Multiple interdependent sequence elements control splicing of a fibroblast growth factor receptor 2 alternative exon.

The fibroblast growth factor receptor 2 gene contains a pair of mutually exclusive alternative exons, one of which (K-SAM) is spliced specifically in epithelial cells. We have described previously (F. Del Gatto and R. Breathnach, Mol. Cell. Biol. 15:4825-4834, 1995) some elements controlling K-SAM exon splicing, namely weak exon splice sites, an exon-repressing sequence, and an intron-activating sequence. We identify here two additional sequences in the intron downstream from the K-SAM exon which activate splicing of the exon. The first sequence (intron-activating sequence 2 [IAS2]) lies 168 to 186 nucleotides downstream from the exon's 5' splice site. The second sequence (intron-activating sequence 3 [IAS3]) lies 933 to 1,052 nucleotides downstream from the exon's 5' splice site. IAS3 is a complex region composed of several parts, one of which (nucleotides 963 to 983) can potentially form an RNA secondary structure with IAS2. This structure is composed of two stems separated by an asymmetric bulge. Mutations which disrupt either stem decrease activation, while compensatory mutations which reestablish the stem restore activation, either completely or partially, depending on the mutation. We present a model for K-SAM exon splicing involving the intervention of multiple, interdependent pre-mRNA sequence elements.     

Polypyrimidine tract-binding protein represses splicing of a fibroblast growth factor receptor-2 gene alternative exon through exon sequences

The fibroblast growth factor receptor (FGFR)-2 gene contains two mutually exclusive exons, K-SAM and BEK. We made a cell line designed to become drug-resistant on repression of BEK exon splicing. One drug-resistant derivative of this line carried an insertion within the BEK exon of a sequence containing at least two independent splicing silencers. One silencer was a pyrimidine-rich sequence, which markedly increased binding of polypyrimidine tract-binding protein to the BEK exon. The BEK exon binds to polypyrimidine tract-binding protein even in the silencer's absence. Several exonic pyrimidine runs are required for this binding, and they are also required for overexpression of polypyrimidine tract-binding protein to repress BEK exon splicing. These results show that binding of polypyrimidine tract-binding protein to exon sequences can repress splicing. In epithelial cells, the K-SAM exon is spliced in preference to the BEK exon, whose splicing is repressed. Mutation of the BEK exon pyrimidine runs decreases this repression. If this mutation is combined with the deletion of a sequence in the intron upstream from the BEK exon, a complete switch from K-SAM to BEK exon splicing ensues. Binding of polypyrimidine tract binding protein to the BEK exon thus participates in the K-SAM/BEK alternative splicing choice.     

Fox-2 splicing factor binds to a conserved intron motif to promote inclusion of protein 4.1R alternative exon 16

Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins to silencer elements in the exon and that down-regulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This article demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.     

The expression of two isoforms of the human fibroblast growth factor receptor (flg) is directed by alternative splicing.

Structural analysis of the cDNA for the human fibroblast growth factor receptor (flg) revealed the existence of a larger and a shorter isoform of the receptor. The larger form has three extracellular immunoglobulin-like domains. On the other hand, the shorter form deletes the first (the most external) immunoglobulin-like domain region. Two consecutive amino acids (Arg Met) between the first and second immunoglobulin-like domains are sometimes deleted from the shorter form. In this paper, we isolated and analyzed the gene for the human fibroblast growth factor receptor. Organization of the gene revealed that the isoforms are produced by two different types of alternative splicing (the cassette and internal donor types) from the common gene. In human placenta, the shorter form is expressed as the major isoform.     

A novel intronic cis element, ISE/ISS-3, regulates rat fibroblast growth factor receptor 2 splicing through activation of an upstream exon and repression of a downstream exon containing a noncanonical branch point sequence

Mutually exclusive splicing of fibroblast growth factor receptor 2 (FGFR2) exons IIIb and IIIc yields two receptor isoforms, FGFR2-IIIb and -IIIc, with distinctly different ligand binding properties. Several RNA cis elements in the intron (intron 8) separating these exons have been described that are required for splicing regulation. Using a heterologous splicing reporter, we have identified a new regulatory element in this intron that confers cell-type-specific inclusion of an unrelated exon that mirrors its ability to promote cell-type-specific inclusion of exon IIIb. This element promoted inclusion of exon IIIb while at the same time silencing exon IIIc inclusion in cells expressing FGFR2-IIIb; hence, we have termed this element ISE/ISS-3 (for "intronic splicing enhancer-intronic splicing silencer 3"). Silencing of exon IIIc splicing by ISE/ISS-3 was shown to require a branch point sequence (BPS) using G as the primary branch nucleotide. Replacing a consensus BPS with A as the primary branch nucleotide resulted in constitutive splicing of exon IIIc. Our results suggest that the branch point sequence constitutes an important component that can contribute to the efficiency of exon definition of alternatively spliced cassette exons. Noncanonical branch points may thus facilitate cell-type-specific silencing of regulated exons by flanking cis elements.     

hnRNP H and hnRNP F complex with Fox2 to silence fibroblast growth factor receptor 2 exon IIIc

The heterogeneous nuclear ribonucleoprotein H (hnRNP) family of proteins has been shown to activate exon inclusion by binding intronic G triplets. Much less is known, however, about how hnRNP H and hnRNP F silence exons. In this study, we identify hnRNP H and hnRNP F proteins as being novel silencers of fibroblast growth factor receptor 2 exon IIIc. In cells that normally include this exon, we show that the overexpression of either hnRNP H1 or hnRNP F resulted in the dramatic silencing of exon IIIc. In cells that normally skip exon IIIc, skipping was disrupted when RNA interference was used to knock down both hnRNP H and hnRNP F. We show that an exonic GGG motif overlapped a critical exonic splicing enhancer, which was predicted to bind the SR protein ASF/SF2. Furthermore, the expression of ASF/SF2 reversed the silencing of exon IIIc caused by the expression of hnRNP H1. We show that hnRNP H and hnRNP F proteins are present in a complex with Fox2 and that the presence of Fox allows hnRNP H1 to better compete with ASF/SF2 for binding to exon IIIc. These results establish hnRNP H and hnRNP F as being repressors of exon inclusion and suggest that Fox proteins enhance their ability to antagonize ASF/SF2.     

Regulation of alternative RNA splicing by exon definition and exon sequences in viral and mammalian gene expression

Intron removal from a pre-mRNA by RNA splicing was once thought to be controlled mainly by intron splicing signals. However, viral and other eukaryotic RNA exon sequences have recently been found to regulate RNA splicing, polyadenylation, export, and nonsense-mediated RNA decay in addition to their coding function. Regulation of alternative RNA splicing by exon sequences is largely attributable to the presence of two majorcis-acting elements in the regulated exons, the exonic splicing enhancer (ESE) and the suppressor or silencer (ESS). Two types of ESEs have been verified from more than 50 genes or exons: purine-rich ESEs, which are the more common, and non-purine-rich ESEs. In contrast, the sequences of ESSs identified in approximately 20 genes or exons are highly diverse and show little similarity to each other. Through interactions with cellular splicing factors, an ESE or ESS determines whether or not a regulated splice site, usually an upstream 3 splice site, will be used for RNA splicing. However, how these elements function precisely in selecting a regulated splice site is only partially understood. The balance between positive and negative regulation of splice site selection likely depends on thecis-element's identity and changes in cellular splicing factors under physiological or pathological conditions.