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Molecular and Cellular Biology, February 2005, p. 879-887, Vol. 25, No. 3
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.3.879-887.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Identification of Putative New Splicing Targets for ETR-3 Using Sequences Identified by Systematic Evolution of Ligands by Exponential Enrichment

Nuno André Faustino1,2 and Thomas A. Cooper1,3*

Departments of Pathology,1 Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas,3 Graduate Program in Basic and Applied Biology, ICBAS, University of Oporto, Oporto, Portugal2

Received 3 August 2004/ Returned for modification 30 August 2004/ Accepted 1 November 2004

ETR-3 (also know as BRUNOL3, NAPOR, and CUGBP2) is one of six members of the CELF (CUG-BP1- and ETR-3-like factor) family of splicing regulators. ETR-3 regulates splicing by direct binding to the pre-mRNA. We performed systematic evolution of ligands by exponential enrichment (SELEX) to identify the preferred binding sequence of ETR-3. After five rounds of SELEX, ETR-3 selected UG-rich sequences, in particular UG repeats and UGUU motifs. Either of these selected motifs was able to restore ETR-3 binding and responsiveness to a nonresponsive splicing reporter in vivo. Moreover, this effect was not specific to ETR-3 since minigenes containing either of the two motifs were responsive to two other CELF proteins (CUG-BP1 and CELF4), indicating that different members of the CELF family can mediate their effects via a common binding site. Using the SELEX-identified motifs to search the human genome, we identified several possible new ETR-3 targets. We created minigenes for two of these genes, the CFTR and MTMR1 genes, and confirmed that ETR-3 regulates their splicing patterns. For the CFTR minigene this regulation was demonstrated to be dependent on the presence of the putative binding site identified in our screen. These results validate this approach to search for new targets for RNA processing proteins.


* Corresponding author. Mailing address: One Baylor Pl., Dept. of Pathology, Room 268B, Houston, TX 77030. Phone: (713) 798-3141. Fax: (713) 798-5838. E-mail: tcooper{at}bcm.tmc.edu.


Molecular and Cellular Biology, February 2005, p. 879-887, Vol. 25, No. 3
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.3.879-887.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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