Alternative splicing is the major source of proteome diversity in humans and thus is highly relevant to disease and therapy. For example, recent work suggests that the long-sought-after target of the analgesic acetaminophen is a neural-specific, alternatively spliced isoform of cyclooxygenase 1 (COX-1). Several important diseases, such as cystic fibrosis, have been linked with mutations or variations in either cis-acting elements or trans-acting factors that lead to aberrant splicing and abnormal protein production. Correction of erroneous splicing is thus an important goal of molecular therapies. Recent experiments have used modified oligonucleotides to inhibit cryptic exons or to activate exons weakened by mutations, suggesting that these reagents could eventually lead to effective therapies.

Alternative splicing of fibroblast growth factor receptor 2 (FGFR2) transcripts occurs in a cell-type-specific manner leading to the mutually exclusive use of exon IIIb in epithelia or exon IIIc in mesenchyme. Epithelial cell-specific exon choice is dependent on (U)GCAUG elements, which have been shown to bind Fox protein family members. In this paper we show that FGFR2 exon choice is regulated by (U)GCAUG elements and Fox protein family members. Fox-2 isoforms are differentially expressed in IIIb ؉ cells in comparison to IIIc ؉ cells, and expression of Fox-1 or Fox-2 in the latter led to a striking alteration in FGFR2 splice choice from IIIc to IIIb. This switch was absolutely dependent on the (U)GCAUG elements present in the FGFR2 pre-mRNA and required critical residues in the C-terminal region of Fox-2. Interestingly, Fox-2 expression led to skipping of exon 6 among endogenous Fox-2 transcripts and formation of an inactive Fox-2 isoform, which suggests that Fox-2 can regulate its own activity. Moreover, the repression of exon IIIc in IIIb There are four well-characterized fibroblast growth factor receptors (FGFRs), which contain a single transmembrane domain, an intracellular tyrosine kinase domain, and an extracellular FGF binding domain composed of two or three immunoglobulin (Ig)-like domains. The transcripts encoding three FGFRs (FGFR1, -2, and -3) are alternatively spliced to produce isoforms that contain one of two different Ig-III domains. Alternative splicing of FGFR2 transcripts results in the production of two receptors that differ in the carboxy-terminal half of the Ig-III domain. This hemidomain is determined by the tissue-specific inclusion of either exon IIIb or exon IIIc, which ultimately controls ligand binding specificity (7,14,27,52). FGFR2(IIIb) primarily binds FGF10 and FGF7 and is the isoform of choice in epithelial cells, whereas FGFR2(IIIc) binds FGF2 and is exclusively expressed in cells of mesenchymal origin (36, 49). FGF/FGFR2 signaling governs epithelialmesenchymal interactions that are required for organogenesis in mouse embryos (3, 15, 16); therefore, it is critical for normal development to maintain the proper cell-type-specific expression of each receptor isoform. Mutations that alter the ligand binding specificity of FGFR2(IIIc) or those that lead to the inappropriate expression of exon IIIb in mesenchyme have been linked to developmental disorders in humans (3,16,35,54). The importance of FGFR2 isoform choice is underscored by studies demonstrating a switch from FGFR2(IIIb) to FGFR2(IIIc) during the progression of prostate carcinomas (4, 49), where the loss of FGFR2(IIIb) appears to be required for this progression (51).The regulation of FGFR2 alternative splicing depends on a complex interplay between cis-acting elements in the FGFR2 pre-mRNA and trans-acting factors, with some of the transacting factors appearing to be cell type specific. To study this regulation, we have employed two cell lines derived from Dunning rat prostate tumors. The DT3 cell line is a well-differenti...

