Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.
Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany. Bioanalytics Group, Institute for Clinical Chemistry, University Medical Center Göttingen, D-37075 Göttingen, Germany.
Science. 2016 Mar 25;351(6280):1416-20. doi: 10.1126/science.aad2085. Epub 2016 Feb 18.
The U4/U6.U5 triple small nuclear ribonucleoprotein (tri-snRNP) is a major spliceosome building block. We obtained a three-dimensional structure of the 1.8-megadalton human tri-snRNP at a resolution of 7 angstroms using single-particle cryo-electron microscopy (cryo-EM). We fit all known high-resolution structures of tri-snRNP components into the EM density map and validated them by protein cross-linking. Our model reveals how the spatial organization of Brr2 RNA helicase prevents premature U4/U6 RNA unwinding in isolated human tri-snRNPs and how the ubiquitin C-terminal hydrolase-like protein Sad1 likely tethers the helicase Brr2 to its preactivation position. Comparison of our model with cryo-EM three-dimensional structures of the Saccharomyces cerevisiae tri-snRNP and Schizosaccharomyces pombe spliceosome indicates that Brr2 undergoes a marked conformational change during spliceosome activation, and that the scaffolding protein Prp8 is also rearranged to accommodate the spliceosome's catalytic RNA network.
U4/U6.U5 三重小核核糖核蛋白（tri-snRNP）是剪接体的主要组成部分。我们使用单颗粒冷冻电镜（cryo-EM）获得了分辨率为 7 埃的 1.8 兆道尔顿人源 tri-snRNP 的三维结构。我们将所有已知的 tri-snRNP 组分的高分辨率结构拟合到 EM 密度图中，并通过蛋白质交联进行了验证。我们的模型揭示了 Brr2 RNA 解旋酶的空间组织如何防止在分离的人源 tri-snRNPs 中过早解开 U4/U6 RNA，以及泛素 C 末端水解酶样蛋白 Sad1 如何将解旋酶 Brr2 固定在其预激活位置。与酿酒酵母 tri-snRNP 和裂殖酵母剪接体的 cryo-EM 三维结构的比较表明，Brr2 在剪接体激活过程中发生显著的构象变化，支架蛋白 Prp8 也进行了重排以适应剪接体的催化 RNA 网络。
