Regulation of alternative splicing by the cellular microenvironment

ANABELLA SREBROW; FEDERICO PELISCH; LEANDRO QUADRANA; MATÍAS BLAUSTEIN

Buenos Aires, Argentina

Congreso; VIII Jornadas Multidisciplinarias de la Sociedad Argentina de Biología – SAB; 2006

Alternative splicing is considered the most important source of protein diversity in vertebrates and its regulation by extracellular cues is a key event in the control of gene expression. We use mouse mammary cell lines and two different genes, fibronectin and Rac1, to study the linkage between the cellular microenvironment and the splicing machinery. We found that a laminin-rich basement membrane down-regulates the inclusion of two fibronectin alternative regions, EDA and IIICS, through a JNK-dependent pathway in epithelial cells. Using a cell culture system that simulates mammary epithelial-stromal communication we found that soluble factors from a mammary mesenchymal cell-conditioned medium as well as different growth factors up-regulate the inclusion of these two alternative regions via PI 3-kinase. The laminin-rich basement membrane blocks the effect of the mammary mesenchymal cell-conditioned medium, extending the already proposed antagonism between these two pathways to the field of alternative splicing. These results highlight the fact that the splicing pattern of a single transcript is the read out of an intricate network of different signaling cascades, strengthening the view that the control of alternative splicing is as complex and relevant as transcriptional control, together accounting for the spatiotemporal requirements of gene expression. We also found that the signaling pathway triggered by soluble factors not only affect mRNA splicing but also alter translation of reporter mRNAs containing a fibronectin EDA exonic splicing enhancer. These effects on splicing and translation are dependent on SR proteins and can be duplicated by over-expressing a constitutively active Akt, a downstream target of PI 3-kinase. These results show how SR protein activity is modified in response to extracellular cues leading to a concerted regulation of splicing and translation. Expression of Rac1b, a splicing isoform of Rac1, is induced by exposure of normal mouse mammary epithelial cells to the matrix metalloprotease MMP-3, and Rac1b activity is required for MMP-3-triggered epithelial to mesenchymal transition, a phenomenon characterized by the loss of intact E-cadherin, increased motility and invasiveness, down-modulation of epithelial markers and up-regulation of mesenchymal ones. Rac1b is generated by inclusion of a 57-nucleotide exon, accumulates in tumors and shows transforming properties when over-expressed in cultured cells. We are currently investigating the cis-acting elements and trans-acting factors involved in the regulation of Rac1 alternative splicing by MMP-3.