Mapping and engineering RNA-driven architecture of the multiphase nucleolus

Sofia A. Quinodoz, Lifei Jiang, Aya A. Abu-Alfa, Troy J. Comi, Hongbo Zhao, Qiwei Yu, Lennard W. Wiesner, Jordy F. Botello, Anita Donlic, Elizabeth Soehalim, Prashant Bhat, Christiane Zorbas, Ludivine Wacheul, Andrej Košmrlj, Denis L. J. Lafontaine (), Sebastian Klinge () and Clifford P. Brangwynne ()
Additional contact information
Sofia A. Quinodoz: Princeton University
Lifei Jiang: Princeton University
Aya A. Abu-Alfa: Princeton University
Troy J. Comi: Princeton University
Hongbo Zhao: Princeton University
Qiwei Yu: Lewis-Sigler Institute for Integrative Genomics
Lennard W. Wiesner: Princeton University
Jordy F. Botello: Princeton University
Anita Donlic: Princeton University
Elizabeth Soehalim: Princeton University
Prashant Bhat: California Institute of Technology
Christiane Zorbas: Université libre de Bruxelles (ULB)
Ludivine Wacheul: Université libre de Bruxelles (ULB)
Andrej Košmrlj: Department of Mechanical and Aerospace Engineering
Denis L. J. Lafontaine: Université libre de Bruxelles (ULB)
Sebastian Klinge: The Rockefeller University
Clifford P. Brangwynne: Princeton University

Nature, 2025, vol. 644, issue 8076, 557-566

Abstract: Abstract Biomolecular condensates are key features of intracellular compartmentalization1,2. 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 subunits3–5. 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.

Date: 2025
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DOI: 10.1038/s41586-025-09207-4

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