GENERAL PRE-MRNA SPLICING FACTORS IN DROSOPHILA

本提案的目的是了解 参与保守的丝氨酸-丝氨酸（R/S）的功能 含结构域的前mRNA剪接因子U2 snRNP辅助 黑腹果蝇（Drosophila melanogaster）中的U2 AF因子。 这 该提案利用了果蝇作为一种 实验生物，并允许多学科的方法， 生物问题。 利用遗传学分子生物学 生物学和生物化学，我们将阐明的机制， 其中R/S结构域在前体mRNA剪接中起作用。 R/S域 在许多哺乳动物和果蝇中发现， 调节剪接因子，但体内实验分析 它们在生理水平上的功能尚未发挥。 我们已经分离出编码果蝇U2 AF和 同源物，已经表明重组dU 2AF 50具有所有的RNA hU 2AF 65的结合和剪接活性，并表达了 dU_2AF_38蛋白在大肠杆菌中的表达。杆菌 细胞遗传学和生殖系 我们实验室进行的转化实验表明， 先前鉴定的在14 C1 -2位的致命突变， X染色体在一个未表征的互补组称为 9-21对应于U2 AF大亚基dU 2AF 50。 我们还 表明dU 2AF 38小亚基基因位于 第二染色体，并已确定致命的dU 2AF 38突变。 细胞遗传学实验已经将dU 2AF 38基因定位于 第二条染色体上的位置21 B-C，我们现在已经分离出 dU 2AF 38小亚基基因中的致命突变表明， 基因是必不可少的。 这些观察提供了基础 测定必需前mRNA突变形式的功能 剪接因子在转基因后生动物有机体中的作用。 已经到那里了 存在许多用于U2 AF功能的生物化学测定 突变U2 AF衍生物的任何体内表型可以直接 与体外生物化学活动的改变有关。 期间 在这段时间里，为了实现这一目标， 建议，我们将1）进行野生型的遗传分析， 体内突变体U2 AF大亚基和小亚基衍生物; 2） 定义U2 AF异二聚体相互作用结构域在体内和体内 3）使用粟酒裂殖酵母和序列信息 突变的prp-2等位基因产生温度敏感的dU 2AF 50 突变体并在果蝇中测试其功能; 4）测定野生型 和突变U2 AF异二聚体的剪接活性和剪接 复合物体外组装; 5）RNA的结构研究 性致死与dU 2AF 50的结合域及其相互作用 dU 2AF异二聚体的结构域。 很明显，许多人 癌基因通过替代性前体mRNA差异表达 拼接 这些癌基因蛋白质异构体可能具有不同的 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，是由前体mRNA缺陷引起的 剪接，很可能是选择性剪接缺陷可能导致 到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.