Remodeling an RNA processing machine Splicing of precursor messenger RNA (pre-mRNA) is carried out by the spliceosome, a highly dynamic, supramolecular complex that undergoes assembly, activation, catalysis, and disassembly. These essential spliceosome remodeling events are driven by a conserved family of adenosine triphosphatase (ATPase)/helicases. In the presence of its coactivator Spp2, the ATPase/helicase Prp2 associates with the activated spliceosome and translocates the single-stranded pre-mRNA toward its 3′ end. Bai et al. now report the cryo–electron microscopy structures of Prp2 both before and after recruitment into the activated spliceosome. These structures and the associated biochemical analysis reveal how Prp2 remodels the activated spliceosome and how Spp2 safeguards the function of Prp2. Science, this issue p. eabe8863 A cryo-EM study of the yeast spliceosome reveals how RNA helicase Prp2 ensures the unidirectionality of pre-mRNA translocation towards its 3′ end. INTRODUCTION Splicing of precursor messenger RNA (pre-mRNA) is carried out by the spliceosome. During each splicing cycle, the spliceosome undergoes assembly, activation, catalysis, and disassembly. The assembled spliceosome exists in eight major functional states. Spliceosome remodeling between neighboring states is driven by conserved adenosine triphosphatase (ATPase)/helicases. Together with its coactivator Spp2, the ATPase/helicase Prp2 harnesses the energy of adenosine 5′-triphosphate (ATP) binding and hydrolysis to translocate 3′-to-5′ on the single-stranded pre-mRNA, thus remodeling the activated spliceosome (known as the Bact complex) into the catalytically activated spliceosome (the B* complex). The B* complex catalyzes the branching reaction. Since 2015, cryo–electron microscopy (cryo-EM) structures of all major states of the assembled spliceosome have been determined at near-atomic resolutions, generating crucial information on the active site and overall organization of the spliceosome. How spliceosome remodeling occurs, however, remains poorly understood because none of the ATPase/helicases in the presence of the spliceosome has been visualized in atomic details owing to limited resolution. In particular, it remains largely unknown how Prp2 remodels the Bact complex and why Prp2 requires its coactivator Spp2. RATIONALE To address these questions, we need to examine the detailed structural features of Prp2 and Spp2 in the Bact complex and compare these features with those of Prp2 and Spp2 in isolation. Atomic resolution is required, which may need improvement in sample preparation. Structure-guided biochemical analyses may be needed to corroborate the conclusions. RESULTS We used galactose-inducible expression of an ATPase-defective Prp2 mutant to stall remodeling of the endogenous Bact complex. This strategy resulted in marked enrichment of the Bact complex in the final cryo-EM sample. We determined the cryo-EM structure of the S. cerevisiae Bact complex at 2.5 Å—the highest resolution achieved for an intact spliceosome to date—which allows atomic identification of 12 new proteins, including Prp2 and Spp2 (see the figure). Prp2 weakly associates with the