Quinodoz Sofia A, Jiang Lifei, Abu-Alfa Aya A, Comi Troy J, Zhao Hongbo, Yu Qiwei, Wiesner Lennard W, Botello Jordy F, Donlic Anita, Soehalim Elizabeth, Bhat Prashant, Zorbas Christiane, Wacheul Ludivine, Košmrlj Andrej, Lafontaine Denis L J, Klinge Sebastian, Brangwynne Clifford P
Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ, USA.
Howard Hughes Medical Institute, Chevy Chase, MD, USA.
Nature. 2025 Jul 2. doi: 10.1038/s41586-025-09207-4.
Biomolecular condensates are key features of intracellular compartmentalization. As the most prominent nuclear condensate in eukaryotes, the nucleolus is a multiphase liquid-like structure in which ribosomal RNAs (rRNAs) are transcribed and processed, undergoing multiple maturation steps to form the small (SSU) and large (LSU) ribosomal subunits. However, how rRNA processing is coupled to the layered organization of the nucleolus is poorly understood owing to a lack of tools to precisely monitor and perturb nucleolar rRNA processing dynamics. Here we developed two complementary approaches to spatiotemporally map rRNA processing and engineer de novo nucleoli. Using sequencing in parallel with imaging, we found that rRNA processing steps are spatially segregated, with sequential maturation of rRNA required for its outward movement through nucleolar phases. By generating synthetic nucleoli in cells using an engineered rDNA plasmid system, we show that defects in SSU processing can alter the ordering of nucleolar phases, resulting in inside-out nucleoli and preventing rRNA outflux, while LSU precursors are necessary to build the outermost layer of the nucleolus. These findings demonstrate how rRNA is both a scaffold and substrate for the nucleolus, with rRNA acting as a programmable blueprint for the multiphase architecture that facilitates assembly of an essential molecular machine.
生物分子凝聚物是细胞内区室化的关键特征。作为真核生物中最突出的核凝聚物，核仁是一种多相类液体结构，核糖体RNA（rRNA）在其中进行转录和加工，经过多个成熟步骤形成小（SSU）和大（LSU）核糖体亚基。然而，由于缺乏精确监测和扰动核仁rRNA加工动态的工具，人们对rRNA加工如何与核仁的分层组织相耦合了解甚少。在这里，我们开发了两种互补的方法，用于在时空上绘制rRNA加工过程并构建从头核仁。通过将测序与成像并行使用，我们发现rRNA加工步骤在空间上是分离的，rRNA向外穿过核仁阶段的移动需要其顺序成熟。通过使用工程化的rDNA质粒系统在细胞中生成合成核仁，我们表明SSU加工缺陷会改变核仁阶段的顺序，导致核仁由内向外翻转并阻止rRNA外流，而LSU前体对于构建核仁的最外层是必需的。这些发现证明了rRNA如何既是核仁的支架又是底物，rRNA作为多相结构的可编程蓝图，促进了一种基本分子机器的组装。
