SWAP AND SR PROTEIN REGULATION OF ALTERNAIVE SPLICING

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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.