INTERACTION BETWEEN SUBUNITS OF HETERODIMERIC SPLICING FACTOR U2AF ISESSENTIAL IN-VIVO

The heterodimeric pre-mRNA splicing factor, U2AF (U2 snRNP auxiliary f actor), plays a critical role in 3' splice site selection. Although th e U2AF subunits associate in a tight complex, biochemical experiments designed to address the requirement for both subunits ire splicing hav e yielded conflicting results. We have taken a genetic approach to ass ess the requirement for the Drosophila U2AF heterodimer in vivo. We de veloped a novel Escherichia coli copurification assay to map the domai n on the Drosophila U2AF large subunit (dU2AF(50)) that interacts with the Drosophila small subunit (dU2AF(38)). A 28-amino-acid fragment on dU2AF(50) that is both necessary and sufficient for interaction with dU2AF(38) was identified, Using the copurification assay, we scanned t his 28-amino-acid interaction domain for mutations that abrogate heter odimer formation. A collection of these dU2AF(50) point mutants was th en tested in vivo, for genetic complementation of a recessive lethal d U2AF(50) allele. A mutation that completely abolished interaction with dU2AF(38) was incapable of complementation, whereas dU2AF(50) mutatio ns that did not effect heterodimer formation rescued the recessive let hal dU2AF(50) allele, Analysis of heterodimer formation in embryo extr acts derived from these interaction mutant lines revealed a perfect co rrelation between the efficiency of subunit association and the abilit y to complement the dU2AF(50) recessive lethal allele. These data indi cate that Drosophila U2AF heterodimer formation is essential for viabi lity in vivo, consistent with a requirement for both subunits in splic ing in vitro.