Splicing machine shifts into gear Spliceosome activation involves extensive protein exchanges and RNA rearrangements that lead to the formation of a catalytically active U2/U6 RNA structure called Bact. Previously, little was known about the pathway leading to the U2/U6 active site and how proteins aid in folding the U2/U6 RNA. Using cryo–electron microscopy to determine structures of two human pre-Bact complexes, Townsend et al. uncovered an intricate cascade of coordinated structural changes involving mutually exclusive interactions that facilitate the directionality of the activation process. These structures reveal the assembly pathway of the U2/U6 catalytic RNA and the mechanism whereby proteins facilitate its folding. Science, this issue p. eabc3753 A cryo–electron microscopy study reveals the structure of human pre-Bact spliceosomes. INTRODUCTION The spliceosome, including its catalytic center, is formed anew on each pre-mRNA intron, through a pathway involving multiple, successive assembly intermediates. Spliceosome activation involves extensive protein exchanges and RNA rearrangements that lead to the formation of a catalytically active U2/U6 RNA structure. As of now, little is known about the assembly pathway of the latter and the mechanism whereby proteins aid its proper folding. RATIONALE To elucidate the complex rearrangements that occur during transformation of a spliceosomal B complex into an activated spliceosome (i.e., Bact complex), we blocked spliceosome assembly at previously uncharacterized, intermediate stages of activation and determined the structure of purified human pre-Bact complexes. RESULTS The cryo–electron microscopy (cryo-EM) structures of two distinct, human pre-Bact complexes (denoted pre-Bact-1 and pre-Bact-2) that lack a mature catalytic U2/U6 RNA structure were obtained at core resolutions of 3.9 and 4.2 Å, and a pseudo-atomic model of each complex was generated using an integrative structural biology approach. Their composition and molecular architecture indicate that pre-Bact-1 is a precursor to pre-Bact-2, and chase experiments demonstrate that they are functional spliceosome intermediates. The pre-Bact-1 and pre-Bact-2 structures, aided by biochemical analyses, provide new insight into the order of protein exchanges during Bact formation. They also elucidate a number of mutually exclusive protein-protein and protein-RNA interactions that ensure a productive pathway of ribonucleoprotein (RNP) rearrangements needed to form the U2/U6 catalytic RNA and reveal new roles for the so-called B-specific proteins. They show that there is a stepwise repositioning of BRR2 and U2 small nuclear RNP during activation, the latter being a prerequisite for bringing U2 and U6 small nuclear RNA (snRNA) into sufficiently close proximity to form U2/U6 helix I. Furthermore, they indicate that the proteins TCERG1, WBP11, CTNNBL1, and KIN17, which interact transiently with the spliceosome, stabilize intermediate RNP conformational states of the pre-Bact complexes. The pre-Bact-1 and pre-Bact-2 cryo-EM structures reveal that a U6 internal stem-loop (ISL) with a distinctive conformation, which is stabilized by WBP11 in pre-Bact-1, and U2/U6 helix Ib are initially formed, followed by U2/U6 helix Ia and the U6 catalytic triplex. Facilitated by several B-specific proteins, the scaffold protein PRP8 retains its open conformation in both pre-Bact complexes, thereby providing sufficient three-dimensional (3D) space for the formation of the U6 ISL and helix Ib, which are docked already to their cognate PRP8 binding sites in the pre-Bact complexes. Other spliceosomal proteins accommodating the U2/U6 network are largely in place in both pre-Bact complexes and are thus poised to aid PRP8 in folding the U2 and U6 snRNAs. Structural comparisons with mature Bact complexes reveal the molecular mechanism whereby formation of a catalytically active U2/U6 RNA network is facilitated by spliceosomal proteins, with a conformational change in the scaffold protein PRP8 playing a key role in facilitating its final 3D folding. CONCLUSION The cryo-EM structures of two human pre-Bact complexes presented here reveal an intricate cascade of highly coordinated structural changes during the activation phase of the human spliceosome, involving mutually exclusive interactions that facilitate the directionality of the activation process. In addition, they provide new insights into the strategy used by the spliceosome to assemble its catalytic RNA network and how a conformational rearrangement in PRP8 facilitates the 3D folding of the catalytically active U2/U6 RNA. Protein-guided 3D folding of U2/U6 RNA during spliceosome activation. Structural organization of U2 and U6 snRNA and the proteins cradling them in pre-Bact-2 and Bact spliceosomal complexes. A PRP8 conformational change moves U2/U6 RNA elements docked on PRP8 toward the U6 ISL and also repositions PRP8-docked proteins, restricting space within the now closed PRP8 cavity and thereby aiding the final tertiary folding of the U2/U6 catalytic RNA. Single letters followed by numbers indicate nucleotides. Spliceosome activation involves extensive protein and RNA rearrangements that lead to formation of a catalytically active U2/U6 RNA structure. At present, little is known about the assembly pathway of the latter and the mechanism whereby proteins aid its proper folding. Here, we report the cryo–electron microscopy structures of two human, activated spliceosome precursors (that is, pre-Bact complexes) at core resolutions of 3.9 and 4.2 angstroms. These structures elucidate the order of the numerous protein exchanges that occur during activation, the mutually exclusive interactions that ensure the correct order of ribonucleoprotein rearrangements needed to form the U2/U6 catalytic RNA, and the stepwise folding pathway of the latter. Structural comparisons with mature Bact complexes reveal the molecular mechanism whereby a conformational change in the scaffold protein PRP8 facilitates final three-dimensional folding of the U2/U6 catalytic RNA.

