HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 280, Issue 14, 14017-14027, April 8, 2005

This Article Full Text Full Text (PDF) All Versions of this Article: 280/14/14017    most recent M414492200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wagner, E. J. Articles by Garcia-Blanco, M. A. Search for Related Content PubMed PubMed Citation Articles by Wagner, E. J. Articles by Garcia-Blanco, M. A. Social Bookmarking                      What's this?

Characterization of the Intronic Splicing Silencers Flanking FGFR2 Exon IIIb^*

Eric J. Wagner¶||, Andrew P. Baraniak||**, October M. Sessions**, David Mauger**, Eric Moskowitz, and Mariano A. Garcia-Blanco¶¶

From the Department of Molecular Genetics and Microbiology, Program in Molecular Cancer Biology, and Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710

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.

Received for publication, December 22, 2004

^* This work was supported in part by United States Public Health Service Grant GM63090 (to M. A. G.-B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Present address: Dept. of Biochemistry and Biophysics, and Program in Molecular Biology and Biotechnology, CB 7100, University of North Carolina, Chapel Hill, North Carolina 27599.

^|| Supported by a Department of Defense predoctoral fellowship.

^** Recipient of United States Public Health Service training grant.

Present address: 1025 Walnut St., Philadelphia, PA 19107.

^¶¶ To whom correspondence should be addressed. E-mail: garci001{at}mc.duke.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				Z. Wang and C. B. Burge Splicing regulation: From a parts list of regulatory elements to an integrated splicing code RNA, May 1, 2008; 14(5): 802 - 813. [Abstract] [Full Text] [PDF]

				P. Seth, H. B. Miller, E. L. Lasda, J. L. Pearson, and M. A. Garcia-Blanco Identification of an Intronic Splicing Enhancer Essential for the Inclusion of FGFR2 Exon IIIc J. Biol. Chem., April 11, 2008; 283(15): 10058 - 10067. [Abstract] [Full Text] [PDF]

				G. Ohno, M. Hagiwara, and H. Kuroyanagi STAR family RNA-binding protein ASD-2 regulates developmental switching of mutually exclusive alternative splicing in vivo Genes & Dev., February 1, 2008; 22(3): 360 - 374. [Abstract] [Full Text] [PDF]

				K. Fukumura, A. Kato, Y. Jin, T. Ideue, T. Hirose, N. Kataoka, T. Fujiwara, H. Sakamoto, and K. Inoue Tissue-specific splicing regulator Fox-1 induces exon skipping by interfering E complex formation on the downstream intron of human F1{gamma} gene Nucleic Acids Res., August 13, 2007; 35(16): 5303 - 5311. [Abstract] [Full Text] [PDF]

				M. A. Caligiuri, R. Briesewitz, J. Yu, L. Wang, M. Wei, K. J. Arnoczky, T. B. Marburger, J. Wen, D. Perrotti, C. D. Bloomfield, et al. Novel c-CBL and CBL-b ubiquitin ligase mutations in human acute myeloid leukemia Blood, August 1, 2007; 110(3): 1022 - 1024. [Abstract] [Full Text] [PDF]

				D. Das, T. A. Clark, A. Schweitzer, M. Yamamoto, H. Marr, J. Arribere, S. Minovitsky, A. Poliakov, I. Dubchak, J. E. Blume, et al. A correlation with exon expression approach to identify cis-regulatory elements for tissue-specific alternative splicing Nucleic Acids Res., July 10, 2007; (2007) gkm485v1. [Abstract] [Full Text] [PDF]

				M. Simarro, D. Mauger, K. Rhee, M. A. Pujana, N. L. Kedersha, S. Yamasaki, M. E. Cusick, M. Vidal, M. A. Garcia-Blanco, and P. Anderson Fas-activated serine/threonine phosphoprotein (FAST) is a regulator of alternative splicing PNAS, July 3, 2007; 104(27): 11370 - 11375. [Abstract] [Full Text] [PDF]

				V. I. Bonano, S. Oltean, R. M. Brazas, and M. A. Garcia-Blanco Imaging the alternative silencing of FGFR2 exon IIIb in vivo RNA, December 1, 2006; 12(12): 2073 - 2079. [Abstract] [Full Text] [PDF]

				S. Oltean, B. S. Sorg, T. Albrecht, V. I. Bonano, R. M. Brazas, M. W. Dewhirst, and M. A. Garcia-Blanco Alternative inclusion of fibroblast growth factor receptor 2 exon IIIc in Dunning prostate tumors reveals unexpected epithelial mesenchymal plasticity PNAS, September 19, 2006; 103(38): 14116 - 14121. [Abstract] [Full Text] [PDF]

				A. P. Baraniak, J. R. Chen, and M. A. Garcia-Blanco Fox-2 Mediates Epithelial Cell-Specific Fibroblast Growth Factor Receptor 2 Exon Choice Mol. Cell. Biol., February 15, 2006; 26(4): 1209 - 1222. [Abstract] [Full Text] [PDF]

				R. H. Hovhannisyan, C. C. Warzecha, and R. P. Carstens Characterization of sequences and mechanisms through which ISE/ISS-3 regulates FGFR2 splicing Nucleic Acids Res., January 12, 2006; 34(1): 373 - 385. [Abstract] [Full Text] [PDF]

				M.J. DYE, N. GROMAK, D. HAUSSECKER, S. WEST, and N.J. PROUDFOOT Turnover and Function of Noncoding RNA Polymerase II Transcripts Cold Spring Harb Symp Quant Biol, January 1, 2006; 71(0): 275 - 284. [Abstract] [PDF]