Hackl W, Fischer U, Lührmann R
Institut für Molekularbiologie and Tumorforschung, Philipps Universität Marburg, Federal Republic of Germany.
J Cell Biol. 1994 Feb;124(3):261-72. doi: 10.1083/jcb.124.3.261.
The biogenesis of the spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4, and U5 involves: (a) migration of the snRNA molecules from the nucleus to the cytoplasm; (b) assembly of a group of common proteins (Sm proteins) and their binding to a region on the snRNAs called the Sm-binding site; and (c) translocation of the RNP back to the nucleus. A first prerequisite for understanding the assembly pathway and nuclear transport of the snRNPs in more detail is the knowledge of all the snRNP proteins that play essential roles in these processes. We have recently observed a previously undetected 69-kD protein in 12S U1 snRNPs isolated from HeLa nuclear extracts under non-denaturing conditions that is clearly distinct from the U1-70K protein. The following evidence indicates that the 69-kD protein is a common, rather than a U1-specific, protein, possibly associating with the snRNP core particles by protein-protein interaction. (a) Antibodies raised against the 69-kD protein, which did not cross-react with any of the Sm proteins B'-G, precipitated not only U1 snRNPs, but also the other spliceosomal snRNPs U2, U4/U6 and U5, albeit to a lower extent. (b) U1, U2, and U5 core RNP particles reconstituted in vitro contain the 69-kD protein. (c) Xenopus laevis oocytes contain an immunologically related homologue of the human 69-kD protein. When U1 snRNA as well as a mutant U1 snRNA, that can bind the Sm core proteins but lacks the capacity to bind the U1-specific proteins 70K, A, and C, were injected into Xenopus oocytes to allow assembly in vivo, they were recognized by antibodies specific against the 69-kD protein in the ooplasm and in the nucleus. The 69-kD protein is under-represented, if present at all, in purified 17S U2 and in 25S [U4/U6.U5] tri-snRNPs, isolated from HeLa nuclear extracts. Our results are consistent with the working hypothesis that this protein may either play a role in the cytoplasmic assembly of the core domain of the snRNPs and/or in the nuclear transport of the snRNPs. After transport of the snRNPs into the nucleus, it may dissociate from the particles as for example in the case of the 17S U2 or the 25S [U4/U6.U5] tri-snRNP, which bind more than 10 different snRNP specific proteins each in the nucleus.
剪接体小核核糖核蛋白（snRNP）U1、U2、U4和U5的生物合成涉及：（a）snRNA分子从细胞核迁移到细胞质；（b）一组共同蛋白（Sm蛋白）的组装及其与snRNA上一个称为Sm结合位点的区域结合；以及（c）核糖核蛋白（RNP）转运回细胞核。更详细地了解snRNP的组装途径和核运输的首要前提是了解在这些过程中起关键作用的所有snRNP蛋白。我们最近在非变性条件下从HeLa细胞核提取物中分离出的12S U1 snRNP中观察到一种先前未检测到的69-kD蛋白，它与U1-70K蛋白明显不同。以下证据表明69-kD蛋白是一种共同蛋白，而非U1特异性蛋白，可能通过蛋白质-蛋白质相互作用与snRNP核心颗粒结合。（a）针对69-kD蛋白产生的抗体，与任何Sm蛋白B'-G均无交叉反应，它不仅沉淀了U1 snRNP，还沉淀了其他剪接体snRNP U2、U4/U6和U5，尽管程度较低。（b）体外重构的U1、U2和U5核心RNP颗粒含有69-kD蛋白。（c）非洲爪蟾卵母细胞含有与人类69-kD蛋白免疫相关的同源物。当将U1 snRNA以及一种突变的U1 snRNA（其能结合Sm核心蛋白但缺乏结合U1特异性蛋白70K、A和C的能力）注射到非洲爪蟾卵母细胞中以在体内组装时，它们在卵质和细胞核中被针对69-kD蛋白的特异性抗体识别。在从HeLa细胞核提取物中分离出的纯化17S U2和25S [U4/U6.U5]三snRNP中，69-kD蛋白即使存在也含量不足。我们的结果与以下工作假设一致，即该蛋白可能在snRNP核心结构域的细胞质组装和/或snRNP的核运输中发挥作用。在snRNP转运到细胞核后，它可能会从颗粒上解离，例如在17S U2或25S [U4/U6.U5]三snRNP的情况下，它们在细胞核中各自结合超过10种不同的snRNP特异性蛋白。