Alternative splicing of fibroblast growth factor receptor 2 (FGFR2) occurs in a cell-type-specific manner with the mutually exclusive use of exon IIIb or exon IIIc. Specific inclusion of exon IIIb is observed in epithelial cells, whereas exon IIIc inclusion is seen in mesenchymal cells. Epithelium-specific activation of exon IIIb and repression of exon IIIc are coordinately regulated by intronic activating sequence 2 (IAS2) and intronic splicing activator and repressor (ISAR) elements in FGFR2 pre-mRNA. Previously, it has been suggested that IAS2 and a 20-nucleotide core sequence of ISAR form a stem structure that allows for the proper regulation of FGFR2 alternative splicing. Replacement of IAS2 and the ISAR core with random sequences capable of stem formation resulted in the proper activation of exon IIIb and repression of exon IIIc in epithelial cells. Given the high degree of phylogenetic conservation of the IAS2-ISAR core structure and the fact that unrelated stem-forming sequences could functionally substitute for IAS2 and ISAR elements, we postulated that the stem structure facilitated the approximation of intronic control elements. Indeed, deletion of the entire stem-loop region and juxtaposition of sequences immediately upstream of IAS2 with sequences immediately downstream of the ISAR core maintained proper cell-type-specific inclusion of exon IIIb. These data demonstrate that IAS2 and the ISAR core are dispensable for the cell-type-specific activation of exon IIIb; thus, the major, if not the sole, role of the IAS2-ISAR stem in exon IIIb activation is to approximate sequences upstream of IAS2 with sequences downstream of the ISAR core. The downstream sequence is very likely a highly conserved GCAUG element, which we show was required for efficient exon IIIb activation.Fibroblast growth factor receptor 2 (FGFR2) contains a single transmembrane domain, an intracellular tyrosine kinase domain, and an extracellular fibroblast growth factor (FGF) binding domain, which is composed of immunoglobulin (Ig)-like domains II and III. Alternative splicing of FGFR2 transcripts produces two variants of the Ig-III domain with different carboxy-terminal halves, which lead to distinct ligand binding specificity. The two forms of the Ig-III domain are derived from the tissue-specific inclusion of either exon IIIb or exon IIIc (36,44). FGFR2(IIIb) primarily binds FGF10 and FGF7 and is the isoform of choice in epithelial cells, whereas FGFR2(IIIc) binds FGF2 with high affinity and is predominantly expressed in mesenchyme (36, 51). Proper cell-typespecific expression of each isoform is essential for maintaining FGF/FGFR2 signaling, which governs epithelial-mesenchymal interactions required for organogenesis in mouse embryos (17,22). Mutations that alter the ligand specificity of FGFR2(IIIc) or those that lead to inappropriate expression of exon IIIb in mesenchyme have been linked to several developmental syndromes in humans (22,43,52). The physiological importance of regulating FGFR2 isoform choice is highlighted further b...

Although multiple regulatory elements and protein factors are known to regulate the non-neuronal pathway of alternative processing of the calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA, the mechanisms controlling the neuron-specific pathway have remained elusive. Here we report the identification of Fox-1 and Fox-2 proteins as novel regulators that mediate the neuron-specific splicing pattern. Fox-1 and Fox-2 proteins function to repress exon 4 inclusion, and this effect depends on two UGCAUG elements surrounding the 3 splice site of the calcitonin-specific exon 4. In neuron-like cells, mutation of a subset of UGCAUG elements promotes the non-neuronal pattern in which exon 4 is included. In HeLa cells, overexpression of Fox-1 or Fox-2 protein decreases exon 4 inclusion. Fox-1 and Fox-2 proteins interact with the UGCAUG elements specifically and regulate splicing by blocking U2AF 65 binding to the 3 splice site upstream of exon 4. We further investigated the inter-relationship between the UGCAUG silencer elements and the previously identified intronic and exonic splicing regulatory elements and found that exon 4 is regulated by an intricate balance of positive and negative regulation. These results define a critical role for Fox-1 and Fox-2 proteins in exon 4 inclusion of calcitonin/CGRP pre-mRNA and establish a regulatory network that controls the fate of exon 4.Alternative RNA processing is a major contributor to proteomic complexity in eukaryotic organisms. Through this process, the majority of human pre-mRNA molecules generate more than one mRNA molecule, which are then translated into different protein isoforms that have distinct biological activities (6, 27). Tissue-specific alternative RNA processing plays an important role in regulating gene expression. It has been demonstrated that alternative splicing is controlled by a complex interplay between positive and negative splicing regulators that function through binding at their cognate splicing enhancer and silencer elements located in both the alternatively spliced exon and the adjacent introns (27). However, our knowledge of tissue-specific regulation of splicing is very limited, with only a handful of tissue-specific splicing regulators identified to date (6, 40).The human calcitonin/calcitonin gene-related peptide (CGRP) gene is an excellent model to study tissue-specific regulation of alternative splicing. The calcitonin/CGRP gene contains six exons. In neurons, CGRP mRNA production results from joining of exons 1 to 3 to exons 5 to 6 accompanied by usage of a distal polyadenylation signal located at the 3Ј end of the sixth exon (3, 35). In thyroid C cells, calcitonin mRNA is produced by joining exons 1 to 3 to exon 4, accompanied by usage of the proximal polyadenylation site located at the 3Ј end of exon 4 (Fig. 1A) (38). Alternative processing of calcitonin/ CGRP pre-mRNA is subject to complex control involving multiple cis-acting regulatory elements and trans-acting factors. Studies to date have identified several RNA sequence elements ...