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Molecular and Cellular Biology, February 2004, p. 1387-1400, Vol. 24, No. 3
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.3.1387-1400.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

RNA Folding Affects the Recruitment of SR Proteins by Mouse and Human Polypurinic Enhancer Elements in the Fibronectin EDA Exon

Emanuele Buratti, Andrés F. Muro, Maurizio Giombi, Daniel Gherbassi,{dagger} Alessandra Iaconcig, and Francisco E. Baralle*

International Centre for Genetic Engineering and Biotechnology, I-34012 Trieste, Italy

Received 21 July 2003/ Returned for modification 22 September 2003/ Accepted 24 October 2003

In humans, inclusion or exclusion of the fibronectin EDA exon is mainly regulated by a polypurinic enhancer element (exonic splicing enhancer [ESE]) and a nearby silencer element (exonic splicing silencer [ESS]). While human and mouse ESEs behave identically, mutations introduced into the homologous mouse ESS sequence result either in no change in splicing efficiency or in complete exclusion of the exon. Here, we show that this apparently contradictory behavior cannot be simply accounted for by a localized sequence variation between the two species. Rather, the nucleotide differences as a whole determine several changes in the respective RNA secondary structures. By comparing how the two different structures respond to homologous deletions in their putative ESS sequences, we show that changes in splicing behavior can be accounted for by a differential ESE display in the two RNAs. This is confirmed by RNA-protein interaction analysis of levels of SR protein binding to each exon. The immunoprecipitation patterns show the presence of complex multi-SR protein-RNA interactions that are lost with secondary-structure variations after the introduction of ESE and ESS variations. Taken together, our results demonstrate that the sequence context, in addition to the primary sequence identity, can heavily contribute to the making of functional units capable of influencing pre-mRNA splicing.

* Corresponding author. Mailing address: ICGEB, Padriciano 99, I-34012 Trieste, Italy. Phone: (39) 040-3757337. Fax: (39) 040-3757361. E-mail: baralle{at}icgeb.org.

{dagger} Present address: Institut für Anatomie und Zellbiologie III, Universität Heidelberg, 69120 Heidelberg, Germany.

Molecular and Cellular Biology, February 2004, p. 1387-1400, Vol. 24, No. 3
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.3.1387-1400.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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