The multi-layered nucleolus serves as the primary site of ribosome biogenesis1,2, where successive maturation of small (SSU)3,4 and large (LSU)5 ribosomal subunit precursors occur. However, the spatio-functional relationship between pre-rRNA processing and nucleolar substructures and how this adapts to changing cellular physiological demands have remained incompletely understood6,7. Here, our spatiotemporal ***yses revealed a compartment-specific ribosomal subunit processing in human nucleoli, with SSU processomes maintained in fibrillar center/dense fibrillar component/periphery dense fibrillar component (FC/DFC/PDFC) domains while LSU pre-rRNAs largely transited to PDFC/granular component (GC) regions. Slow proliferating cells exhibited unexpected 5′ external transcribed space (5′ ETS)-centered SSU processing impairment, accompanied by FC/DFC structural remodeling and retarded SSU outflux. Direct 5′ ETS processing perturbation at least partially recapitulated these FC/DFC alterations, supporting the functional interdependence between SSU processing and nucleolar architecture. Notably, anamniote bipartite nucleoli with merged FC/DFC compartments8,9 exhibited distinct 5′ ETS distribution and slower pre-rRNA flux compared to multi-layered nucleoli in amniotes. Introducing a FC/DFC interface to bipartite nucleoli enhanced processing efficiency, indicating that evolutionary emergence of nested FC/DFC may have optimized pre-rRNA processing. Collectively, depicting the spatiotemporal distribution of pre-rRNAs revealed an essential role of 5′ ETS-centered SSU processing in maintaining nucleolar substructures and suggested a possible evolutionary advantage of the multi-layered structure in amniotes.
