Multiple Interdependent Sequence Elements Control Splicing of a Fibroblast Growth Factor Receptor 2 Alternative Exon

Abstract

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.Cells very often make several related yet distinct proteins from a single gene by alternative splicing of the corresponding pre-mRNA (36). This strategy can be exploited to express one form of a protein in some cell types and a different form in other cell types. One example of this behavior is provided by the fibroblast growth factor receptor 2 (FGFR-2) gene (28). The extracellular part of the receptor encoded by this gene is made up of three immunoglobulin-like domains. Two alternative exons, K-SAM and BEK, code for the carboxy-terminal half of the third, membrane-proximal, immunoglobulin-like domain (11). Splicing of the K-SAM exon generates a highaffinity receptor for FGF-1 and FGF-7, while splicing of the BEK exon generates a high-affinity receptor for FGF-1 and FGF-2 (38, 56). The splicing choice is strictly controlled, as certain cell types splice essentially only the K-SAM exon, while others splice essentially only the BEK exon (11,38). As the splicing choice conditions the response of a cell to certain growth factors in its environment, the importance of strict control of splicing is evident during both development and adult life (40,43).Many other cases of alternative exon splicing have been described. For example, mouse c-src pre-mRNA contains two exons spliced only in neurons (7,12,39). The avian troponin T gene contains an optional exon spliced in the embryonic but not the adult heart (41, 54). Alpha-and beta-tropomyosin genes contain pairs of mutually exclusive internal exons spliced with strict tissue specificity (5,14,24,25,32). The pre-mRNA encoding calcitonin and calcitonin gene-related peptide contains six exons, one of which (exon 4) is skipped in a few cell types, including neuronal cell...

Mutation screening in patients with syndromic craniosynostoses indicates that a limited number of recurrent FGFR2 mutations accounts for severe forms of Pfeiffer syndrome

“…This complex process may involved increased binding of ribonuclear proteins (RNP) to regulatory intronic sequences controlling exon 9 (IIIb) splicing. 41,42 Interestingly, it has been previously demonstrated that Alu-element insertion upstream or within exon 10 (IIIc) of FGFR2-induced ectopic expression of the IIIb isoform in one Apert patient. 43 The same effects were observed in four patients with PS carrying the splice mutation 940 -2A4G/T.…”

MAZ Elements Alter Transcription Elongation and Silencing of the Fibroblast Growth Factor Receptor 2 Exon IIIb

“…RNase Protection Assay-pB68.8 (described above) was linearized with BamHI, and [␣- 32 P]UTP-labeled probes were generated with T7 RNA polymerase (Ambion). Approximately 5,000 -10,000-cpm probe and 10 -15 g of each sample RNA were coprecipitated and resuspended in 20 l of 40 mM PIPES pH 6.4, 500 mM NaCl, 1 mM EDTA, and 80% formamide.…”

“…In DT3 cells, activation of exon IIIb is required to overcome the silencing of this exon. Exon IIIb is activated, and IIIc is repressed by IAS2 and the intronic splicing activator and repressor (ISAR, also called IAS3) (32)(33)(34). Mutation of either IAS2 or ISAR results in the default state of exon IIIb silencing, even in cells that would normally include this exon (32,33).…”

TIA-1 or TIAR Is Required for DT40 Cell Viability

“…RT-PCR was carried out as described elsewhere (34,35). Primers used were 5Ј-ggaagacgacgggcagcc-3Ј from tiar exon 1, with 5Ј-gcaaaggctgacttgata-3Ј from tiar exon 5; 5Ј-caatcacttccacgtgttcg-3Ј from tiar exon 5, with 5Ј-gttagcccagaagcaat-3Ј from tiar exon 8; 5Ј-gtactcatgggaggccag-3Ј from tiar exon 7 with 5Ј-aggctgagcaccaaatccacccat-3Ј from tiar exon 12.…”