Abstract
NPM1 is an abundant nucleolar chaperone that, in addition to facilitating ribosome biogenesis, contributes to nucleolar stress responses and tumor suppression through its regulation of the p14 Alternative Reading Frame tumor suppressor protein (p14ARF). Oncogenic stress induces p14ARF to inhibit MDM2, stabilize p53 and arrest the cell cycle. Under non-stress conditions, NPM1 stabilizes p14ARF in nucleoli, preventing its degradation and blocking p53 activation. However, the mechanisms underlying the regulation of p14ARF by NPM1 are unclear because the structural features of the p14ARF-NPM1 complex were elusive. Here we show that p14ARF assembles into a gel-like meso-scale network upon phase separation with NPM1. This assembly is mediated by intermolecular contacts formed by hydrophobic residues in an α-helix and β-strands within a partially folded N-terminal portion of p14ARF. These hydrophobic interactions promote phase separation with NPM1, enhance p14ARF nucleolar partitioning, restrict NPM1 diffusion within condensates and nucleoli, and reduce cellular proliferation. Our structural analysis provides insights into the multifaceted chaperone function of NPM1 in nucleoli by mechanistically linking the nucleolar localization of p14ARF to its partial folding and meso-scale assembly upon phase separation with NPM1.
| Original language | English |
|---|---|
| Article number | 9531 |
| Journal | Nature Communications |
| Volume | 15 |
| Issue number | 1 |
| DOIs | |
| State | Published - Dec 2024 |
Funding
The authors gratefully acknowledge Aaron Taylor, George Campbell & Victoria Frohlich of the Cell and Tissue Imaging core facility for support of fluorescence microscopy; the members of the St. Jude Children’s Research Hospital Center for Advanced Genome Engineering for genome engineering; Dr. Richard Ashmun and the staff of the Flow Cytometry core facility for instructions on the single-cell cloning and cell sorting; the Vector Development and Production Shared Resource for lentiviral production; the staff of the Hartwell Center for Sanger DNA sequencing and STR profiling. The St. Jude Children’s Research Hospital core facilities are supported by NCI Cancer Center Support Grant P30 CA021765 and by ALSAC. We acknowledge funding support, as follows: NIGMS grant 5R35GM131891 to R.K., Research Collaborative grant on the Biophysics of RNP Granules from St. Jude Children’s Research Hospital to R.K., NIGMS grant 1F32GM133078 to E.B.G., and a Gephardt Endowed Postdoctoral Fellowship from St. Jude Children’s Research Hospital to E.B.G. A portion of this research used resources at the Spallation Neutron Source, a DOE Office of Science user facility operated by the Oak Ridge National Laboratory.