Characterization of the intronic splicing silencers flanking FGFR2 exon IIIb

Eric Wagner, Andrew P. Baraniak, October M. Sessions, David Mauger, Eric Moskowitz, Mariano Garcia-Blanco

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)14017-14027
Number of pages11
JournalJournal of Biological Chemistry
Volume280
Issue number14
DOIs
StatePublished - Apr 8 2005
Externally publishedYes

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Exons
Polypyrimidine Tract-Binding Protein
Protein Binding
Binding Sites
RNA Splice Sites
Trans-Activators
RNA Precursors
Alternative Splicing
Redundancy
Substitution reactions
Nucleotides
Chemical activation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Characterization of the intronic splicing silencers flanking FGFR2 exon IIIb. / Wagner, Eric; Baraniak, Andrew P.; Sessions, October M.; Mauger, David; Moskowitz, Eric; Garcia-Blanco, Mariano.

In: Journal of Biological Chemistry, Vol. 280, No. 14, 08.04.2005, p. 14017-14027.

Research output: Contribution to journalArticle

Wagner, Eric ; Baraniak, Andrew P. ; Sessions, October M. ; Mauger, David ; Moskowitz, Eric ; Garcia-Blanco, Mariano. / Characterization of the intronic splicing silencers flanking FGFR2 exon IIIb. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 14. pp. 14017-14027.
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