p14ARF forms meso-scale assemblies upon phase separation with NPM1

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 (p14^ARF). Oncogenic stress induces p14^ARF to inhibit MDM2, stabilize p53 and arrest the cell cycle. Under non-stress conditions, NPM1 stabilizes p14^ARF in nucleoli, preventing its degradation and blocking p53 activation. However, the mechanisms underlying the regulation of p14^ARF by NPM1 are unclear because the structural features of the p14^ARF-NPM1 complex remain elusive. Here we show that NPM1 sequesters p14^ARF within phase-separated condensates, facilitating the assembly of p14^ARF into a gel-like meso-scale network. This assembly is mediated by intermolecular contacts formed by hydrophobic residues in an α-helix and β-strands within a partially folded N-terminal domain of p14^ARF. Those hydrophobic interactions promote phase separation with NPM1, enhance nucleolar partitioning of p14^ARF, restrict p14^ARF and NPM1 diffusion within condensates and in nucleoli, and reduce cell viability. Our structural model provides novel insights into the multifaceted chaperone function of NPM1 in nucleoli by mechanistically linking the nucleolar localization of p14^ARF to its partial folding and meso-scale assembly upon phase separation with NPM1.

Fig. 1.. p14^ARF Exhibits Local and Long-Range Ordering within Condensates with NPM1.

A) NPM1 structural features, including the secondary structure calculated from PDB:4N8M (OD), and PDB:2LLH (NTD) using DSSP, and the linear net charge per residue (LNCPR) and linear hydropathy (Hydro.) calculated using CIDER. B) p14^ARF structural features, including PSI-PRED secondary structure prediction (2°Struc.; β-strands are indicated with arrows and an α-helix with a cylinder), CIDER linear net charge per residue (LNCPR) and linear hydropathy (Hydro.), sequence conservation (Cons.) based on multi-sequence alignment using MUSCLE, and Rosetta steric zipper propensity energy (R. Energy) calculated using ZipperDB. C) CV-SANS curves for the p14^ARF-NPM1 condensed phase, which reveal the spatial organization of NPM1 (green trace), p14^ARF (blue trace) and the p14^ARF-NPM1 complex (grey trace). All curves are offset for clarity, with points shown as the average and standard deviation. Correlation peaks at ~200 Å and ~400 Å correspond to meso-scale organization of p14^ARF. D) 2D CC-DARR spectrum of [¹³C,¹⁵N]-p14^ARF within the condensed phase. E) Secondary ¹³C chemical shifts for [¹³C,¹⁵N]-p14^ARF within the condensed phase. Assigned residues are highlighted in grey. The secondary structure prediction from panel B is shown at the top.

Fig. 2.. Structural Model for the p14^ARF Component of the p14^ARF-NPM1 Condensed Phase.

A) Intramolecular C_α-C_α distances for the p14^ARF ensemble. B) Comparison of the ensemble and experimental polymer scaling factors. C) Comparison of the ensemble and experimental C_α chemical shifts. D) Comparison of the ensemble and experimental C_β chemical shifts. E) Representative conformers from the p14^ARF ensemble. F) Comparison of the experimental p14^ARF CV-SANS curve (light blue scatter points) and the p14^ARF ensemble model (blue trace). Points represent the average and standard deviation. G) Ensemble model for the p14^ARF meso-scale assembly.

Fig. 3.. The NPM1 IDR Retains Disorder and Experiences Attenuated Backbone Motions within the Condensed Phase with p14^ARF.

A) 2D ¹H-¹⁵N TROSY-HSQC spectrum of [¹³C,¹⁵N]-NPM1 within the p14^ARF-NPM1 condensed phase, displaying signals from the NPM1 IDR. B) Linear net charge per residue (LNCPR) for the NPM1 IDR. ¹H-¹⁵N heteronuclear NOE, R₁ and R₂ transverse relaxation profiles for NPM1 in solution (blue) and within the p14^ARF-NPM1 condensed phase (red), which show a restriction of IDR backbone motions on the ps-ns timescale. Exchange broadening rates R_ex for condensed NPM1 are shown on the bottom. C) ¹⁵N-CPMG relaxation dispersion profiles for Ala186, A201 and T199 collected at 800 MHz, with fits to a two-state model. D) Upon phase separation with p14^ARF the NPM1 IDR exchanges slowly between multiple conformations on the μs-ms timescale. All error bars represent the standard deviations.

Fig. 4.. Substitution of p14^ARF Hydrophobic Residues Blocks p14^ARF Meso-Scale Ordering and Restores NPM1 Mobility within Condensates.

A) p14^ARF structural features, including PSI-PRED4.0 secondary structure (2°Struc.) prediction, CIDER linear net charge per residue (LNCPR) and CIDER linear hydropathy (Hydro.). CIDER analysis for p14^ARFΔH1-3 is shown on the bottom. B) Confocal fluorescence micrographs of p14^ARF-NPM1 condensates (top) and p14^ARFΔH1-3-NPM1 condensates (bottom). Scale bars = 10 μm. C) Phase diagrams for condensates shown in panel B quantified using the index of dispersion. D) CV-SANS curves for the p14^ARFΔH1-3-NPM1 condensates; NPM1 (green trace), p14^ARFΔH1-3 (blue trace), p14^ARFΔH1-3-NPM1 complex (grey trace). All curves are offset for clarity, with points shown as the average and standard deviation. E) FRAP of NPM1-AF488 within condensates shows that substitution of p14^ARF hydrophobic residues to Gly/Ser spacer residues restores NPM1 mobility. F) FRAP recovery curves for p14^ARF-NPM1 and p14^ARFΔH1-3-NPM1 condensates (n=10 for each condition, Wilcoxon rank-sum test). G) NPM1-AF488 D_App values extracted from the FRAP recovery curves in panel F (n=10, Wilcoxon rank-sum test). For panels F and G, (***) p < 0.001.

Fig. 5.. p14^ARF Reduces Nucleolar NPM1 Diffusion in a Concentration Dependent Manner.

A) Schematic constant-temperature and pressure phase diagram for p14^ARF-NPM1. Single phase regions are shown in white; coexistence regions are shown in gray. The curved arrow represents a concentration vector that crosses through the coexistence regions, initially sampling a liquid-like NPM1-rich phase, followed by a gel-like p14^ARF-NPM1 phase, terminating in a solid-like p14^ARF-rich phase. B) Fluorescence microscopy images of live B11 cells before and 48 hours after doxycycline induced p14^ARF-iRFP expression. Scale bars = 2 μm. C) Z-score analysis of NPM1-GFP and p14^ARF-iRFP levels in DLD-1^NPM1–G cells, showing that p14^ARF and NPM1 levels are anti-correlated (two-sided Mann-Whitney U-test, n = 2272, 122, 54), (*) p < 0.05, (****) p < 0.0001. D) Representative single-cell FRAP for two cells selected from the DLD-1 population shown in C. The curves on the left are from a cell expressing a high level of nucleolar NPM1 and low level of p14^ARF. The curves on the right are from a cell expressing a low level of nucleolar NPM1 and a high level of p14^ARF. E) The D_App and F) the mobility for nucleolar NPM1-GFP is reduced as nucleolar p14^ARF-iRFP levels increase (small, transparent markers) and as the duration of p14^ARF-iRFP expression is extended (large, opaque markers). These correlated reductions in dynamics are consistent with the assembly of large molecular weight p14^ARF-NPM1 complexes. For panels E and F error bars represent the standard deviation.