Description: Ribosome is the evolutionarily conserved molecular machine responsible for synthesis of proteins. In eukaryotic cells, ribosome is produced in a biophysically distinct subnuclear compartment, the nucleolus, through a cascade of energy-driven events. This process underlies a number of genetic diseases and is a major target for anti-cancer and anti-neurodegeneration therapeutics. However, the physical interactions of the network required for this process remain largely uncharacterized. This application will characterize a key molecular complex called R2TP that acts early in ribosome production by facilitating assembly of several ribosome production enzymes. R2TP has a wide client base, collaborates with a general protein folding chaperone, heat shock protein 90 (Hsp90), and delivers clients to the nucleolus. The Li laboratory uses a combination of structural biology, biochemical, and genetics methods to provide functional insights on R2TP and its impact on ribosome production. Exceptionally detailed three-dimensional views of R2TP itself and those R2TP acts upon will be obtained and analyzed along with the available functional data. Two specific aims are designed to 1) establish the structure and function cycle of R2TP; 2) elucidate the physiological role of R2TP. Results of this study will identify new molecular sites for developing anti-cancer and anti-neurodegenerative drugs through controls of ribosome production and degradation. The Li laboratory has assembled a team of scientists with complementary expertise in x-ray crystallography, high-throughput electron cryomicroscopy, mass spectrometry, biophysics, protein biochemistry, and yeast genetics in order to maximize the chance of successes while mitigating risks. Relevance: Aberrant nucleolar morphology and function have been linked to cancers and Alzheimer's disease in human. Correspondingly, R2TP and its client proteins have been identified as key regulators of nucleolar morphology. Whereas more than fifteen types of anti-cancer drugs have been investigated or in clinical trials that target molecules involved in this pathway, none is known for anti-neurodegeneracy. Though effective in cell-death assays, the pharmacological basis of many compounds remains to be explained and some develop resistance in cells. The proposed research provides platforms for explaining the actions of the existing drugs while discovering new ones.