Polar Effect in Alternative Splicing Regulation

EZEQUIEL PETRILLO; JUAN PABLO FEDEDA; ALBERTO RODOLFO KORNBLIHTT

Iguazú, MIsiones, Argentina

Congreso; SAIB, XL Reunión Anual, SOCIEDAD ARGENTINA DE INVESTIGACIÓN, BIOQUÍMICA Y BIOLOGÍA MOLECULAR; 2004

About 60% of human genes are alternatively spliced and ~30% ofthem have more than one alternative region. The fibronectin gene,comprises three alternative regions (EDII, EDI & IIICS). Toevaluate if there is a link between the way in which two alternativeevents are processed in the same transcript, we transfectedmammalian cells with minigenes carrying two alternative exonsseparated by three constitutive exons and analyzed the rate of exoninclusion into mature mRNA by RT-PCR. In a tandem constructwith two EDI exons we found that the proximal splicing eventinfluences the distal one. Disruption of the proximal EDI splicingenhancer (ESE) not only prevents its own inclusion, but favorsskipping of the distal EDI exon. The effect shows polarity, sincedisruption of the distal EDI ESE does not affect the proximal one.The polar effect was observed in the endogenous fibronectin gene:in cultured murine embryonic fibroblasts (MEFs) thatconstitutively include EDI, the isoform of total inclusion of IIICSwas underepresented in comparison to MEFs which always excludeEDI. Using promoter swapping, we demonstrated that the effectis dependent on the promoter structure. These results provide thefirst evidence of regulation of one alternative splicing event byanother-one located upstream in the same transcript, and thepossible coordination between this regulation and transcription.