Fibronectin, a large extracellular glycoprotein, is heterogeneous in structure. This heterogeneity is largely due to differences in the primary sequence of the protein arising from the alternative splicing of at least three exons (IIIB, IIIA, and V) during processing of a common primary transcript. Previous studies have shown that the splicing patterns of fibronectin mRNAs change during chondrogenesis. Specifically, all of the fibronectin mRNA present in prechondrogenic limb mesenchyme contains both exon IIIB and exon IIIA (B+A+), whereas all of the fibronectin mRNA in cartilage contains exon IIIB but does not contain exon IIIA (B+A-). Therefore, the splicing patterns of exon IIIA appear to be modulated during chondrogenesis. The goals of the experiments outlined in this proposal are to characterize the molecular signals (both cis- and trans- acting) necessary for regulating the tissue-specific splicing of exon IIIA of the chick fibronectin primary transcript during cartilage differentiation.
The first aim describes the preparation of genomic minigenes to be utilized in subsequent aims for examining the mechanism(s) regulating the alternative splicing of exon IIIA during chondrogenesis. Similar minigenes will be prepared for exon III-9 to be utilized as a control for constitutive splicing. Transfection of the minigenes into appropriate cells in culture will determine whether the minigenes contain all of the necessary cis-acting elements to result in faithful tissue-specific splicing of exon IIIA during chondrogenesis. These minigenes will then be added to HeLa cell nuclear extracts for in vitro splicing. Tissue-specific extracts from chick limb mesenchymal cells and vertebral chondrocytes will be utilized to substitute entirely for the HeLa extracts or to supplement the HeLa extracts in order to achieve tissue-specific in vitro splicing (Aim 2). The actual cis- acting elements necessary for the regulation of exon IIIA alternative splicing during chondrogenesis will be identified using chimeras between the exon IIIA and exon III-9 minigenes and various mutant minigenes in transfection assays and in the tissue-specific in vitro splicing assay (Aim 3). The addition of fractionated limb mesenchymal cells and mature chondrocytes to the tissue-specific in vitro splicing assays in combination with the use of RNA gel retardation assays, RNA-protein UV cross linking experiments and competition assays will identify tissue specific trans-acting factors necessary for the regulation of exon IIIA alternative splicing (Aim 4). Finally, the possibility that known general splicing factors are regulated during chondrogenesis and affect the regulation of exon IIIA alternative splicing will be examined by Northern hybridization analysis of RNAs isolated from limb mesenchymal cells and mature chondrocytes (Aim 5). The information resulting from these experiments will provide increased insight into the exon-skipping mechanism of alternative splicing as well as the signals necessary for alternative splicing events occurring during normal cartilage differentiation.
