A Polar Mechanism Coordinates Different Regions Of Alternative Splicing Within A Single Gene

JUAN PABLO FEDEDA; EZEQUIEL PETRILLO; MIKHAIL S. GELFAND; ANDREI D. NEVEROV; ALBERTO RODOLFO KORNBLIHTT

CSHL, CSH-NY, USA

Congreso; Eukaryotic mRNA Processing Meeting; 2005

About 60% of human genes are alternatively spliced and ~30%of them have more than one alternative region. The fibronectin gene, comprises three alternative regions (EDII,EDI & IIICS). To evaluate if there is a link between the way in which two alternative events are processed in thesame transcript, we transfected mammalian cells with minigenes carrying two alternative exons separated by threeconstitutive exons and analyzed the rate of exon inclusion into mature mRNA by RT-PCR. In a tandem constructwith two EDI exons we found that the proximal splicing event influences the distal one. Disruption of the proximalEDI splicing enhancer (ESE) not only prevents its own inclusion, but favors skipping of the distal EDI exon. Theeffect shows polarity, since disruption of the distal EDI ESE does not affect the proximal one. The polar effect wasobserved in the endogenous fibronectin gene: in cultured murine embryonic fibroblasts (MEFs) that constitutively include EDI, the isoform of total inclusion of IIICS was underepresented in comparison to MEFs which always excludeEDI. Using promoter swapping, we demonstrated that the effect is dependent on the promoter structure. Theseresults provide the first evidence of regulation of one alternative splicing event by another-one located upstream inthe same transcript, and the possible coordination between this regulation and transcription.