The proposed study aims to determine how phase separation regulates the ARF protein, a tumor suppressor that frequently experiences loss of function in human cancers. During oncogenic stress, ARF sequesters essential proteins in the nucleolus in order to arrest the cell cycle or induce apoptosis. Specifically, ARF sequesters HDM2 in the nucleolus, which activates p53-dependent cell cycle arrest or apoptosis. ARF also sequesters NPM1 in the nucleolus, disrupting ribosome assembly, which also induces cell cycle arrest independently of p53. However, the mechanism by which ARF sequesters its targets in the nucleolus is not understood. Recent observations have demonstrated that nucleoli possess liquid-like properties, which manifest through phase separation of their constituent proteins and RNAs. Recently, our lab along with the Brangwynne group, showed that the liquid like features of the nucleolus depends in-part on the phase separation properties of NPM1. I hypothesize that p14ARF disrupts the liquid-like properties of the granular component through hydrophobic self-association and multivalent interactions of its multiple arginine-rich motifs with NPM1, resulting in inhibition of ribosome biogenesis. This study will determine (1) how p14ARF?s physicochemical properties allow it to sequester its target proteins within phase separated bodies, (2) the structure and dynamics of phase separated p14ARF-NPM1 complexes, and (3) the effect of p14ARF expression on NPM1 dynamics, ribosome biogenesis and growth in live cells. Results from this study will provide novel insights into ARF?s nucleolar tumor suppressor function, and in a broader context, how the fluid-features of nucleoli are altered during oncogenic stress events.
It has been known for over 100 years that cancer cells exhibit more and larger nucleoli than normal cells, resulting in increased protein synthesis that supports cancer cell growth. Recently, the nucleolus has emerged as a target for cancer therapy. p14ARF is a nucleolar protein that plays key roles in maintaining protein synthetic homeostasis and preventing malignant transformation, thus insights gained from this study may propel novel therapeutic approaches targeting the nucleolus in the future.
