Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences and School of Medicine, Tsinghua University, Beijing 100084, China.
Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences and School of Medicine, Tsinghua University, Beijing 100084, China; Technology Center for Protein Sciences, Ministry of Education Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China.
Cell. 2019 Apr 4;177(2):339-351.e13. doi: 10.1016/j.cell.2019.02.006. Epub 2019 Mar 14.
Pre-mRNA splicing is executed by the spliceosome. Structural characterization of the catalytically activated complex (B) is pivotal for understanding the branching reaction. In this study, we assembled the B complexes on two different pre-mRNAs from Saccharomyces cerevisiae and determined the cryo-EM structures of four distinct B complexes at overall resolutions of 2.9-3.8 Å. The duplex between U2 small nuclear RNA (snRNA) and the branch point sequence (BPS) is discretely away from the 5'-splice site (5'SS) in the three B complexes that are devoid of the step I splicing factors Yju2 and Cwc25. Recruitment of Yju2 into the active site brings the U2/BPS duplex into the vicinity of 5'SS, with the BPS nucleophile positioned 4 Å away from the catalytic metal M2. This analysis reveals the functional mechanism of Yju2 and Cwc25 in branching. These structures on different pre-mRNAs reveal substrate-specific conformations of the spliceosome in a major functional state.
前体 mRNA 剪接由剪接体执行。对于理解分支反应，催化激活复合物 (B) 的结构特征至关重要。在这项研究中，我们在来自酿酒酵母的两种不同前体 mRNA 上组装了 B 复合物，并在整体分辨率为 2.9-3.8Å 的情况下确定了四个不同 B 复合物的 cryo-EM 结构。在缺乏步骤 I 剪接因子 Yju2 和 Cwc25 的三个 B 复合物中，U2 小核 RNA (snRNA) 和分支点序列 (BPS) 之间的双链离散地远离 5'-剪接位点 (5'SS)。Yju2 招募到活性位点使 U2/BPS 双链进入 5'SS 的附近，BPS 亲核试剂定位于催化金属 M2 4Å 之外。该分析揭示了 Yju2 和 Cwc25 在分支中的功能机制。这些不同前体 mRNA 上的结构揭示了剪接体在主要功能状态下的底物特异性构象。
