Pre-rRNA spatial distribution and functional organization of the nucleolus.

The multilayered nucleolus is the primary site of ribosome biogenesis, where successive maturation of small (SSU) and large (LSU) ribosomal subunit precursors occurs. However, the spatiofunctional relationship between pre-rRNA processing and nucleolar substructures and how this adapts to changing cellular physiological demands have remained incompletely understood. Here our spatiotemporal analyses revealed a compartment-specific ribosomal subunit processing in human nucleoli, with SSU processomes maintained in fibrillar centre (FC)-dense fibrillar component (DFC)-periphery dense fibrillar component (PDFC) domains while LSU pre-rRNAs largely transited to PDFC-granular component regions. Slowly proliferating cells exhibited unexpected 5' external transcribed spacer (5' ETS)-centred SSU processing impairment, accompanied by structural remodelling of FC-DFC units and retarded SSU outflux. Direct 5' ETS processing perturbation at least partially recapitulated these FC-DFC unit alterations, supporting the functional interdependence between SSU processing and nucleolar architecture. Notably, anamniote bipartite nucleoli with merged FC-DFC compartments exhibited distinct 5' ETS distribution and slower pre-rRNA flux compared with multilayered nucleoli in amniotes. Introducing a FC/DFC interface to bipartite nucleoli enhanced processing efficiency, indicating that the evolutionary emergence of nested FC-DFC units may have optimized pre-rRNA processing. Collectively, depicting the spatiotemporal distribution of pre-rRNAs reveals an essential role of 5' ETS-centred processing in maintaining nucleolar substructures and suggests a possible evolutionary advantage of the multilayered structure in amniotes.