核糖体是一种由大小亚基组成的核酸蛋白复合物，几乎存在于所有细胞中，是蛋白质合成的场所。真核核糖体的成熟是一个极其复杂同时又高度分级的过程，包括核糖体蛋白质的合成与组装以及rRNAs的转录、修饰、折叠与加工。大约有300种因子参与核糖体的组装过程，这些因子包括蛋白质和snoRNPs。核糖体大亚基的功能中心包括中央凸起（CP）、肽基转移酶中心（PTC）以及新生肽链出口隧道（NPET），这些功能中心在进化上是高度保守的。真核核糖体大亚基的前体从核仁到核质的组装过程中，结构会发生重大变化，其中包括组成中央凸起的5S RNP的旋转、组成肽基转移酶中心的H69的结构重排以及Nog1碳末端探入新生肽链出口隧道。在此阶段，细胞是否以及如何将这些重塑事件偶联在一起，在很大程度上尚待探索。Rpf2是重要的核糖体大亚基组装因子，在进化上是高度保守的，它与Rrs1形成的异源二聚体，对于5S RNP锚定到核糖体大亚基前体上是必需的。核糖体蛋白uL4隧道结构域以及组装因子Nog1碳末端是新生肽链出口隧道的重要组成部分，它们对于新生肽链出口隧道的完整性至关重要。我们利用冷冻电镜技术解析了rpf2△255-344突变体、Sda1-depletion突变体、ul4Δ63-87突变体以及nog1Δ595-647 rei1Δ341-393 reh1Δ380-432突变体中Nog2-TAP pre-60S样品。发现Rpf2碳末端的缺失会影响rRNA螺旋H68-70的折叠，继而影响5S RNP的旋转以及肽基转移酶中心和新生肽链出口隧道的组装。由此，我们提出Rpf2-Rrs1复合体稳定锚定到pre-60S亚基帮助H68-70的正确折叠是核糖体大亚基成熟过程中的一个重要的质量控制检验点。此外，我们发现uL4和Nog1的通道结构域缺失时，会造成新生肽链出口隧道的组成部分Helix74的构象变化。继而发现NPET的组装与5S RNP的旋转是通过H74相互偶联的，H74接收NPET组装缺陷的信号并阻止CP的组装。我们的结构也揭示了uL4 TD、Nog1 CTD以及H74三者的相互作用对于NPET的组装至关重要。并且组成NPET的uL4的突变会产生一系列处于不同组装阶段的有缺陷的前体，表明NPET的组装是核糖体大亚基前体核内成熟中的另一个重要的质量控制检验点。综上所述，我们的研究将真核核糖体大亚基功能模块的组装过程偶联在一起了，为深入理解核糖体大亚基的组装过程提供了丰富的结构基础。
Ribosome is a nucleic acid-protein complex composed of large and small subunits. It exists in almost all cells and is the site of protein synthesis. The maturation of eukaryotic ribosomes is an extremely sophisticated and highly hierarchical pathway, including the synthesis and assembly of ribosomal proteins and the transcription, modification, folding and processing of rRNAs. Approximately 300 factors involved in the assembly process of ribosome, including proteins and snoRNPs.The functional centers of the large ribosomal subunit include central protuberance, peptidyl transferase center and nascent polypeptide exit tunnel，which are evolutionarily conserved. Eukaryotic large ribosomal subunit will undergo significant conformational changes in structures, including the rotation of the 5S RNP that forms the central protuberance, the rearrangement of H69 that constitutes the peptidyl transferase center, and the insertion of the Nog1 CTD into the nascent polypeptide exit tunnel, as they transition from the nucleolus to the nucleoplasm. During this stage, whether and how these remodeling events are coupled remains largely unexplored.Rpf2 is an important large ribosomal subunit assembly factor and is highly conserved in evolution. The heterodimer formed by Rpf2-Rrs1 is necessary for the anchoring of 5S RNP to pre-60S. The tunnel domain of the ribosomal protein uL4 and the C-terminal domain of assembly factor Nog1 are important components of the nascent polypeptide exit tunnel, and they are critical to the integrity of the nascent polypeptide exit tunnel.We used cryo-EM to solve the sructure of Nog2-TAP pre-60S samples obtained from rpf2Δ255-344 mutants, Sda1-depletion mutants, ul4Δ63-87 mutant and nog1Δ595-647 rei1Δ341-393 reh1Δ380-432 mutant. It was found that the absence of Rpf2 CTD would affect the folding of rRNA Helix H68-70, which in turn affected the rotation of 5S RNP and the assembly of PTC and NPET. Therefore, we propose that the stable anchoring of the Rpf2-Rrs1 complex to the pre-60S to help the correct folding of H68-70 is an important checkpoint during the maturation of the large ribosomal subunit. In addition, We found that the deletion of the tunnal domain of uL4 and Nog1 will cause the conformational changes of Helix74, a component of NPET. Therefore, we found that the assembly of NPET and rotation of 5S RNP were coupled to each other through Helix74. H74 received the signal of NPET assembly defect and prevented the assembly of CP. Our structures also reveal that the interaction between uL4 TD, Nog1 CTD and H74 is essential for the assembly of NPET. Moreover, the mutation of uL4 that composes NPET will produce a series of defective precursors at different assembly stages, indicating that the assembly of NPET is another important checkpoint in the nuclear maturation of the large ribosomal subunit.In summary, our research illustrates the conformational coupling of the maturation of different functional centers on the eukaryotic large ribosomal subunit, and provides structural basis for in-depth understanding of the quality control of assembly process of ribosomal large subunit production.
