Regulation of Rac1 alternative splicing in mammary epithelial cells

FEDERICO PELISCH; GUILLERMO RISSO; DEREK RADISKY; ANABELLA SREBROW

Cracovia, Polonia

Conferencia; First International EURASNET (European Alternative Splicing Network) Meeting on Alternative Splicing; 2008

EURASNET

Rac1 is a Rho-GTPase involved in cell division, survival, motility and adhesion. Inclusion of an alternative exon “3b” gives rise to the splice variant Rac1b. This isoform is constitutively active, is found in breast and colorectal tumors and promotes cellular transformation. So far, the mechanism controlling its induction has not been elucidated. Matrix metalloproteinase-3 (MMP-3) was found to cause epithelial-mesenchymal transition (EMT) and malignant transformation in cultured cells. Exposure of mammary epithelial cells to MMP-3 induces Rac1b expression, and siRNA-mediated knockdown of this isoform prevents MMP-3-induced EMT. We proposed to investigate the cis-acting elements and trans-acting factors responsible for the regulation of Rac1 splicing in normal vs. cancer cells, and by MMP-3. RNA affinity chromatography/mass spec revealed that hnRNP A1 and A2 interact with Rac1 exon 3b. RNA mobility shift assays indicated that less protein complex is assembled on 3b RNA when cells are treated with MMP-3. Preliminary results indicate that over-expression of hnRNP A1 sligltly diminishes the MMP-3 effect, knocking down of hnRNP A1 by RNAi is not enough to induce 3b exon inclusion but it potentiates the stimulatory effect of MMP-3. We are currently trying to unravel other factor/s involved as well as other extracellular cues that may regulate Rac1 splicing, an ultimately, how its regulation impacts on cellular transformation.