中文翻译：

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剪接体激活过程中活性位点 U2/U6 RNA 的蛋白质引导折叠机制

剪接机换档 剪接体激活涉及广泛的蛋白质交换和 RNA 重排，导致形成称为 Bact 的具有催化活性的 U2/U6 RNA 结构。以前，人们对通往 U2/U6 活性位点的途径以及蛋白质如何帮助折叠 U2/U6 RNA 知之甚少。Townsend 等人使用冷冻电子显微镜确定两种人类 pre-Bact 复合物的结构。揭示了一系列复杂的协调结构变化，涉及促进激活过程方向性的相互排斥的相互作用。这些结构揭示了 U2/U6 催化 RNA 的组装途径和蛋白质促进其折叠的机制。科学，这个问题 p。eabc3753 一项冷冻电子显微镜研究揭示了人类 pre-Bact 剪接体的结构。引言 剪接体（包括其催化中心）在每个前 mRNA 内含子上通过涉及多个连续组装中间体的途径重新形成。剪接体激活涉及广泛的蛋白质交换和 RNA 重排，导致形成具有催化活性的 U2/U6 RNA 结构。到目前为止，对后者的组装途径以及蛋白质帮助其正确折叠的机制知之甚少。基本原理为了阐明在剪接体 B 复合体转化为激活的剪接体（即 Bact 复合体）期间发生的复杂重排，我们在之前未表征的激活中间阶段阻断了剪接体组装，并确定了纯化的人类 pre-Bact 复合体的结构。结果 在核心分辨率下获得了两种不同的人类 pre-Bact 复合物（表示为 pre-Bact-1 和 pre-Bact-2）的冷冻电子显微镜 (cryo-EM) 结构，这些复合物缺乏成熟的催化 U2/U6 RNA 结构3.9 和 4.2 Å，每个复合物的伪原子模型是使用综合结构生物学方法生成的。它们的组成和分子结构表明 pre-Bact-1 是 pre-Bact-2 的前体，追逐实验证明它们是功能性剪接体中间体。在生化分析的帮助下，pre-Bact-1 和 pre-Bact-2 结构提供了对 Bact 形成过程中蛋白质交换顺序的新见解。他们还阐明了许多相互排斥的蛋白质 - 蛋白质和蛋白质 - RNA 相互作用，确保形成 U2/U6 催化 RNA 所需的核糖核蛋白 (RNP) 重排的生产途径，并揭示所谓的 B 特异性蛋白质的新作用。他们表明，在激活过程中 BRR2 和 U2 小核 RNP 会逐步重新定位，后者是使 U2 和 U6 小核 RNA (snRNA) 足够接近以形成 U2/U6 螺旋 I 的先决条件。此外，他们表明与剪接体瞬时相互作用的蛋白质 TCERG1、WBP11、CTNNBL1 和 KIN17 稳定了 pre-Bact 复合物的中间 RNP 构象状态。pre-Bact-1 和 pre-Bact-2 低温电镜结构表明 U6 内部茎环 (ISL) 具有独特的构象，它在 pre-Bact-1 中由 WBP11 稳定，最初形成 U2/U6 螺旋 Ib，然后是 U2/U6 螺旋 Ia 和 U6 催化三链体。在几种 B 特异性蛋白的促进下，支架蛋白 PRP8 在两个 pre-Bact 复合物中保留其开放构象，从而为 U6 ISL 和螺旋 Ib 的形成提供足够的三维 (3D) 空间，它们已经停靠在它们的pre-Bact 复合物中的同源 PRP8 结合位点。其他适应 U2/U6 网络的剪接体蛋白主要存在于两个 pre-Bact 复合物中，因此准备帮助 PRP8 折叠 U2 和 U6 snRNA。与成熟 Bact 复合物的结构比较揭示了剪接体蛋白促进催化活性 U2/U6 RNA 网络形成的分子机制，支架蛋白 PRP8 的构象变化在促进其最终 3D 折叠方面发挥关键作用。结论 这里介绍的两种人类 pre-Bact 复合物的冷冻电镜结构揭示了人类剪接体激活阶段高度协调的结构变化的复杂级联，涉及促进激活过程方向性的互斥相互作用。此外，他们对剪接体用于组装其催化 RNA 网络的策略以及 PRP8 中的构象重排如何促进具有催化活性的 U2/U6 RNA 的 3D 折叠提供了新的见解。剪接体激活过程中 U2/U6 RNA 的蛋白质引导 3D 折叠。U2 和 U6 snRNA 的结构组织以及在 pre-Bact-2 和 Bact 剪接体复合物中包含它们的蛋白质。PRP8 构象变化将停靠在 PRP8 上的 U2/U6 RNA 元件移向 U6 ISL，并重新定位 PRP8 停靠的蛋白质，限制现在关闭的 PRP8 腔内的空间，从而帮助 U2/U6 催化 RNA 的最终三级折叠。单个字母后跟数字表示核苷酸。剪接体激活涉及广泛的蛋白质和 RNA 重排，导致形成具有催化活性的 U2/U6 RNA 结构。目前，对后者的组装途径以及蛋白质帮助其正确折叠的机制知之甚少。在这里，我们以 3.9 和 4.2 埃的核心分辨率报告了两种人类激活的剪接体前体（即 pre-Bact 复合物）的冷冻电子显微镜结构。这些结构阐明了激活过程中发生的众多蛋白质交换的顺序、确保形成 U2/U6 催化 RNA 所需的核糖核蛋白重排正确顺序的相互排斥的相互作用，以及后者的逐步折叠途径。与成熟 Bact 复合物的结构比较揭示了分子机制，其中支架蛋白 PRP8 的构象变化促进了 U2/U6 催化 RNA 的最终三维折叠。