In mammalian cells, the nucleolus is the central site for ribosome biogenesis. It is composed of distinct subcompartments: the fibrillar center (FC), the dense fibrillar component (DFC), the periphery of the DFC (PDFC), and the granular component (GC), each executing specific steps in the transcription and processing of rRNA precursors (pre-rRNAs) and in ribosomal subunit assembly.

Nucleolar dysfunction is implicated in various diseases such as cancer, neurodegeneration, and developmental disorders. The traditional view of the ribosome biogenesis process is that rRNA synthesis and processing occur in the FC and DFC units, while ribosome assembly occurs in the GC. However, how these compartments functionally coordinate to support efficient ribosome production has long been unclear.

In a study published in Nature on July 23, a team led by Prof. CHEN Lingling from the Center for Excellence in Molecular Cell Science (Shanghai Institute of Biochemistry and Cell Biology) of the Chinese Academy of Sciences revealed a spatiotemporal separation in the processing of distinct pre-rRNAs within the nucleolus, offering a new perspective on how nucleolar architecture is functionally organized.

By integrating metabolic labeling, single-molecule RNA imaging, super-resolution microscopy, and quantitative proteomics, the researchers systematically mapped the spatiotemporal distribution of pre-rRNA processing in the nucleolus. 

They revealed that small subunit (SSU) pre-rRNA is predominantly processed within the inner FC–PDFC regions and its processing is largely completed within the first 30 minutes of nascent RNA labeling. In contrast, large subunit (LSU) pre-rRNA is enriched in the outer PDFC–GC regions and matures gradually after 30 minutes. This spatial separation contradicts traditional models that place both processing steps within the GC, and it redefines the specialized functions within nucleolar subdomains.

Such compartmentalized processing is functionally significant. In slow-proliferation cells, nucleoli exhibit fewer but enlarged FC-DFC units, with accompanying less-efficient SSU processing and accumulated SSU pre-rRNAs near the inner nucleolar regions, ultimately reducing the production of ribosomes. To explore the relationship between pre-rRNA processing and nucleolar organization, the researchers introduced a geometric parameter, the Relative FC–DFC interface, to characterize the FC-DFC interface under varying conditions. This parameter provides a way to link individual nested FC–DFC structures to SSU processing efficiency.

Antisense oligonucleotides targeting the 5' external transcribed space (5' ETS) region of SSU pre-rRNA were used to inhibit pre-rRNA processing, recapitulating the structural defects observed in slow-proliferation cells. The findings revealed the essential role of 5' ETS-centered SSU processing in maintaining nucleolar substructures.

Notably, evolutionary comparisons of pre-rRNA processing kinetics further support a model of interdependence between nucleolar structure and function. Bipartite nucleoli in anamniotes such as zebrafish, which lack a separated FC–DFC interface, exhibit distinct 5′ ETS distribution and much slower pre-rRNA diffusion compared to multilayered nucleoli in amniotes. The introduction of an artificial FC–DFC interface into bipartite nucleoli led to enhanced processing efficiency, suggesting that the emergence of multilayered nucleolar organization may have conferred evolutionary advantages in ribosome production.

This study establishes a direct link between molecular-level function and micron-level nucleolar architecture, revealing the dynamic coordination between pre-rRNA processing and nucleolar substructure. These results suggest potential strategies for nucleolus-related disease treatment by targeting key steps of ribosome biogenesis within nucleolar substructures.