Rudner D Z, Kanaar R, Breger K S, Rio D C
Department of Molecular and Cell Biology, University of California, Berkeley 94720-3204, USA.
Mol Cell Biol. 1998 Apr;18(4):1765-73. doi: 10.1128/MCB.18.4.1765.
The heterodimeric pre-mRNA splicing factor, U2AF (U2 snRNP auxiliary factor), plays a critical role in 3' splice site selection. Although the U2AF subunits associate in a tight complex, biochemical experiments designed to address the requirement for both subunits in splicing have yielded conflicting results. We have taken a genetic approach to assess the requirement for the Drosophila U2AF heterodimer in vivo. We developed a novel Escherichia coli copurification assay to map the domain on the Drosophila U2AF large subunit (dU2AF50) that interacts with the Drosophila small subunit (dU2AF38). A 28-amino-acid fragment on dU2AF50 that is both necessary and sufficient for interaction with dU2AF38 was identified. Using the copurification assay, we scanned this 28-amino-acid interaction domain for mutations that abrogate heterodimer formation. A collection of these dU2AF50 point mutants was then tested in vivo for genetic complementation of a recessive lethal dU2AF50 allele. A mutation that completely abolished interaction with dU2AF38 was incapable of complementation, whereas dU2AF50 mutations that did not effect heterodimer formation rescued the recessive lethal dU2AF50 allele. Analysis of heterodimer formation in embryo extracts derived from these interaction mutant lines revealed a perfect correlation between the efficiency of subunit association and the ability to complement the dU2AF50 recessive lethal allele. These data indicate that Drosophila U2AF heterodimer formation is essential for viability in vivo, consistent with a requirement for both subunits in splicing in vitro.
异源二聚体前体mRNA剪接因子U2AF（U2 snRNP辅助因子）在3'剪接位点选择中起关键作用。尽管U2AF亚基以紧密复合物的形式结合，但旨在研究剪接过程中两个亚基需求的生化实验却得出了相互矛盾的结果。我们采用遗传学方法来评估果蝇U2AF异源二聚体在体内的需求。我们开发了一种新颖的大肠杆菌共纯化测定法，以绘制果蝇U2AF大亚基（dU2AF50）上与果蝇小亚基（dU2AF38）相互作用的结构域。在dU2AF50上鉴定出一个28个氨基酸的片段，该片段对于与dU2AF38相互作用既是必需的也是足够的。使用共纯化测定法，我们在这个28个氨基酸的相互作用结构域中扫描了消除异源二聚体形成的突变。然后在体内测试了这些dU2AF50点突变体的集合，以检测隐性致死dU2AF50等位基因的遗传互补情况。一个完全消除与dU2AF38相互作用的突变无法互补，而不影响异源二聚体形成的dU2AF50突变则挽救了隐性致死dU2AF50等位基因。对源自这些相互作用突变体系的胚胎提取物中异源二聚体形成的分析表明，亚基结合效率与互补dU2AF50隐性致死等位基因的能力之间存在完美的相关性。这些数据表明，果蝇U2AF异源二聚体的形成对于体内生存能力至关重要，这与体外剪接中两个亚基的需求一致。
