PROJECT SUMMARY/ABSTRACT Ribosomes are complex ribonucleoprotein particles that catalyze protein synthesis in almost all cells in nature. The long-term goal of this project is to understand how the 80 proteins and four rRNAs comprising eukaryotic ribosomes are assembled in vivo. We use the yeast Saccharomyces cerevisiae to facilitate molecular genetic approaches, and cultured Hela cells to enable us to visualize the nucleolus, the cellular compartment where ribosomes are made. Production of ribosomes is tightly linked to cell growth and proliferation. Consequently, dysregulation of ribosome biogenesis and nucleolar integrity is linked to many diseases such as cancer, neurodegenerative diseases, or developmental disorders. Because pathways of ribosome biogenesis are very conserved, our studies in yeast will help understand mechanisms of regulation and dysregulation of ribosome production in humans. Ribosome biogenesis requires a dynamic series of remodeling steps in which protein and RNA interactions are established and reconfigured. These steps are made more efficient and more accurate by the activities of more than 200 assembly factors present in nascent yeast ribosomes, which are required for their assembly, and conserved across eukaryotes. To enable in-depth studies of mechanisms driving ribosome assembly, we focus on one interval: just prior to, during, and immediately after exit of large ribosomal subunit precursors from the nucleolus into the nucleoplasm. During this stage, several domains of ribosomal RNA are rearranged, numerous assembly factors complete their functions and exit from pre-ribosomes, and new assembly factors enter the particles. We want to learn how these dynamic remodeling steps are powered forward by energy-consuming assembly factors present at this stage. We are also investigating interconnections between ribosome assembly and the nucleolus, the prominent biomolecular condensate thought to be formed through multivalent interactions between pre-ribosomes and other nucleolar components. However, it is not clear exactly how ribosome assembly creates a nucleolus, nor how material properties of the nucleolus enable efficient ribosome assembly. We propose experiments to address the following questions: (1) How do the RNA helicases/ATPases Drs1, Has1, Dbp10, and Spb4 power maturation of pre-60S subunits during mid to late nucleolar stages of assembly? (2) How does the structure and composition of pre- ribosomes enable them to be retained in the nucleolus? (3) How does ribosome assembly contribute to the morphology and fluidity of the nucleolus?