PROJECT SUMMARY A wide range of ribonucleoprotein (RNP) granules forms in response to a variety of cellular stress. An exciting new paradigm of liquid-liquid phase transitions postulates that RNP granules phase-separate to create organized subcellular compartments with discrete functions, ranging from mRNA storage to protein processing. Diseases such as tumor formation and neurodegeneration have been linked to abnormal granule formation, but further studies of these RNP complexes are necessary to uncover their biogenesis and physiological roles and lead to potential therapeutic targets. We have recently shown that low oxygen (hypoxia) selectively triggers the reorganization of core glycolytic enzymes into a “glycolytic body”, or G body, in yeast and human cell culture. We established that G bodies function to promote glycolysis and cell survival in hypoxia, as well as identified G body constituent proteins and the genes required for G body formation. Our evidence additionally suggests that RNA serves an important role to preserve the integrity of G bodies. However, the mechanisms of G body formation and function remain unknown. We hypothesize that G bodies are evolutionarily conserved RNP granules that form via phase separation to facilitate glycolysis and fuel associated metabolic functions in hypoxia. To test this hypothesis, we will further define the composition, biophysical properties, dynamics and regulation of formation, and physiological roles of G bodies in yeast and mammalian cell culture. We will also evaluate the role that RNA plays in G body formation and structural integrity and define the key signaling events required for G body formation. Finally, we will investigate the function of G bodies in an innovative 3D culture system as a model for solid tumor growth. The outcome of this proposal will define the importance of a novel RNP granule and establish a robust system with which to study glycolysis in a disease-centered context.

PROJECT NARRATIVE The formation of phase-separated granules within cells has emerged as a fundamental theme in cell biology and in a diverse range of diseases. We have discovered a novel metabolic granule, the G body, which forms in response to hypoxia and serves to increase glycolytic function in yeast. With this proposal, we aim to explore the biophysical and structural components of G bodies in yeast and mammalian cell culture, as well as further define the signaling required for formation and the physiological role of G bodies as an adaptive response to hypoxic stress. !