2. Academic Validation
  3. Mapping and engineering RNA-driven architecture of the multiphase nucleolus

Mapping and engineering RNA-driven architecture of the multiphase nucleolus

  • Nature. 2025 Aug;644(8076):557-566. doi: 10.1038/s41586-025-09207-4.
Sofia A Quinodoz # 1 2,  Lifei Jiang # 3,  Aya A Abu-Alfa 3,  Troy J Comi 4,  Hongbo Zhao 1 4,  Qiwei Yu 5,  Lennard W Wiesner 1,  Jordy F Botello 3,  Anita Donlic 1,  Elizabeth Soehalim 4,  Prashant Bhat 6 7,  Christiane Zorbas 8,  Ludivine Wacheul 8,  Andrej Košmrlj 9 10,  Denis L J Lafontaine 11,  Sebastian Klinge 12,  Clifford P Brangwynne 13 14 15 16 17 18
Affiliations

Affiliations

  • 1 Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ, USA.
  • 2 Howard Hughes Medical Institute, Chevy Chase, MD, USA.
  • 3 Department of Molecular Biology, Princeton University, Princeton, NJ, USA.
  • 4 Omenn-Darling Bioengineering Institute, Princeton University, Princeton, NJ, USA.
  • 5 Lewis-Sigler Institute for Integrative Genomics, Princeton, NJ, USA.
  • 6 Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.
  • 7 David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA.
  • 8 RNA Molecular Biology, Fonds de la Recherche Scientifique (F.R.S./FNRS), Université libre de Bruxelles (ULB), Gosselies, Belgium.
  • 9 Department of Mechanical and Aerospace Engineering, Princeton, NJ, USA.
  • 10 Princeton Materials Institute, Princeton, NJ, USA.
  • 11 RNA Molecular Biology, Fonds de la Recherche Scientifique (F.R.S./FNRS), Université libre de Bruxelles (ULB), Gosselies, Belgium. denis.lafontaine@ulb.be.
  • 12 Laboratory of Protein and Nucleic Acid Chemistry, The Rockefeller University, New York, NY, USA. klinge@rockefeller.edu.
  • 13 Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ, USA. cbrangwy@princeton.edu.
  • 14 Howard Hughes Medical Institute, Chevy Chase, MD, USA. cbrangwy@princeton.edu.
  • 15 Department of Molecular Biology, Princeton University, Princeton, NJ, USA. cbrangwy@princeton.edu.
  • 16 Omenn-Darling Bioengineering Institute, Princeton University, Princeton, NJ, USA. cbrangwy@princeton.edu.
  • 17 Lewis-Sigler Institute for Integrative Genomics, Princeton, NJ, USA. cbrangwy@princeton.edu.
  • 18 Princeton Materials Institute, Princeton, NJ, USA. cbrangwy@princeton.edu.
  • # Contributed equally.
PMID: 40604277 DOI: 10.1038/s41586-025-09207-4
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.

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