Understanding Phase Separation in Biology and Disease - SUMMARY The nucleolus is a nuclear membraneless organelle with three sub-compartments, the FC, DFC, and GC. While first observed in the 1830s, how nucleoli form remained elusive until the discovery of biomolecular phase separation (PS). Brangwynne & Hyman reported in 2011 that nucleoli are active liquids governed by the laws of fluid physics. However, this report did not elucidate the molecular mechanisms underlying biomolecular PS in the nucleolus. Several reports in 2016 established molecular mechanisms governing the fluid behavior of nucleoli. Brangwynne, Pappu & Kriwacki reported that the DFC and GC are fluid layers separated by differences in surface tension (Feric, et al., Cell, 2016). Also in 2016, Kriwacki & co-workers showed how different structural domains within NPM1, a GC scaffold protein, promote PS with ribosomal proteins and RNA (r-proteins & rRNA) and proposed how phase separation mediates ribosome assembly (Mitrea, et al., Elife, 2016). Subsequent studies by the Kriwacki team revealed that acidic and basic tracts in an intrinsically disordered region (IDR) in NPM1 mediate homotypic PS that buffers heterotypic interactions with r-proteins and rRNA (Mitrea, et al., and Ferrolino, et al., Nat. Commun., 2018). We developed a thermodynamic “molecular hand-off” model describing NPM1’s role in ribosome subunit assembly (Mitrea, et al., Nat. Commun., 2018) that was later validated with the Brangwynne lab (Riback, et al., Nature, 2020). This latter study established key concepts: 1) multicomponent biomolecular PS, and 2) PS-enabled, thermodynamically-driven efflux of ribosomal subunits from the nucleolus. In the 2018 studies, we showed that the ribosomal assembly factor, SURF6, phase separates with NPM1 and serves as a GC co-scaffold. Our current findings show that SURF6 forms a GC sub-phase with rRNA, which is surrounded by a NPM1-rich sub-phase, creating granularity in the GC. We propose that the SURF6-rRNA and NPM1 GC sub-phases are an assembly line for maturation of ribosomal subunits and will test this hypothesis in our future studies. The Kriwacki lab has investigated the p14ARF tumor suppressor protein (ARF) since the 2000s and discovered in 2016 that ARF phase separates with NPM1 in the nucleolar GC (Mitrea, et al., Nat. Commun., 2016). Reports in 2020 and 2024 (Gibbs, et al., J. Magn. Reson., 2020; and Nat. Commun., 2024) suggested that ARF exerts it tumor suppressor function in part by binding and immobilizing NPM1 in the GC, inhibiting cell viability. Unpublished results also show that ARF binds 80S monosomes in the cytoplasm, imposing a state of ribosome dormancy and inhibiting translation. Our future studies are investigating dual modes of ARF-dependent tumor suppression: 1) inhibition of ribosome biogenesis through disturbance of the nucleolar GC, and 2) induction of ribosome dormancy through interactions with 80S monosomes. Collectively, our findings have established new paradigms regarding relationships between biomolecular phase separation and biological function. Our future studies will continue to advance understanding in the exciting field of biomolecular condensates.
