hnRNP A1 está involucrada en la regulación del splicing alternativo de Rac1 en células epiteliales mamarias

FEDERICO PELISCH; GUILLERMO RISSO; DEREK RADISKY; ANABELLA SREBROW

Córdoba, Argentina

Congreso; Reunión Anual de la Sociedad Argentina de Investigaciones Bioquímicas; 2008

SAIB

Rac1 is a Rho-GTPase involved in cell division, survival, motility and adhesion. Inclusion of an alternative exon “3b” gives rise to Rac1b splice variant, which is constitutively active, is found in breast and colorectal tumors and promotes cellular transformation. The mechanism controlling its induction has not been elucidated. Matrix metalloproteinase-3 (MMP-3) causes 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 cis-acting elements and trans-acting factors responsible for the regulation of Rac1 splicing by MMP-3 and to define how its regulation impacts on cellular transformation. RNA affinity chromatography/mass spec revealed that hnRNP A1 and A2, two splicing inhibitory factors, 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. UV-crosslinking/immunoprecipitation suggest that MMP-3 treatment decreases the amount of hnRNP A1 bound to exon 3b. Knocking down hnRNP A1 by siRNA is not enough to induce 3b inclusion but potentiates MMP-3 stimulatory effect. We are mapping hnRNP A1 binding site within exon 3b and trying to unravel other factor/s involved in Rac1 splicing regulation.