Jurica Melissa S, Sousa Duncan, Moore Melissa J, Grigorieff Nikolaus
Howard Hughes Medical Institute, Rosenstiel Basic Medical Sciences Research Center, Department of Biochemistry, Brandeis University, 415 South Street, Waltham, Massachusetts 02454, USA.
Nat Struct Mol Biol. 2004 Mar;11(3):265-9. doi: 10.1038/nsmb728. Epub 2004 Feb 15.
The spliceosome is a multimegadalton RNA-protein machine that removes noncoding sequences from nascent pre-mRNAs. Recruitment of the spliceosome to splice sites and subsequent splicing require a series of dynamic interactions among the spliceosome's component U snRNPs and many additional protein factors. These dynamics present several challenges for structural analyses, including purification of stable complexes to compositional homogeneity and assessment of conformational heterogeneity. We have isolated spliceosomes arrested before the second chemical step of splicing (C complex) in which U2, U5 and U6 snRNAs are stably associated. Using electron microscopy, we obtained images of C complex spliceosomes under cryogenic conditions and determined a three-dimensional structure of a core complex to a resolution of 30 A. The structure reveals a particle of dimensions 27 x 22 x 24 nm with a relatively open arrangement of three primary domains.
剪接体是一种多兆道尔顿的RNA-蛋白质机器，可从新生的前体mRNA中去除非编码序列。剪接体募集到剪接位点以及随后的剪接过程需要剪接体的组成成分U snRNP与许多其他蛋白质因子之间进行一系列动态相互作用。这些动态变化给结构分析带来了几个挑战，包括将稳定复合物纯化至成分均一以及评估构象异质性。我们分离出了在剪接的第二个化学步骤之前停滞的剪接体（C复合物），其中U2、U5和U6 snRNA稳定结合。利用电子显微镜，我们获得了低温条件下C复合物剪接体的图像，并确定了核心复合物的三维结构，分辨率为30埃。该结构揭示了一个尺寸为27×22×24纳米的颗粒，其三个主要结构域排列相对开放。
