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Molecular and Cellular Biology, September 2005, p. 7580-7591, Vol. 25, No. 17
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.17.7580-7591.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

ELAV Multimerizes on Conserved AU4-6 Motifs Important for ewg Splicing Regulation

Matthias Soller* and Kalpana White*

Department of Biology and Volen Center for Complex Systems, MS 008, Brandeis University, Waltham, Massachusetts 02454

Received 23 February 2005/ Returned for modification 21 March 2005/ Accepted 27 May 2005

ELAV is a gene-specific regulator of alternative pre-mRNA processing in Drosophila neurons. Since ELAV/Hu proteins preferentially bind to AU-rich regions that are generally abundant in introns and untranslated regions, it has not been clear how gene specificity is achieved. Here we used a combination of in vitro biochemical experiments together with phylogenetic comparisons and in vivo analysis of Drosophila transgenes to study ELAV binding to the last ewg intron and splicing regulation. In vitro binding studies of ELAV show that ELAV multimerizes on the ewg binding site and forms a defined and saturable complex. Further, sizing of the ELAV-RNA complex and a series of titration experiments indicate that ELAV forms a dodecameric complex on 135 nucleotides in the last ewg intron. Analysis of the substrate RNA requirements for ELAV binding and complex formation indicates that a series of AU4-6 motifs spread over the entire binding site are important, but not a strictly defined sequence element. The importance of AU4-6 motifs, but not spacing between them, is further supported by evolutionary conservation in several melanogaster species subgroups. Finally, using transgenes we demonstrate in fly neurons that ELAV-mediated regulation of ewg intron 6 splicing requires several AU4-6 motifs and that introduction of spacer sequence between conserved AU4-6 motifs has a minimal effect on splicing. Collectively, our results suggest that ELAV multimerization and binding to multiple AU4-6 motifs contribute to target RNA recognition and processing in a complex cellular environment.

* Corresponding author. Mailing address: Biology Department, MS 008, Brandeis University, Waltham, MA 02454. Phone: (781) 736-3177. Fax: (781) 736-3107. E-mail for Matthias Soller: msoller{at}brandeis.edu. E-mail for Kalpana White: white{at}brandeis.edu.

Molecular and Cellular Biology, September 2005, p. 7580-7591, Vol. 25, No. 17
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.17.7580-7591.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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