SWAP AND SR PROTEIN REGULATION OF ALTERNAIVE SPLICING

许多基因的表达受选择性mRNA的调节 通过删除、增加或交换外显子进行拼接。 关于基因是如何被称为 剪接调节因子，在生理过程中调节剪接的变化 细胞活化和分化等过程。这项建议 描述了将检查所涉及的剪接调节因子的研究 在CD45、Fas和纤维连接蛋白的选择性剪接调控中， 与风湿性疾病有关的三个基因。 CD45选择性剪接模式的变化，这是 与记忆细胞表型的发展有关，有 已在系统性红斑狼疮(SLE)患者中发现。 一种可选择的拼接形式的FAS，可产生可溶的 抑制全长Fas功能的编码蛋白质的形式， 也与系统性红斑狼疮有关。改变了拼接 纤维连接蛋白IIICS区，它编码与 α4beta1整合素，已在血管内皮细胞中被描述 类风湿性关节炎患者。这样做的长期目标是 项目是定义定义剪接的细胞内因子 这些选择性剪接基因中的位点在两个生理过程中 以及病理状态。在这项提案中，主要是生理上的 这些基因的调节将通过确定 定义CD45和FAS的剪接位点选择的剪接调控因子 在T细胞的激活和成熟过程中，纤维连接蛋白 肝细胞IIICS区与成纤维细胞和内皮细胞的比较 细胞。两个哺乳动物剪接调节因子，掉期，这是 与果蝇白杏抑制子基因同源， 和SF2已被证明调节选择性剪接 CD45和纤维连接蛋白。SWAP和SF2均增强外显子 跳过CD45，但对纤维连接蛋白外显子有不同的影响 IIICS、SF2增强和互换抑制外显子包涵体。至 对拼接有更全面的了解 CD45、Fas和纤维连接蛋白的调节，实验研究 掉期、SF2和其他剪接调节剂对 提出了这些基因的选择性剪接。的影响 CD45、Fas和Fas上的交换、SR蛋白、PTB和hnRNP A1 纤维连接蛋白将通过共转染实验进行研究。至 在观察到的模式中进一步牵涉到调节基因 选择性剪接，剪接因子的mRNA水平 在主要表达一个或另一个的细胞中分析 另一种拼接形式。最后，为了更清楚地表明 剪接调节器负责观察到的调节， 将进行反义实验，选择性地抑制 剪接中涉及的每个剪接调控因子的表达 CD45、Fas或纤维连接蛋白。

Expression of many genes is regulated by alternative mRNA splicing through the deletion, addition, or exchange of exons. Relatively little is known regarding how genes, referred to as splicing regulators, mediate changes in splicing during physiologic processes such as cell activation and differentiation. This proposal describes studies that will examine the splicing regulators involved in alternative splicing regulation of CD45, fas and fibronectin, three genes that have been implicated in rheumatic diseases. Changes in the pattern of alternative splicing of CD45, which are associated with the development of a memory cell phenotype, have been found in patients with systemic lupus erythematosus (SLE). An alternatively spliced form of fas, which produces a soluble form of the encoded protein that inhibits full length fas function, has also been associated with SLE. Altered splicing of the fibronectin IIICS region, which encodes the binding site for the alpha4beta1 integrin, has been described in endothelial cells from patients with rheumatoid arthritis. The long range goals of this project are to define the intracellular factors that define the splice sites in these alternatively spliced genes during both physiologic and pathologic states. In this proposal primarily physiologic regulation of these genes will be analyzed by determining the splicing regulators that define splice site selection of CD45 and fas during T-cell activation and maturation, and of the fibronectin IIICS region in hepatocytes versus fibroblasts and endothelial cells. Two mammalian splicing regulators, SWAP, which is homologous to the Drosophila suppressor-of-white-apricot gene, and SF2 have been shown to regulate the alternative splicing of CD45 and fibronectin. Both SWAP and SF2 enhance exon skipping of CD45, but has a different effect on fibronectin exon IIICS, SF2 enhance and SWAP inhibits exon inclusion. To develop a more comprehensive understanding of the splicing regulation of CD45, fas, and fibronectin, experiments to study the effect of SWAP, SF2 and other splicing regulators of the alternative splicing of these genes are proposed. The effects of SWAP, SR proteins, PTB and hnRNP A1 on CD45, fas and fibronectin will be studied by contransfection experiments. To further implicate regulator genes in the observed patterns of alternative splicing, mRNA levels of splicing factors will be analyzed in cells expressing primarily one or the other of the alternatively spliced forms. Finally, to more clearly show that a splicing regulator is responsible for the observed regulation, antisense experiments will be carried out to selectively inhibit expression of each splicing regulator implicated in splicing of CD45, fas or fibronectin.