spliceosome and fails to function in the absence of Spp2. Spp2 uses its C-terminal sequences to stably associate with Prp2 and its N-terminal sequences to anchor on the spliceosome, thus tethering Prp2 to the Bact complex and allowing Prp2 to function. In the spliceosome, pre-mRNA is loaded into a featured channel between the N and C halves of Prp2, where Leu536 from the N half and Arg844 from the C half serve as barbed wires to prevent backward sliding of pre-mRNA toward its 5′ end. Conserved residues in the channel make hydrogen bonds mainly to the backbone phosphates, but not the nucleobases, of the pre-mRNA, explaining its sequence-independent recognition by Prp2. We then determined the cryo-EM structures of Prp2 in three related functional states: free Prp2, Spp2-bound Prp2, and Spp2-bound ADP-loaded Prp2. These structures, together with that of Prp2 in the Bact complex and other published information, yield a working mechanism for Prp2. Relative movement between the RecA1 and RecA2 domains of Prp2, driven by ATP binding and hydrolysis, results in pre-mRNA translocation. In step 1, ATP binding to RecA1 is predicted to trigger the movement of RecA2 toward RecA1, allowing Leu536 to push the nucleobase of pre-mRNA to translocate by one nucleotide toward its 3′ end. Because of the translocation, Arg844 loses its association with the departing nucleobase and associates with the newly arrived upstream nucleobase. In step 2, ATP hydrolysis leads to the relaxation of RecA2 back to its original position, allowing Leu536 to shift its interactions to an upstream nucleobase. The movement of RecA2 is safeguarded by Arg844, which may prevent backward sliding of pre-mRNA. Last, ADP is released, and Prp2 is reset for the next cycle. In our model, Leu536 and Arg844 serve as two hands to alternately bind and push pre-mRNA toward its 3′ end. CONCLUSION As a coactivator, Spp2 stably associates with Prp2 and tethers it to Bact, enabling Prp2 function. ATP binding and hydrolysis trigger interdomain movement in Prp2, which allows Leu536 and Arg844 to alternately bind and push pre-mRNA unidirectionally toward its 3′ end. Mechanism of action for the ATPase/helicase Prp2 in the activated spliceosome. (A) Structure of the Bact complex. (B) Spp2 uses its N-terminal sequences to anchor on the spliceosome and its C-terminal sequences to associate with Prp2, tethering Prp2 to the spliceosome and enabling its function. (C) Leu536 (L536) and Arg844 (R844) of Prp2 prevent backward sliding of pre-mRNA by only allowing 5′-to-3′ movement of the RNA bases. Spliceosome remodeling, executed by conserved adenosine triphosphatase (ATPase)/helicases including Prp2, enables precursor messenger RNA (pre-mRNA) splicing. However, the structural basis for the function of the ATPase/helicases remains poorly understood. Here, we report atomic structures of Prp2 in isolation, Prp2 complexed with its coactivator Spp2, and Prp2-loaded activated spliceosome and the results of structure-guided biochemical analysis. Prp2 weakly associates with the spliceosome and cannot function without Spp2, which stably associates with Prp2 and anchors on the spliceosome, thus tethering Prp2 to the activated