistry, we will elucidate the mechanisms by which R/S domains function in pre-mRNA splicing. R/S domains have been found in many mammalian and Drosophila general and regulatory splicing factors but in vivo experiments analyzing their function at physiological levels have not been performed. We have isolated the genes encoding both Drosophila U2AF homologs, have shown that recombinant dU2AF50 has all of the RNA binding and splicing activities of hU2AF65 and have expressed the dU2AF38 in protein in E. coli. Cytogenetic and germline transformation experiments carried out in our lab have shown that previously identified lethal mutations at position 14C1-2 on the X chromosome in an uncharacterized complementation group called 9-21 correspond to the large U2AF subunit, dU2AF50. We have also shown that the dU2AF38 small subunit gene is located on the second chromosome and have identified lethal dU2AF38 mutations. Cytogenetic experiments have localized the dU2AF38 gene to position 21B-C on the second chromosome and we have now isolated lethal mutations in the dU2AF38 small subunit gene demonstrating the gene is essential. These observations provide the basis assaying the function of mutant forms of an essential pre-mRNA splicing factor in a transgenic metazoan organism. There already exist a number of biochemical assays for U2AF function and thus any in vivo phenotype of mutant U2AF derivatives can be directly correlated with altered biochemical activities in vitro. During this funding period in order to accomplish the goals of this proposal, we will 1) Carry out genetic analysis of wild type and mutant U2AF large and small subunit derivatives in vivo; 2) Define the U2AF heterodimer interaction domains in vivo and in vitro; 3) Use Schizosaccharomyces pombe and sequence information for mutant prp-2 alleles to create temperature-sensitive dU2AF50 mutants and test their function in Drosophila; 4) Assay wild type and mutant U2AF heterodimers for splicing activity and splicing complex assembly in vitro; and 5) Structure studies on the RNA binding domain of Sex-lethal and dU2AF50 and the interaction domain of the dU2AF heterodimer. It is clear that many human oncogenes are expressed differentially by alternative pre-mRNA splicing. These oncogene protein isoforms may have different functions in different tissues. Because many human diseases, such as beta-thalassemia, results from defects in pre-mRNA splicing, it is likely that alternative splicing defects may lead to oncogenic transformation of certain somatic cell types.