Ohi Melanie D, Ren Liping, Wall Joseph S, Gould Kathleen L, Walz Thomas
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Proc Natl Acad Sci U S A. 2007 Feb 27;104(9):3195-200. doi: 10.1073/pnas.0611591104. Epub 2007 Feb 20.
The spliceosome is a dynamic macromolecular machine that catalyzes the excision of introns from pre-mRNA. The megadalton-sized spliceosome is composed of four small nuclear RNPs and additional pre-mRNA splicing factors. The formation of an active spliceosome involves a series of regulated steps that requires the assembly and disassembly of large multiprotein/RNA complexes. The dynamic nature of the pre-mRNA splicing reaction has hampered progress in analyzing the structure of spliceosomal complexes. We have used cryo-electron microscopy to produce a 29-A density map of a stable 37S spliceosomal complex from the genetically tractable fission yeast, Schizosaccharomyces pombe. Containing the U2, U5, and U6 snRNAs, pre-mRNA splicing intermediates, U2 and U5 snRNP proteins, the Nineteen Complex (NTC), and second-step splicing factors, this complex closely resembles in vitro purified mammalian C complex. The density map reveals an asymmetric particle, approximately 30 x 20 x 18 nm in size, which is composed of distinct domains that contact each other at the center of the complex.
剪接体是一种动态的大分子机器，可催化从前体mRNA中切除内含子。兆道尔顿大小的剪接体由四个小核核糖核蛋白颗粒和其他前体mRNA剪接因子组成。活性剪接体的形成涉及一系列受调控的步骤，这需要大型多蛋白/RNA复合物的组装和拆卸。前体mRNA剪接反应的动态性质阻碍了剪接体复合物结构分析的进展。我们利用冷冻电子显微镜技术，获得了来自遗传背景易于研究的裂殖酵母粟酒裂殖酵母的稳定37S剪接体复合物的29埃密度图。该复合物包含U2、U5和U6小核RNA、前体mRNA剪接中间体、U2和U5小核核糖核蛋白颗粒蛋白、十九复合物（NTC）以及第二步剪接因子，与体外纯化的哺乳动物C复合物极为相似。密度图显示该复合物是一个不对称颗粒，大小约为30×20×18纳米，由在复合物中心相互接触的不同结构域组成。
