REGULATION OF RAC1 ALTERNATIVE SPLICING

PELISCH, F.; BLAUSTEIN, M.; QUADRANA, L.; RADISKY, D.; SREBROW, A.

THE QUEEN'S COLLEGE, Oxford, United Kingdom

Conferencia; Gordon Research Conference:The Biology of Post-Trancriptional Gene Regulation; 2006

Gordon Research Conferences

REGULATION OF RAC1 ALTERNATIVE SPLICING Federico Pelisch1, Matias Blaustein1, Leandro Quadrana1, Derek Radisky2 and Anabella Srebrow1. 1 Laboratorio de Fisiología y Biología Molecular, IFIBYNE-CONICET, FCEyN-UBA, Buenos Aires, Argentina. 2 Mayo Clinic Cancer Center, Jacksonville, FL, USA. Rac1 is a member of the Ras superfamily of small GTPases involved in the formation of lamellipodia, cell proliferation and activation of the JNK/SAPK cascade. Rac1b, an alternatively spliced variant of Rac1, containing a 57-nucleotide insertion (exon 3b), was identified in malignant colorectal tumors and shown to promote cellular transformation in culture. 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 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 3b exon. Both SRp40- and SF2/ASF-dependent enhancers were 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.