Multivalent 28S rRNA Is the Organizer of the Nucleolus’s Multi-layered Architecture

The nucleolus widely exists in all eukaryotic species. Throughout evolution, two types of nucleoli have emerged: bipartite, which has two-layered subcompartments, and tripartite, which features three nested sub-compartments: granular component (GC), dense fibrillar component (DFC), and fibrillar center (FC). FC and DFC form a core-shell architecture and are immersed by a large GC. However, factors that mediated the formation of the three-layered architecture remain largely unknown. Here, we discovered that rRNAs have specific localizations within the nucleolus, with 28S rRNA forming assemblies surrounding the DFC. Multivalent interactions of 28S rRNA drove the formation of the hollow shell structure of DFC, which mediated the development of a three-layered structure resembling nucleolar GC-DFC-FC. Moreover, in organisms with a tripartite nucleolus, 28S rRNA has evolved to acquire a higher degree of multivalency. Our data implies that the emergence of multivalency of 28S rRNA facilitates the transition of bipartite to tripartite nucleolus.