Regulation of Rac1 alternative splicing and cellular transformation

FEDERICO PELISCH; MATÍAS BLAUSTEIN; LEANDRO QUADRANA; GUILLERMO RISSO; DEREK RADISKY; ANABELLA SREBROW

Bariloche, Argentina

Congreso; Combined Meetings: "Gene Expression and RNA Processing" (ICGEB) and "Cell Biology, Signaling and Alternative Splicing" (EURASNET); 2007

ICGEB y EURASNET

Rac1 is a member of the Ras superfamily of small GTPases. When bound to GTP, Rac1 activates signaling pathways that lead to alterations in cytoskeletal organization, cell proliferation and mitosis. Rac1b is an alternatively spliced variant of Rac1 that contains a 57-nt insertion (exon 3b) leading to an in frame addition of 19 aa into the Rac1 sequence. Rac1b functions as a constitutively active isoform, is found in malignant breast and colorectal tumors and promotes cellular transformation in culture. So far, the mechanism controlling its induction has not been identified. Stromelysin-1/matrix metalloproteinase-3 (MMP-3), a stromal enzyme up-regulated in many breast tumors, 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 proposed to investigate the cis-acting elements and trans-acting factors responsible for the regulation of Rac1 splicing in normal vs. cancer cells and, in particular, by MMP-3. RNA affinity chromatography followed by mass spectrometry revealed that heterogeneous nuclear ribonucleoproteins (hnRNPs) A1 and A2 interact with Rac1 exon 3b. This finding was confirmed by UV crosslinking and RNA mobility shift assays. Preliminary results indicate that at least hnRNP A1 represses exon inclusion. We are looking for changes in RNA-binding ability, expression leveles, sub-cellular localization, and/or post-translational modifications of the identified splicing regulators. Our goal is to unravel the mechanism by which Rac1 splicing is regulated and ultimately, its impact on cell transformation.