CONICET | Buscador de Institutos y Recursos Humanos

Rac1 is a member of the Ras superfamily of small GTPases. When activated, the Rac1 GTPase stimulates alterations in the organization of the cytoskeleton, cell proliferation and mitosis, and the activation of many signal pathways. Rac1b, an alternatively spliced variant of Rac1 containing a 57-nt insertion (exon 3b), is a constitutively active isoform and was identified in malignant breast and colorectal tumors and shown to promote cellular transformation in culture, although no mechanism for controlling its induction had been identified. Stromelysin-1/matrix metalloproteinase-3 (MMP-3), a stromal enzyme upregulated in many breast tumours, was found to cause epithelial-mesenchymal transition (EMT) and malignant transformation in cultured cells. Exposure of normal mouse mammary epithelial cells to MMP-3 induces the expression of Rac1b, and siRNA-mediated knockdown of this isoform prevents MMP-3-induced EMT. We decided to elucidate the cis-acting elements and trans-acting factors responsible for the regulation of Rac1 splicing by MMP-3. We investigated the role of several SR and hnRNP proteins by over-expression and siRNA strategies. To gain insight into the sequences regulating exon 3b splicing we constructed a Rac1b minigene under the control of the CMV promoter. This enabled us to perform specific mutations to eliminate putative exonic splicing enhancers predicted by the ESE-finder within exon 3b. Both SRp40- and SF2/ASF-dependent enhancers are predicted by the software and we found that SRp40 over-expression drastically inhibits the inclusion of this exon. We are also analyzing the involvement of different signaling cascades on Rac1 splicing. Our goal is to unravel the mechanism by which the MMP-3-mediated signaling impacts on the splicing machinery to regulate Rac1 splicing and ultimately, cell transformation.