PROJECT SUMMARY / ABSTRACT Ribosome biogenesis occurs in the nucleolus, and disruptions to nucleolar proteins cause ribosomopathies such as Treacher Collins syndrome and contribute to a range of proliferative and degenerative diseases. Nucleoli contain three nested sub-compartments that are phase-separated from each other and differ in composition, density, and function. The inner-most fibrillar centers (FCs) are surrounded by dense fibrillar components (DFCs). Transcription of ribosomal RNA (rRNA) occurs at the interface between the FC and DFC, and maturation of rRNA occurs in the DFC. In the outermost phase, the granular component (GC), rRNA incorporates ribosomal proteins to create ribosomal subunits. The equilibrium nature of phase separation implies that the relevant factors concentrate themselves into specific phases without energy input. This coupled with the nested architecture of the nucleolus, sets up thermodynamically controlled fluxes across the distinct layers. Recent studies have described a molecular handoff model for setting up the inward flux of ribosomal proteins into the granular component. However, ribosomal assembly requires an outward flux of rRNA, and how this is set up, whether this is under thermodynamic control, and the key molecular players that set up such a flux remain entirely unknown. My goal is to uncover the mechanisms that set up the flux of rRNA into and through the granular component of the nucleolus. In my preliminary work I determined the critical factors involved in setting up rRNA flux and began reconstituting this process in vitro. I used bioinformatics analysis and in vivo localization studies to identify the protein nucleolin as the key protein that sets up rRNA flux. My reconstitutions show that nucleolin mediates rRNA flux out of the DFC and into the GC. Recent studies in the literature and my preliminary studies show that nucleolin helps set up the flux of rRNA through the GC. My preliminary data leads to the hypothesis that ribosome biogenesis relies on the thermodynamic flux of rRNA into and through the granular component (GC) and that this is primarily mediated by nucleolin. This hypothesis will drive my specific aims: 1 - Determine which biophysical features of nucleolin (NCL) and rRNA complexes are required to set up rRNA fluxes from the dense fibrillar component (DFC) into the granular component (GC) and 2 - measure a series of linked phase equilibria of nucleolin, nucleophosmin, rRNA, and rProtein to determine if they contribute to rRNA flux through the granular component