spliceosome and allowing Prp2 to function. Pre-mRNA is loaded into a featured channel between the N and C halves of Prp2, where Leu536 from the N half and Arg844 from the C half prevent backward sliding of pre-mRNA toward its 5′-end. Adenosine 5′-triphosphate binding and hydrolysis trigger interdomain movement in Prp2, which drives unidirectional stepwise translocation of pre-mRNA toward its 3′-end. These conserved mechanisms explain the coupling of spliceosome remodeling to pre-mRNA splicing.

中文翻译：

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ATPase/解旋酶 Prp2 及其辅激活因子 Spp2 对剪接体重塑的机制

重塑 RNA 加工机器前体信使 RNA (pre-mRNA) 的剪接是由剪接体进行的，剪接体是一种高度动态的超分子复合物，经过组装、激活、催化和分解。这些重要的剪接体重塑事件是由保守的三磷酸腺苷酶 (ATPase)/解旋酶家族驱动的。在其共激活剂 Spp2 的存在下，ATPase/解旋酶 Prp2 与激活的剪接体结合，并将单链前体 mRNA 向其 3' 末端转移。白等人。现在报告 Prp2 在募集到激活的剪接体之前和之后的冷冻电子显微镜结构。这些结构和相关的生化分析揭示了 Prp2 如何重塑激活的剪接体以及 Spp2 如何保护 Prp2 的功能。科学，这个问题 p。eabe8863 酵母剪接体的冷冻电镜研究揭示了 RNA 解旋酶 Prp2 如何确保前体 mRNA 向其 3' 末端的单向易位。前体信使RNA（pre-mRNA）的剪接由剪接体进行。在每个剪接周期中，剪接体经历组装、活化、催化和分解。组装的剪接体以八种主要功能状态存在。相邻状态之间的剪接体重塑是由保守的三磷酸腺苷酶 (ATPase)/解旋酶驱动的。ATP 酶/解旋酶 Prp2 与其共激活剂 Spp2 一起利用 5'-三磷酸腺苷 (ATP) 结合和水解的能量将单链前 mRNA 上的 3'-5' 易位，从而将活化的剪接体（称为 Bact 复合体）重塑为催化活化的剪接体（B* 复合体）。B* 络合物催化支化反应。自 2015 年以来，组装的剪接体所有主要状态的冷冻电子显微镜 (cryo-EM) 结构都已在近原子分辨率下被确定，从而产生了关于剪接体的活性位点和整体组织的关键信息。然而，人们对剪接体重构如何发生仍然知之甚少，因为由于分辨率有限，剪接体存在下的任何 ATP 酶/解旋酶都没有在原子细节中进行可视化。特别是，Prp2 如何重塑 Bact 复合物以及为什么 Prp2 需要其辅激活剂 Spp2 仍然很大程度上未知。基本原理 为了解决这些问题，我们需要检查 Bact 复合体中 Prp2 和 Spp2 的详细结构特征，并将这些特征与 Prp2 和 Spp2 的特征进行单独比较。需要原子分辨率，这可能需要改进样品制备。可能需要结构引导的生化分析来证实结论。结果我们使用半乳糖诱导的 ATPase 缺陷 Prp2 突变体的表达来阻止内源性 Bact 复合物的重塑。该策略导致最终冷冻电镜样品中 Bact 复合物的显着富集。我们确定了 2.5 Å 酿酒酵母 Bact 复合物的冷冻电镜结构——迄今为止完整剪接体实现的最高分辨率——这允许原子识别 12 种新蛋白质，包括 Prp2 和 Spp2（见图）。Prp2 与剪接体弱相关，并且在没有 Spp2 的情况下无法发挥作用。Spp2 使用其 C 端序列与 Prp2 稳定结合，其 N 端序列锚定在剪接体上，从而将 Prp2 连接到 Bact 复合物并允许 Prp2 发挥作用。在剪接体中，pre-mRNA 被加载到 Prp2 的 N 和 C 半之间的一个特征通道中，其中来自 N 半的 Leu536 和来自 C 半的 Arg844 作为带刺的铁丝，以防止前 mRNA 向其 5' 向后滑动结尾。通道中的保守残基主要与前体 mRNA 的磷酸骨架形成氢键，而不是与核碱基形成氢键，这解释了 Prp2 的序列独立识别。然后，我们确定了 Prp2 在三种相关功能状态下的冷冻电镜结构：游离 Prp2、Spp2 结合的 Prp2 和 Spp2 结合的 ADP 加载的 Prp2。这些结构，连同 Bact 复合体中 Prp2 的结构和其他已发表的信息，产生了 Prp2 的工作机制。Prp2 的 RecA1 和 RecA2 域之间的相对运动，由 ATP 结合和水解驱动，导致前体 mRNA 易位。在步骤 1 中，ATP 与 RecA1 的结合预计会触发 RecA2 向 RecA1 的移动，从而允许 Leu536 推动前 mRNA 的核碱基向其 3' 末端移位一个核苷酸。由于易位，Arg844 失去了与离开的核碱基的关联，并与新到达的上游核碱基关联。在步骤 2 中，ATP 水解导致 RecA2 松弛回到其原始位置，允许 Leu536 将其相互作用转移到上游核碱基。RecA2 的运动由 Arg844 保护，这可以防止pre-mRNA向后滑动。最后，ADP 被释放，Prp2 被重置为下一个周期。在我们的模型中，Leu536 和 Arg844 作为两只手交替结合并将前 mRNA 推向其 3' 末端。结论作为共激活剂，Spp2 与 Prp2 稳定结合并将其束缚在 Bact 上，从而启用 Prp2 功能。ATP 结合和水解触发 Prp2 中的域间运动，这允许 Leu536 和 Arg844 交替结合并将前 mRNA 单向推向其 3' 末端。活化剪接体中 ATP 酶/解旋酶 Prp2 的作用机制。(A) Bact 复合体的结构。(B) Spp2 使用其 N 端序列锚定在剪接体上，其 C 端序列与 Prp2 相关联，将 Prp2 连接到剪接体并启用其功能。(C) Prp2 的 Leu536 (L536) 和 Arg844 (R844) 通过仅允许 RNA 碱基的 5' 到 3' 移动来防止前 mRNA 向后滑动。剪接体重塑由保守的三磷酸腺苷酶 (ATPase)/解旋酶（包括 Prp2）执行，使前体信使 RNA（pre-mRNA）能够剪接。然而，ATPase/解旋酶功能的结构基础仍然知之甚少。在这里，我们单独报告 Prp2 的原子结构，Prp2 与其共激活剂 Spp2 复合，以及负载 Prp2 的激活剪接体以及结构引导的生化分析结果。Prp2 与剪接体弱结合，没有 Spp2 就不能发挥作用，Spp2 稳定地与 Prp2 结合并锚定在剪接体上，从而将 Prp2 拴在激活的剪接体上并允许 Prp2 发挥作用。Pre-mRNA 被加载到 Prp2 的 N 和 C 半之间的一个特征通道中，其中来自 N 半的 Leu536 和来自 C 半的 Arg844 防止前 mRNA 向其 5' 端向后滑动。腺苷 5'-三磷酸结合和水解触发 Prp2 中的域间运动，从而驱动前体 mRNA 向其 3' 末端单向逐步易位。这些保守的机制解释了剪接体重塑与前 mRNA 剪接的耦合。