Alternative splicing of pre-mRNA increases the diversity of protein functions. Here we show that about half of all active alternative splicing events in ovarian and breast tissues are changed in tumors, and many seem to be regulated by a single factor; sequence analysis revealed binding sites for the RNA binding protein FOX2 downstream of one-third of the exons skipped in cancer. High-resolution analysis of FOX2 binding sites defined the precise positions relative to alternative exons at which the protein may function as either a silencer or an enhancer. Most of the identified targets were shifted in the same direction by FOX2 depletion in cell lines as they were in breast and ovarian cancer tissues. Notably, we found expression of FOX2 itself is downregulated in ovarian cancer and its splicing is altered in breast cancer samples. These results suggest that the decreased expression of FOX2 in cancer tissues modulates splicing and controls proliferation.

Alternative splicing is a key mechanism regulating gene expression, and it is often used to produce antagonistic activities particularly in apoptotic genes. Heterogeneous nuclear ribonucleoparticle (hnRNP) proteins form a family of RNA-binding proteins that coat nascent pre-mRNAs. Many but not all major hnRNP proteins have been shown to participate in splicing control. The range and specificity of hnRNP protein action remain poorly documented, even for those affecting splice site selection. We used RNA interference and a reverse transcription-PCR screening platform to examine the implications of 14 of the major hnRNP proteins in the splicing of 56 alternative splicing events in apoptotic genes. Out of this total of 784 alternative splicing reactions tested in three human cell lines, 31 responded similarly to a knockdown in at least two different cell lines. On the other hand, the impact of other hnRNP knockdowns was cell line specific. The broadest effects were obtained with hnRNP K and C, two proteins whose role in alternative splicing had not previously been firmly established. Different hnRNP proteins affected distinct sets of targets with little overlap even between closely related hnRNP proteins. Overall, our study highlights the potential contribution of all of these major hnRNP proteins in alternative splicing control and shows that the targets for individual hnRNP proteins can vary in different cellular contexts.Alternative splicing is a critical process that ensures the production of a multitude of proteins from a limited set of mammalian genes (7, 41). Alternative splicing decisions are regulated by a large collection of RNA-binding proteins (RBPs) that bind to pre-mRNAs in the nucleus (5). The heterogeneous nuclear ribonucleoparticle (hnRNP) proteins are among the most abundant of such proteins, and more than 20 of them have been characterized and given alphabetical names based on size from hnRNP A1 to hnRNP U (17). These proteins have been implicated in a variety of biological processes including telomere biogenesis, translation, and RNA stability, and several (e.g., hnRNP A1, A2, F, H, I [PTB], G, and L) have documented roles in splicing (34, 38). hnRNP A1 has been implicated in the splicing control of many genes, including the A1 gene itself, the caspase-2 gene, c-src, and the SMN2 gene (12), and several exons of human immunodeficiency virus type 1; the very similar hnRNP A2 protein (68% identity) appears to display comparable activity (4,10,29,42). While the related hnRNP F and H proteins play a role in splicing control of many genes, including c-src, Bcl-x, cystathionine ␤-synthase, and several HIV alternative exons (38), it is unclear whether F and H have completely redundant activities. hnRNP I (PTB) is another well-known splicing regulator that has been mostly associated with splicing repression (54,59). A recent global analysis of PTB and its neural paralogue nPTB has revealed their role in the control of murine neuron-specific splicing (8). hnRNP G (RBM-X) has been implicated in the splicing...

Several apoptotic regulators, including Bcl-x, are alternatively spliced to produce isoforms with opposite functions. We have used an RNA interference strategy to map the regulatory landscape controlling the expression of the Bcl-x splice variants in human cells. Depleting proteins known as core (Y14 and eIF4A3) or auxiliary (RNPS1, Acinus, and SAP18) components of the exon junction complex (EJC) improved the production of the proapoptotic Bcl-x S splice variant. This effect was not seen when we depleted EJC proteins that typically participate in mRNA export (UAP56, Aly/Ref, and TAP) or that associate with the EJC to enforce nonsense-mediated RNA decay (MNL51, Upf1, Upf2, and Upf3b). Core and auxiliary EJC components modulated Bcl-x splicing through different cis-acting elements, further suggesting that this activity is distinct from the established EJC function. In support of a direct role in splicing control, recombinant eIF4A3, Y14, and Magoh proteins associated preferentially with the endogenous Bcl-x pre-mRNA, interacted with a model Bcl-x pre-mRNA in early splicing complexes, and specifically shifted Bcl-x alternative splicing in nuclear extracts. Finally, the depletion of Y14, eIF4A3, RNPS1, SAP18, and Acinus also encouraged the production of other proapoptotic splice variants, suggesting that EJC-associated components are important regulators of apoptosis acting at the alternative splicing level.

