This application addresses the molecular basis of the liquid-like features of the nucleolus, a membrane-less nuclear organelle that mediates ribosome biogenesis and certain types of stress signaling (e.g., involving p53). The nucleolus exhibits three structural regions, the fibrillar center (FC), dense fibrillar component (DFC), and granular component (GC), that are the locations of various steps in ribosomal RNA (rRNA) and protein processing and assembly during ribosome biogenesis. Recently, Brangwynne, et al., showed that the GC of the nucleolus exhibits liquid-like features, similar to those of other punctate, membrane-less organelles. Furthermore, Brangwynne previously showed that punctate P granules form liquid-like structures through phase separation of their components. More recently, Rosen demonstrated that multi-valency of binding domains and motifs within interacting proteins is associated with phase separation, and McKnight has shown that multi-valent, low complexity protein sequences experience phase separation with RNA. These and other recent studies have given birth to a new area of structural biology: phase separation phenomena that drive formation of membrane-less organelles with liquid-like structural features. The multi-functional phospho-protein, Nucleophosmin 1 (NPM1; termed Npm here), is a major constituent of the GC of the nucleolus and we hypothesize is a main driver of the phase separation that gives rise to the GC's liquid-like features. We recently described the structure of the pentameric, N-terminal domain of human Npm (N130) and the molecular basis for its interactions with known nucleolar binding partners, including ribosomal proteins. N130 exhibits two acidic tracts, one within its pentamer structure (termed A1) and another within a 10 residue-long disordered C-terminal segment (termed A2); within the N130 pentamer, A1 and A2 create multi-valency. We additionally showed that many of Npm's nucleolar binding partners exhibit disordered regions containing multiple Arg residue-containing motifs (termed R motifs); the multiple R motifs in Npm's binding partners also exhibit multi-valency. In currently unpublished studies, we have shown that N130 forms liquid-like droplets upon binding to various R motif-containing peptides derived from Npm partners. Npm also exhibits a folded nucleic binding domain at its C-terminus, providing an additional level of multi-valency for interactions with rRNA in the nucleolus. We hypothesize that Npm transiently and promiscuously interacts with proteins and rRNA in the nucleolus, nucleating its liquid-like features and organizing the molecular functions that drive ribosome biogenesis.
It has been known for more than 100 years that cancer cells exhibit more and larger nucleoli than normal cells, allowing heightened protein synthesis to support cancer cell growth. The studies proposed here address the structural basis of the granular component (GC) region of the nucleolus. We are providing the first-ever molecular description of the liquid-like features of the GC, which will serve as a basis in the future for th development of new therapeutics against over-active nucleoli in cancer cells.
