The response of cells to environmental signals, as well as their differentiation, death or malignant transformation, involves changes in gene expression. For many years, the modulation of gene expression was thought to be restricted to, or at least dominated by, the control of transcription. According to the Jacob-Monod-Lwoff paradigm, established in bacteria and logically extended to eukaryotes, what made a cell a cell was the combinatorial turning on and off of genes. The discovery of introns and the process of splicing, which eliminates them from the precursor mRNA, introduced an exciting but also perturbing factor in our simplified conceptions of the flow of gene expression in eukaryotes. Introns must be excised precisely to generate bona fide mRNA molecules; otherwise, translational frame shifts would be introduced. Introns are much longer than exons and most of their nucleotide sequence seems to be irrelevant except for the 'donor' and 'acceptor' consensus sequences located at their extremities, and the branching sequence that precedes the acceptor sequence. The spliceosome, a sophisticated nuclear machine comprising five types of small nuclear ribonucleoprotein (snRNP) and hundreds of auxiliary proteins, takes care of intron excision and exon joining repeatedly at each and every one of the seven splicing events that occur on average per human pre-mRNA molecule (Lander et al., 2001).'What is all this for?', a teleologist might ask. We know now that the high adaptive value of introns resides in the fact that they permitted exon shuffling in the past and alternative splicing in the present (Sharp, 1994). However, the apparent 'irrationality of introns' (I. Eperon, http://www.eurasnet.info/ ian_eperon.shtml) was questioned early on following the discovery that mutations that affect splicing, quantitatively or qualitatively, are a widespread source of hereditary diseases. No matter how absurd or energy consuming a biological process appears to be, if its disruption or perturbation causes disease, it must be important, and its conservation in evolution is paramount. The links between splicing and human disease have been extensively reviewed lately (Caceres and Kornblihtt, 2002;Cartegni et al., 2002;Faustino and Cooper, 2003;Pagani and Baralle, 2004). The specific association between splicing and cancer has received less attention (Brinkman, 2004;Venables, 2006), perhaps because the field is still emerging. Here we examine this connection, focusing not only on mutations in cis-acting splicing sequences that are associated with cancer but also on the variations in normal splicing processes and the signals that may affect them in cancer cells. The basic language of splicingSplice sites, branch sites, splicing enhancers and splicing silencers Splicing occurs when a group of proteins and RNPs recognize specific RNA sequences conserved at the boundaries of introns (Fig. 1A). The classical spliceosome, which acts on >99% of introns from genes transcribed by RNA polymerase II (Pol II), recognizes a 5Ј donor splice site...

Serine/arginine-rich (SR) proteins are important regulators of mRNA splicing. Several postsplicing activities have been described for a subset of shuttling SR proteins, including regulation of mRNA export and translation. Using the fibronectin gene to study the links between signal-transduction pathways and SR protein activity, we show that growth factors not only modify the alternative splicing pattern of the fibronectin gene but also alter translation of reporter messenger RNAs in an SR protein-dependent fashion, providing two coregulated levels of isoform-specific amplification. These effects are inhibited by specific small interfering RNAs against SR proteins and are mediated by the AKT kinase, which elicits opposite effects to those evoked by overexpressing SR protein kinases Clk and SRPK. These results show how SR protein activity is modified in response to extracellular stimulation, leading to a concerted regulation of splicing and translation.

Dengue virus NS5 protein plays multiple functions in the cytoplasm of infected cells, enabling viral RNA replication and counteracting host antiviral responses. Here, we demonstrate a novel function of NS5 in the nucleus where it interferes with cellular splicing. Using global proteomic analysis of infected cells together with functional studies, we found that NS5 binds spliceosome complexes and modulates endogenous splicing as well as minigene-derived alternative splicing patterns. In particular, we show that NS5 alone, or in the context of viral infection, interacts with core components of the U5 snRNP particle, CD2BP2 and DDX23, alters the inclusion/exclusion ratio of alternative splicing events, and changes mRNA isoform abundance of known antiviral factors. Interestingly, a genome wide transcriptome analysis, using recently developed bioinformatics tools, revealed an increase of intron retention upon dengue virus infection, and viral replication was improved by silencing specific U5 components. Different mechanistic studies indicate that binding of NS5 to the spliceosome reduces the efficiency of pre-mRNA processing, independently of NS5 enzymatic activities. We propose that NS5 binding to U5 snRNP proteins hijacks the splicing machinery resulting in a less restrictive environment for viral replication.

A dynamic and reciprocal flow of information between cells and the extracellular matrix contributes significantly to the regulation of form and function in developing systems. Signals generated by the extracellular matrix do not act in isolation. Instead, they are processed within the context of global signalling hierarchies whose constituent inputs and outputs are constantly modulated by all the factors present in the cell's surrounding microenvironment. This is particularly evident in the mammary gland, where the construction and subsequent destruction of such a hierarchy regulates changes in tissue-specific gene expression, morphogenesis and apoptosis during each developmental cycle of pregnancy, lactation and involution.

