Document Detail

Characterization of the intronic splicing silencers flanking FGFR2 exon IIIb.
MedLine Citation:
PMID:  15684416     Owner:  NLM     Status:  MEDLINE    
The cell type-specific alternative splicing of FGFR2 pre-mRNA results in the mutually exclusive use of exons IIIb and IIIc, which leads to critically important differences in receptor function. The choice of exon IIIc in mesenchymal cells involves activation of this exon and repression of exon IIIb. This repression is mediated by the function of upstream and downstream intronic splicing silencers (UISS and DISS). Here we present a detailed characterization of the determinants of silencing function within UISS and DISS. We used a systematic mutational analysis, introducing deletions and substitutions to define discrete elements within these two silencers of exon IIIb. We show that UISS requires polypyrimidine tract-binding protein (PTB)-binding sites, which define the UISS1 sub-element, and an eight nucleotide sequence 5'-GCAGCACC-3' (UISS2) that is also required. Even though UISS2 does not bind PTB, the full UISS can be replaced with a synthetic silencer designed to provide optimal PTB binding. DISS is composed of a 5'-conserved sub-element (5'-CE) and two regions that contain multiple PTB sites and are functionally redundant (DISS1 and DISS2). DISS1 and DISS2 are separated by the activator sequence IAS2, and together these opposing elements form the intronic control element. Deletion of DISS in the FGFR2 exon IIIb context resulted in the near full inclusion of exon IIIb, and insertion of this silencer downstream of a heterologous exon with a weak 5' splice site was capable of repressing exon inclusion. Extensive deletion analysis demonstrated that the majority of silencing activity could be mapped to the conserved octamer CUCGGUGC within the 5'CE. Replacement of 5'CE and DISS1 with PTB-binding elements failed to restore repression of exon IIIb. We tested the importance of the relative position of the silencers and of the subelements within each silencer. Whereas UISS1, UISS2, DISS1, and DISS2 appear somewhat malleable, the 5'CE is rigid in terms of relative position and redundancy. Our data defined elements of function within the ISSs flanking exon IIIb and suggested that silencing of this exon is mediated by multiple trans-acting factors.
Eric J Wagner; Andrew P Baraniak; October M Sessions; David Mauger; Eric Moskowitz; Mariano A Garcia-Blanco
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S.     Date:  2005-01-31
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  280     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  2005 Apr 
Date Detail:
Created Date:  2005-04-04     Completed Date:  2005-06-21     Revised Date:  2012-06-05    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  14017-27     Citation Subset:  IM    
Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
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MeSH Terms
Alternative Splicing*
Base Sequence
Cell Line
Gene Expression Regulation
Molecular Sequence Data
Receptor Protein-Tyrosine Kinases / genetics*
Receptor, Fibroblast Growth Factor, Type 2
Receptors, Fibroblast Growth Factor / genetics*
Sequence Alignment
Sequence Analysis, DNA
Silencer Elements, Transcriptional*
Grant Support
Reg. No./Substance:
0/Receptors, Fibroblast Growth Factor; EC protein, human; EC protein, rat; EC Protein-Tyrosine Kinases; EC, Fibroblast Growth Factor, Type 2

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