Pre-mRNA splicing involves two sequential reactions: branching and exon ligation. The C complex after branching undergoes remodeling to become the C^∗ complex, which executes exon ligation. Here, we report cryo-EM structures of two intermediate human spliceosomal complexes, pre-C^∗-I and pre-C^∗-II, both at 3.6 Å. In both structures, the 3′ splice site is already docked into the active site, the ensuing 3′ exon sequences are anchored on PRP8, and the step II factor FAM192A contacts the duplex between U2 snRNA and the branch site. In the transition of pre-C^∗-I to pre-C^∗-II, the step II factors Cactin, FAM32A, PRKRIP1, and SLU7 are recruited. Notably, the RNA helicase PRP22 is positioned quite differently in the pre-C^∗-I, pre-C^∗-II, and C^∗ complexes, suggesting a role in 3′ exon binding and proofreading. Together with information on human C and C^∗ complexes, our studies recapitulate a molecular choreography of the C-to-C^∗ transition, revealing mechanistic insights into exon ligation.

Pre-mRNA 剪接涉及两个连续反应：分支和外显子连接。分支后的 C 复合体经过重构成为 C ^∗复合体，执行外显子连接。在这里，我们报告了两个中间人剪接体复合物的低温-EM 结构，pre-C ^* -I 和 pre-C ^* -II，均为 3.6 Å。在这两种结构中，3' 剪接位点已经停靠到活性位点，随后的 3' 外显子序列锚定在 PRP8 上，步骤 II 因子 FAM192A 接触 U2 snRNA 和分支位点之间的双链体。在 pre-C ^∗ -I 到 pre-C ^{∗的过渡中}-II，步骤 II 因子 Cactin、FAM32A、PRKRIP1 和 SLU7 被招募。值得注意的是，RNA 解旋酶 PRP22 在 pre-C ^* -I、pre-C ^* -II 和 C ^*复合物中的定位完全不同，这表明它在 3' 外显子结合和校对中起作用。连同有关人类 C 和 C ^*复合物的信息，我们的研究概括了 C 到 C ^*转换的分子编排，揭示了外显子连接的机制见解。