A POLAR MECHANISM COORDINATES DIFFERENT REGIONS OF ALTERNATIVE SPLICING WITHIN A GENE

JUAN PABLO FEDEDA; EZEQUIEL PETRILLO; ALBERTO RODOLFO KORNBLIHTT

Pinamar, Buenos Aires, Argentina

Congreso; SAIB, XLI Reunión Anual, Sociedad Argentina de Investigación Bioquímica y Biología Molecular, SAN, XX Reunión Anual, Sociedad Argentina de Neuroquímica; 2005

About 60% of human genes are alternatively spliced and ~30% of them have more than one alternative region. Toevaluate if there is a link between the way in which two alternative events are processed in the same transcript,we transfected mammalian cells with minigenes carrying two alternative regions separated by three constitutiveexons and analyzed the rate of exon inclusion into mature mRNA by RT-PCR. We found that, mutations whicheither inhibit or stimulate inclusion of the upstream alternative exon deeply affect inclusion of the downstream one.However, similar mutations at the downstream alternative exon have little effect on the upstream one. This polareffect is promoter specific and is enhanced by inhibition of transcriptional elongation. Consistently, cultured murineembryonic fibroblasts from mutant mice with either constitutive or null inclusion of a fibronectin alternative exonrevealed coordination with a second alternative splicing region located far downstream . Using allele-specific RTPCR,we demonstrate that this coordination occurs in cis. Bioinformatics supports the generality of these findings,identifying several genes with nonrandom distribution of mRNA isoforms at two alternative regions.