Multivalent 28S rRNA expansion segments enable reconstitution of multilayered nucleolar architecture

Eukaryotic 28S rRNA contains expansion segments (ESs) that increase dramatically in length during evolution, yet their functions remain elusive. The nucleolus, where rRNA is transcribed, exhibits divergent architectures: three-layered (tripartite) in amniotes but two-layered (bipartite) in other eukaryotes. Here, we identified distinct rRNA localization patterns within nucleolar compartments. Through in vitro reconstitution and simulations, we demonstrated that 28S rRNA induces layered nucleolar-like structures via multivalent RNA-RNA interactions. This structure-forming capacity correlates with the evolutionary expansion of ESs. 28S rRNAs from tripartite-nucleolus organisms possess longer ESs and exhibit enhanced multivalency compared with those from bipartite-nucleolus species. Deletion of specific ESs abolishes human 28S rRNA's ability to induce nucleolar-like structures, whereas transferring these segments to C. elegans 26S rRNA confers this capability in vitro. We conclude that rRNA ESs function as architectural elements that promote nucleolar complexity, providing mechanistic insight into how genomic expansion drives sophisticated cellular organization through enhanced intermolecular interactions.