Nucleolar reorganization on stress depends on physicochemical changes due to nascent rRNA synthesis

Abstract Successive maturation of ribosomal subunits occurs through multilayered phase-separated structures of the cell nucleolus. The spatio-functional relationship between transcription of rRNA and nucleolar substructures and how this adapts to cellular stress remain incompletely understood. In this study, we resolve the sub nucleolar structures using expansion microscopy to reveal ordered structures of fibrillar center (FC) and dense fibrillar component (DFC) domains as nested shells, which is reorganized upon cellular stress like DNA damage or RNA polymerase I (RNAPI) inhibition. Direct visualization of nascent (5’ ETS) and mature (28S) rRNA suggested that rRNA synthesis is the critical regulator of nucleolar size, and organization. Nucleolar reorganization upon stress emerges to be a direct function of nascent rRNA levels. Stress-induced transcription inhibition remodels the sub-nucleolar compartments from a viscoelastic state into solid-like condensates thereby perturbing the nucleolar pH gradient due to the missing rRNA scaffold. We show, that rather than signaling to mediate rDNA repair, nucleolar reorganization naturally arises primarily from reduced rRNA levels and the resultant biophysical restructuring of the nucleolus under cellular stress.