Meister G, Bühler D, Pillai R, Lottspeich F, Fischer U
Max-Planck Institute of Biochemistry, Am Klopferspitz 18a, D-82152 Martinsried, Germany.
Nat Cell Biol. 2001 Nov;3(11):945-9. doi: 10.1038/ncb1101-945.
The spliceosomal snRNPs U1, U2, U4 and U5 contain a common RNP structure termed the Sm core that is formed by the binding of Sm proteins onto the U snRNA. Although isolated Sm proteins assemble spontaneously onto U snRNAs in vitro, there is increasing evidence that SMN and its interactor Gemin2 are involved in this process in vivo. Here, we describe a cell-free assay system for the assembly of U snRNPs that closely reproduces in vivo conditions. Using this system, we show that assembly of U1 snRNP depends on ATP. Immunodepletion of SMN-Gemin2 from the extract abolished assembly even though the extract contained high levels of Sm proteins. An affinity-purified macromolecular SMN complex consisting of 16 components including all Sm proteins restored assembly in the immunodepleted extract. These data provide the first direct evidence that a complex containing SMN and Gemin2 mediates the active assembly of spliceosomal U snRNPs.
剪接体小核核糖核蛋白U1、U2、U4和U5含有一种称为Sm核心的常见核糖核蛋白结构，它由Sm蛋白与U小核RNA结合形成。尽管分离出的Sm蛋白在体外能自发地组装到U小核RNA上，但越来越多的证据表明，运动神经元存活蛋白（SMN）及其相互作用蛋白Gemin2在体内参与了这一过程。在此，我们描述了一种用于组装U小核核糖核蛋白的无细胞检测系统，该系统能紧密重现体内条件。利用这个系统，我们表明U1小核核糖核蛋白的组装依赖于ATP。从提取物中免疫去除SMN-Gemin2后，即使提取物中含有高水平的Sm蛋白，组装也会被消除。由包括所有Sm蛋白在内的16种成分组成的亲和纯化大分子SMN复合物可恢复免疫去除提取物中的组装。这些数据首次直接证明，含有SMN和Gemin2的复合物介导了剪接体U小核核糖核蛋白的活性组装。
