Spliceosome is often assembled across an exon and undergoes rearrangement to span a neighboring intron. Most states of the intron-defined spliceosome have been structurally characterized. However, the structure of a fully assembled exon-defined spliceosome remains at large. During spliceosome assembly, the pre-catalytic state (B complex) is converted from its precursor (pre-B complex). Here we report atomic structures of the exon-defined human spliceosome in four sequential states: mature pre-B, late pre-B, early B, and mature B. In the previously unknown late pre-B state, U1 snRNP is already released but the remaining proteins are still in the pre-B state; unexpectedly, the RNAs are in the B state, with U6 snRNA forming a duplex with 5′-splice site and U5 snRNA recognizing the 3′-end of the exon. In the early and mature B complexes, the B-specific factors are stepwise recruited and specifically recognize the exon 3′-region. Our study reveals key insights into the assembly of the exon-defined spliceosomes and identifies mechanistic steps of the pre-B-to-B transition.

剪接体通常在外显子上组装，并进行重排以跨越邻近的内含子。内含子定义的剪接体的大多数状态已被结构表征。然而，完全组装的外显子定义的剪接体的结构仍然存在。在剪接体组装过程中，预催化状态（B 复合物）从其前体（前 B 复合物）转变而来。在这里，我们报告了外显子定义的人类剪接体在四种连续状态下的原子结构：成熟前 B、晚期前 B、早期 B 和成熟 B。在先前未知的晚期前 B 状态中，U1 snRNP 已经释放，但其余蛋白质仍处于前B状态；出乎意料的是，RNA 处于 B 状态，U6 snRNA 形成具有 5' 剪接位点的双链体，U5 snRNA 识别外显子的 3' 末端。在早期和成熟的 B 复合物中，B 特异性因子被逐步招募并特异性识别外显子 3' 区域。我们的研究揭示了外显子定义的剪接体组装的关键见解，并确定了前 B 到 B 转变的机制步骤。