The epithelial-mesenchymal transition (EMT), a prerequisite for cancer progression and metastasis formation, is regulated not only at the transcriptional but also at the post-transcriptional level, including at the level of alternative pre-mRNA splicing. Several recent studies have highlighted the involvement of splicing factors, including epithelial splicing regulatory proteins (Esrps) and RNA-binding Fox protein 2 (Rbfox2), in this process. Esrps regulate epithelial-specific splicing, and their expression is downregulated during EMT. By contrast, the role of Rbfox2 is controversial because Rbfox2 regulates epithelial as well as mesenchymal splicing events. Here, we have used several established cell culture models to investigate the functions of Rbfox2 during EMT. We demonstrate that induction of an EMT upregulates the expression of Rbfox2, which correlates with an increase in Rbfox2-regulated splicing events in the cortactin (Cttn), Pard3 and dynamin 2 (Dnm2) transcripts. At the same time, however, the epithelial-specific ability to splice the Enah, Slk and Tsc2 transcripts is either reduced or lost completely by Rbfox2, which might be due, in part, to downregulation of the expression of the Esrps cooperative factors. Depletion of Rbfox2 during EMT did not prevent the activation of transforming growth factor-β signaling, the upregulation of mesenchymal markers or changes in cell morphology toward a mesenchymal phenotype. In addition, this depletion did not influence cell migration. However, depletion of Rbfox2 in cells that have completed an EMT significantly reduced their invasive potential. Taken together, our results suggest that during an EMT, Rbfox2-regulated splicing shifts from epithelial-to mesenchymal-specific events, leading to a higher degree of tissue invasiveness.

The Epstein-Barr virus-encoded protein BZLF1 is a member of the basic leucine zipper (bZip) Epstein-Barr virus is a human herpesvirus that infects B lymphocytes and epithelial cells of the nasopharynx (1). Infection of B cells typically results in a latent state, whereas infection of epithelial cells leads to viral replication and cytolysis (2). The switch from latency to the productive (replicative) cycle is induced by various agents such as calcium ionophores, sodium butyrate, and tumor promoters like phorbol 12-myristate 13-acetate, all of which activate expression of the Epstein-Barr virus immediate-early gene product BZLF1 (2). Overexpression of BZLF1 is sufficient to trigger the switch to the lytic cycle (2). Like the c-Jun/c-Fos proteins BZLF1 is a member of the basic leucine zipper (bZIP) transcription factor family (3, 4). BZLF1 binds as a homodimer to DNA (4) and transactivates several early viral promoters required in the cytolytic cycle of the virus. The BZLF1 response elements (ZREs) in these promoters are similar and sometimes identical to AP-1 binding sites (4).Retinoids play an important role in development and differentiation and are well-known inhibitors of cell growth (for review, see ref. 5). Retinoic acid (RA) exerts biological effects by acting through at least two distinct classes of intracellular proteins including the RA receptors (RARs) and the retinoid X receptors (RXRs), both of which are members of the nuclear receptor superfamily (5). While both the RARs and RXRs are effective activators of some genes, RA is also known to repress expression of AP-1-dependent genes (6). RAR has beenThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.shown to inhibit transcriptional activity of the AP-1 complex by a mechanism that is not dependent on the receptor's ability to bind DNA and most likely reflects protein-protein interactions (6). This interaction of distinct regulatory pathways, termed "cross-talk," suggested a means by which nuclear hormone receptors could modulate the action of growthinducing factors. Recent data suggested that RARa is able to repress the BZLF1-mediated transcriptional activation of the BMFR1 promoter via direct protein-protein interactions between RARa and BZLF1 (7).To further extend our understanding of the cross-talk between RAR and members of the bZIP transcription factor family, we were interested in examining the mutual repression effects of RARa and BZLF1 in more detail. In this report, we show that RARa and BZLF1 repress each other's transcriptional activity. Transfection analysis of RARa and BZLF1 mutants reveals that the transactivation domain and the dimerization domain of the BZLF1 and the DNA binding domain (DBD) of RARa are required for repression. Glutathione S-transferase (GST)-pulldown assays and coimmunoprecipitation experiments demonstrate direct protein-protein interactions between BZ...

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