Angiogenesis is characterized by distinct phenotypic changes in vascular endothelial cells (EC). Evidence is provided that the Hox D3 homeobox gene mediates conversion of endothelium from the resting to the angiogenic/invasive state. Stimulation of EC with basic fibroblast growth factor (bFGF) resulted in increased expression of Hox D3, integrin αvβ3, and the urokinase plasminogen activator (uPA). Hox D3 antisense blocked the ability of bFGF to induce uPA and integrin αvβ3 expression, yet had no effect on EC cell proliferation or bFGF-mediated cyclin D1 expression. Expression of Hox D3, in the absence of bFGF, resulted in enhanced expression of integrin αvβ3 and uPA. In fact, sustained expression of Hox D3 in vivo on the chick chorioallantoic membrane retained EC in this invasive state and prevented vessel maturation leading to vascular malformations and endotheliomas. Therefore, Hox D3 regulates EC gene expression associated with the invasive stage of angiogenesis.

The fibronectin (FN) gene has become paradigmatic to illustrate genome evolution by exon shuffling, generation of protein diversity by alternative mRNA splicing, and topological coordination between transcription and splicing. Alternative splicing in three sites of the primary transcript gives rise to multiple FN polypeptides. This process is cell type-, development- and age-regulated. The different FN variants seem to play specific roles in FN dimer secretion, blood clotting, adhesion to lymphoid cells, skin wound healing, atherosclerosis, and liver fibrosis. This review focuses on function assignment to the alternatively spliced segments, as well as on the external signals and cis-acting sequences that control the mechanisms of alternative splicing. We also discuss FN transcriptional regulation in response to viral transformation, growth factors, and cyclic AMP in the light of promoter architecture and its interaction with specific transcription factors. The relevance of FN RNA "tracks" as assembly lines of coordinated transcription and RNA processing is also addressed.

Stromelysin-1 is a member of the metalloproteinase family of extracellular matrix-degrading enzymes that regulates tissue remodeling. We previously established a transgenic mouse model in which rat stromelysin-1 targeted to the mammary gland augmented expression of endogenous stromelysin-1, disrupted functional differentiation, and induced mammary tumors. A cell line generated from an adenocarcinoma in one of these animals and a previously described mammary tumor cell line generated in culture readily invaded both a reconstituted basement membrane and type I collagen gels, whereas a nonmalignant, functionally normal epithelial cell line did not. The matrix metalloproteinases (MMPs) 1 are a family of extracellular matrix (ECM)-degrading enzymes that have been implicated in a variety of normal developmental and pathological processes, including tumorigenesis (1, 2). The MMP family comprises at least 15 members with different, albeit overlapping, substrate specificities. During activation of latent MMPs, their propeptides are cleaved and they are converted to a lower molecular weight form by other enzymes, including serine proteinases, and by autocatalytic cleavage.Among the MMPs, stromelysin-1 (SL1) possesses the broadest substrate specificity (1). Despite increasing knowledge about its enzymatic properties and the regulation of its expression, little is known about its function. We have generated transgenic animals that express an autoactivating mutant of rat SL1 targeted to the epithelial compartment of the mammary gland (3). Phenotypically, SL1 transgenic mice display increased branching morphogenesis and lactogenic differentiation at prepubertal stages and premature involution during late pregnancy (3, 4). Branching morphogenesis requires the invasion of epithelial cells into the adipose tissue, a process reminiscent of invasion of stromal compartments by tumor cells. Strikingly, a large number of SL1 transgenic animals also develop mammary tumors of various histotypes, including invasive adenocarcinomas (5). 2 Because tumor development is a late response of SL1 transgenic mice to overexpression of the transgene, it remains unclear whether SL1 plays a direct role in tumor growth and/or invasion or whether the observed tumors are a consequence of other molecular alterations in the microenvironment of the mammary gland before the onset of tumor growth. 3 Studies performed with synthetic inhibitors of MMP activity and tissue inhibitors of metalloproteinases (TIMPs) have shown that suppression of MMP activity also suppresses tumor growth and metastasis (6, 7). In many cases, the level of SL1 expression in tumors of the mammary gland and other tissues is positively correlated with the degree of malignancy (8, 9). However, the only direct evidence for the nature of the MMPs involved was provided by the demonstration that function-blocking antibodies against gelatinase A and antisense inhibition of matrilysin expression decreased the invasiveness of tumor cells in a reconstituted basement membrane assay (10, 11). The...

Here we investigate the promoter control of alternative splicing by studying two transcriptional activators on templates under replicating conditions. SV40 large T-antigen (T-Ag) activates template replication only 2-fold but transcription 25-fold. T-Ag-mediated replication, reported to inhibit RNA polymerase II elongation, provokes a 10- to 30-fold increase in the inclusion of the fibronectin EDI exon into mature mRNA. The T-Ag effect is exon specific, occurs in cis and depends strictly on DNA replication and not on cell transformation. VP16, an activator of transcriptional initiation and elongation, has a similar effect on transcription but the opposite effect on splicing: EDI inclusion is inhibited by 35-fold. VP16 completely reverts the T-Ag effect, but a VP16 mutant with reduced elongation ability provokes only partial reversion. Both T-Ag and VP16 promote conspicuous co-localization of mRNA with nuclear speckles that contain the SR protein SF2/ASF, a positive regulator of EDI inclusion. Therefore, we conclude that co-localization of transcripts and speckles is not sufficient to stimulate EDI inclusion.