剪接体通过四个连续阶段执行pre-mRNA剪接:组装,激活,催化作用,和拆卸。剪接体的激活,即将预催化剪接体(B复合物)重塑为活化剪接体(Bact复合物)和催化活化剪接体(B*复合物),涉及蛋白质成分和结构重排的主要流动。依靠剪接抑制剂,我们已经捕获了B和B*复合物之间的六个中间状态:pre-Bact,Bact-I,Bact-II,Bact-III,Bact-IV,和事后。他们的低温EM结构,以及催化步骤I剪接体(C络合物)的改进结构,揭示了催化中心如何在U6snRNA的内部茎环周围成熟,分支站点如何接近5'-拼接站点,RNA解旋酶PRP2如何重排以结合pre-mRNA,以及U2snRNP如何经历显著的运动以促进激活。我们确定了PRP2在剪接体激活中先前未被识别的关键作用。我们的研究概括了人类剪接体在催化活化过程中的分子编排。
The spliceosome executes pre-mRNA splicing through four sequential stages: assembly, activation, catalysis, and disassembly. Activation of the spliceosome, namely remodeling of the pre-catalytic spliceosome (B complex) into the activated spliceosome (Bact complex) and the catalytically activated spliceosome (B* complex), involves major flux of protein components and structural rearrangements. Relying on a splicing inhibitor, we have captured six intermediate states between the B and B* complexes: pre-Bact, Bact-I, Bact-II, Bact-III, Bact-IV, and post-Bact. Their cryo-EM structures, together with an improved structure of the catalytic step I spliceosome (C complex), reveal how the catalytic center matures around the internal stem loop of U6 snRNA, how the branch site approaches 5\'-splice site, how the RNA helicase PRP2 rearranges to bind pre-mRNA, and how U2 snRNP undergoes remarkable movement to facilitate activation. We identify a previously unrecognized key role of PRP2 in spliceosome activation. Our study recapitulates a molecular choreography of the human spliceosome during its catalytic activation.