Ferrolino, M. C. | Search bioRxiv

bioRxiv2025

Granular component sub-phases direct ribosome biogenesis in the nucleolus

The hierarchical, multiphase organization of the nucleolus underlies ribosome biogenesis. Ribonucleoprotein particles that regulate ribosomal subunit assembly are heterogeneously disposed in the granular component (GC) of the nucleolus. However, the molecular origins of the GCs spatial heterogeneity and its association with ribosomal subunit assembly remain poorly understood. Here, using super-resolution microscopy, we uncover that key GC biomolecules, including nucleophosmin (NPM1), surfeit locus protein 6 (SURF6), and ribosomal RNA (rRNA), are heterogeneously localized within sub-phases in the GC. In vitro reconstitution showed that these GC biomolecules form multiphase condensates with SURF6/rRNA-rich core and NPM1-rich shell, providing a mechanistic basis for GCs spatial heterogeneity. SURF6s association with rRNA is weakened upon ribosome subunit assembly, enabling NPM1 to extract assembled subunits from condensates--suggesting an assembly-line-like mechanism of subunit efflux from the GC. Our results establish a framework for understanding the heterogeneous structure of the GC and reveal how its distinct sub-phases facilitate ribosome subunit assembly.

biochemistry↗

bioRxiv2019

Composition dependent phase separation underlies directional flux through the nucleolus

Intracellular bodies such as nucleoli, Cajal bodies, and various signaling assemblies, represent membraneless organelles, or condensates, that form via liquid-liquid phase separation (LLPS)1,2. Biomolecular interactions, particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions (IDRs), are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems3-6 have led to the concept that a single fixed saturation concentration (Csat) is a defining feature of endogenous LLPS7-9, and has been suggested as a mechanism for intracellular concentration buffering2,7,8,10. However, the assumption of a fixed Csat remains largely untested within living cells, where the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed Csat. As the concentration of individual components is varied, their partition coefficients change, in a manner that can be used to extract thermodynamic interaction energies, that we interpret within a framework we term polyphasic interaction thermodynamic analysis (PITA). We find that heterotypic interactions between protein and RNA components stabilize a variety of archetypal intracellular condensates, including the nucleolus, Cajal bodies, stress granules, and P bodies. These findings imply that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA processing condensates such as the nucleolus, this stoichiometric self-tuning manifests in selective exclusion of fully-assembled RNP complexes, providing a thermodynamic basis for vectorial ribosomal RNA (rRNA) flux out of the nucleolus. The PITA methodology is conceptually straightforward and readily implemented, and it can be broadly utilized to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deep understanding of the thermodynamics of multi-component intracellular phase behavior and its interplay with nonequilibrium activity characteristic of endogenous condensates.

biophysics↗