Reed R, Griffith J, Maniatis T
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138.
Cell. 1988 Jun 17;53(6):949-61. doi: 10.1016/s0092-8674(88)90489-8.
Mammalian spliceosomes were purified in preparative amounts by gel filtration chromatography and shown to be functional by in vitro complementation experiments. The column fractions containing spliceosomes are enriched in the snRNAs U1, U2, U4, U5, and U6 and a subset of proteins present in the nuclear extract. Splicing intermediates, the entire set of snRNAs, and the enriched proteins can be immunoprecipitated with three different monoclonal antibodies that recognize snRNP determinants. At least one U1 snRNP is present in each spliceosome since the particles are quantitatively immunoprecipitated by an anti-U1 snRNP monoclonal antibody. Examination of the spliceosome fractions by EM revealed a relatively homogeneous population of 40-60 nm particles with a striking morphology. Evidence that these particles are spliceosomes is their sensitivity to micrococcal nuclease, their ATP-dependent assembly, and their immunoprecipitation with a trimethyl cap monoclonal antibody. In addition, pre-mRNA was visualized in the particles by EM.
通过凝胶过滤色谱法以制备量纯化了哺乳动物剪接体，并通过体外互补实验证明其具有功能。含有剪接体的柱级分富含核提取物中存在的snRNA U1、U2、U4、U5和U6以及一部分蛋白质。剪接中间体、整套snRNA和富集的蛋白质可以用三种识别snRNP决定簇的不同单克隆抗体进行免疫沉淀。由于这些颗粒被抗U1 snRNP单克隆抗体定量免疫沉淀，因此每个剪接体中至少存在一个U1 snRNP。通过电子显微镜检查剪接体级分，发现了一群相对均匀的40-60纳米颗粒，其形态引人注目。这些颗粒是剪接体的证据包括它们对微球菌核酸酶的敏感性、它们依赖ATP的组装以及它们与三甲基帽单克隆抗体的免疫沉淀。此外，通过电子显微镜在颗粒中观察到了前体mRNA。
