Selection of the pre-mRNA branch site (BS) by the U2 small nuclear ribonucleoprotein (snRNP) is crucial to prespliceosome (A complex) assembly. The RNA helicase PRP5 proofreads BS selection but the underlying mechanism remains unclear. Here we report the atomic structures of two sequential complexes leading to prespliceosome assembly: human 17S U2 snRNP and a cross-exon pre-A complex. PRP5 is anchored on 17S U2 snRNP mainly through occupation of the RNA path of SF3B1 by an acidic loop of PRP5; the helicase domain of PRP5 associates with U2 snRNA; the BS-interacting stem-loop (BSL) of U2 snRNA is shielded by TAT-SF1, unable to engage the BS. In the pre-A complex, an initial U2–BS duplex is formed; the translocated helicase domain of PRP5 stays with U2 snRNA and the acidic loop still occupies the RNA path. The pre-A conformation is specifically stabilized by the splicing factors SF1, DNAJC8 and SF3A2. Cancer-derived mutations in SF3B1 damage its association with PRP5, compromising BS proofreading. Together, these findings reveal key insights into prespliceosome assembly and BS selection or proofreading by PRP5.

U2 小核核糖核蛋白 (snRNP) 对前 mRNA 分支位点 (BS) 的选择对于前剪接体（A 复合物）的组装至关重要。 RNA 解旋酶 PRP5 校对 BS 选择，但潜在机制仍不清楚。在这里，我们报告了导致前剪接体组装的两个连续复合物的原子结构：人17S U2 snRNP和跨外显子前A复合物。 PRP5 主要通过 PRP5 的酸性环占据 SF3B1 的 RNA 路径而锚定在 17S U2 snRNP 上； PRP5 的解旋酶结构域与 U2 snRNA 结合； U2 snRNA 的 BS 相互作用茎环 (BSL) 被 TAT-SF1 屏蔽，无法与 BS 结合。在 pre-A 复合体中，形成初始 U2-BS 双链体； PRP5 的易位解旋酶结构域保留在 U2 snRNA 中，并且酸性环仍然占据 RNA 路径。前 A 构象由剪接因子 SF1、DNAJC8 和 SF3A2 特异性稳定。 SF3B1 的癌症衍生突变会损害其与 PRP5 的关联，从而损害 BS 校对。总之，这些发现揭示了对前剪接体组装和 BS 选择或 PRP5 校对的重要见解。