Alternative splicing provides a critical and flexible layer of regulation intervening in many biological processes to regulate the diversity of proteins and impact cell phenotype. To identify alternative splicing differences that distinguish epithelial from mesenchymal tissues, we have investigated hundreds of cassette exons using a high-throughput reverse transcription-PCR (RT-PCR) platform. Extensive changes in splicing were noted between epithelial and mesenchymal tissues in both human colon and ovarian tissues, with many changes from mostly one splice variant to predominantly the other. Remarkably, many of the splicing differences that distinguish normal mesenchymal from normal epithelial tissues matched those that differentiate normal ovarian tissues from ovarian cancer. Furthermore, because splicing profiling could classify cancer cell lines according to their epithelial/mesenchymal characteristics, we used these cancer cell lines to identify regulators for these specific splicing signatures. By knocking down 78 potential splicing factors in five cell lines, we provide an extensive view of the complex regulatory landscape associated with the epithelial and mesenchymal states, thus revealing that RBFOX2 is an important driver of mesenchymal tissue-specific splicing.T ransitions from epithelial to mesenchymal (EMT) and mesenchymal to epithelial (MET) states have important roles not only in normal tissue and organ development but in the pathogenesis of diseases including cancer (1). In normal tissues, epithelial cells display a cuboidal morphology and a polar organization maintained by tight cell-cell interconnections. Mesenchymal cells lack these features and display higher motility and invasiveness. During the process of carcinogenesis, EMT is thought to be crucial to elicit migration, resistance to apoptosis, and ultimately invasion and metastasis (1-5). Conversely, the reverse process, MET, is associated with the colonization of secondary sites by cells that have metastasized. Therefore, profiling the molecular differences between the epithelial and mesenchymal states may help us understand the underlying regulatory programs that establish these states and promote their interconversion.The commonly accepted view is that EMT can be induced by growth factors, such as transcription growth factor ␤1 (TGF-␤1), which trigger signaling pathways that ultimately activate a network of transcription regulators, including Snail, Slug, Twist, and others (6). This transcriptional reprogramming elicits the expression of mesenchymal markers (e.g., vimentin) and represses the expression of epithelial ones (e.g., E-cadherin) to impart distinctive properties such as motility and invasion (7-10). Alternative splicing control provides another layer of regulation that can contribute to EMT (11,12). The tyrosine kinase Ron (MST1R) is alternatively spliced to produce an exon 11-lacking version that can promote invasion (13). Three RNA-binding proteins (RBPs), SRSF1, hnRNP H, and hnRNP A2/B1, affect the alternative splicing of Ro...

Breast cancer is the most common cause of cancer death among women under age 50 years, so it is imperative to identify molecular markers to improve diagnosis and prognosis of this disease. Here, we present a new approach for the identification of breast cancer markers that does not measure gene expression but instead uses the ratio of alternatively spliced mRNAs as its indicator. Using a high-throughput reverse transcription-PCR-based system for splicing annotation, we monitored the alternative splicing profiles of 600 cancer-associated genes in a panel of 21 normal and 26 cancerous breast tissues. We validated 41 alternative splicing events that significantly differed in breast tumors relative to normal breast tissues. Most cancer-specific changes in splicing that disrupt known protein domains support an increase in cell proliferation or survival consistent with a functional role for alternative splicing in cancer. In a blind screen, a classifier based on the 12 best cancer-associated splicing events correctly identified cancer tissues with 96% accuracy. Moreover, a subset of these alternative splicing events could order tissues according to histopathologic grade, and 5 markers were validated in a further blind set of 19 grade 1 and 19 grade 3 tumor samples. These results provide a simple alternative for the classification of normal and cancerous breast tumor tissues and underscore the putative role of alternative splicing in the biology of cancer. [Cancer Res 2008;68(22):9525-31]