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 were elusive. Here we show that p14^ARF 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 p14^ARF. These hydrophobic interactions promote phase separation with NPM1, enhance p14^ARF 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 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 the oligomerization domain (OD) PDB 4N8M and the nucleic acid binding domain (NBD) PDB 2LLH, using DSSP (2^o Struc.; β-strands are indicated with arrows and α-helicies are indicated with cylinders). The CIDER linear net charge per residue (LNCPR) and linear hydropathy (Hydro.) are shown for the IDR. B p14^ARF structural features, including PSI-PRED secondary structure prediction (2^o Struc.), CIDER linear net charge per residue (LNCPR), and linear hydropathy (Hydro.). The amino acid sequence conservation (Cons.) is based on a multiple sequence alignment using MUSCLE. The bottom panel shows the Rosetta steric zipper propensity energy (R. Energy) calculated using ZipperDB. C CV-SANS curves for p14^ARF-NPM1 condensates, in 100% D₂O buffer for full scattering intensity (NPM1 and p14^ARF detected; gray trace), in 45% D₂O buffer where p14^ARF is contrast matched ([²H]-NPM1 detected; green trace), and in 85% D₂O buffer where [²H]-NPM1 is contrast matched (p14^ARF detected; blue trace). Correlation peaks at ~200 Å and ~400 Å correspond to meso-scale organization of p14^ARF. All curves are offset for clarity. Scatter points represent the average, the error bars represent the uncertainty derived from the counting statistics of the SANS instrument, as described and cited in the Methods. D Schematic describing NPM1 with extended IDR conformations. E Schematic describing the spatial organization of p14^ARF within p14^ARF-NPM1 condensates. F 2D CC-DARR spectrum of [¹³C,¹⁵N]-p14^ARF within the condensed phase. Select resonance assignments are labeled. G Secondary ¹³C chemical shifts for [¹³C,¹⁵N]-p14^ARF within the condensed phase. Assigned residues are highlighted in gray. The secondary structure prediction from panel B is shown for reference (2^o Struc.; top).

Fig. 2. p14^ARF engages in intra- and intermolecular contacts within the condensed phase with NPM1.

A A CC-DARR spectrum acquired for [¹³C,¹⁵N]-p14^ARF with 20 ms DARR mixing time shows resonances for T8 in two states, including in an expanded p14^ARF conformation (top), and in a collapsed p14^ARF conformation (bottom). B A CC-DARR spectrum acquired with 400 ms DARR mixing time shows additional cross-peaks indicating intramolecular contacts between T8 and H26. C The 2D-NHHC spectrum (gray) of p14^ARF (equal mixture of [¹³C]-p14^ARF and [¹⁵N]-p14^ARF) shows that sidechains within the p14^ARF N-terminus make intermolecular contacts within the condensed phase with NPM1. HNCA (magenta) and 2D-HNCACX (blue) spectra for [¹³C,¹⁵N]-p14^ARF are shown for reference. D Schematic describing possible modes of p14^ARF intra- and intermolecular interaction.

Fig. 3. The NPM1 IDR retains disorder and experiences attenuated backbone motions within the condensed phase with p14^ARF.

A The 2D ¹H-¹⁵N TROSY-HSQC spectrum of [¹³C,¹⁵N]-NPM1 within the p14^ARF-NPM1 condensed phase displays resonances from the NPM1 IDR. B Linear net charge per residue (LNCPR) for the NPM1 IDR. Nuclear spin relaxation for [²H,¹⁵N]-NPM1 in solution (blue scatter points) and condensed phase [¹³C,¹⁵N]-NPM1 (red scatter points), including C ¹H-¹⁵N heteronuclear NOEs, D R₁, and E R₂ transverse relaxation, which shows a restriction of NPM1 IDR backbone motions on the ps-ns timescale. The error bars for R₁ and R₂ transverse relaxation plots represent the standard errors from curve fitting, as described in Methods. F The contributions from exchange broadening, R_ex. G ¹⁵N-CPMG relaxation dispersion profiles for condensed [¹³C,¹⁵N]-NPM1 measured at 800 MHz, including A186, T199, and A201, fit to a two-state model. Scatter points represent the decay rates, and the error bars represent the estimated systematic error, as described in Methods. H Schematic describing NPM1 IDR conformational exchange within condensates with p14^ARF.

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 prediction (2^o Struc.), CIDER linear net charge per residue (LNCPR) and linear hydropathy (Hydro.). The CIDER analysis for p14^ARFΔH1-3 is shown below. B Zoomed in regions from confocal fluorescence micrographs of NPM1-AF488 in condensates with p14^ARF (top) and p14^ARFΔH1-3 (bottom). Scale bars = 10 µm. C Index of dispersion for NPM1 in condensates with p14^ARF (gray boxes, whiskers and trace; derived from n = 6, 6, 6, 7, 6, 6, 7, 6, 6, 6, 7 images) and p14^ARFΔH1-3 (blue boxes and whiskers and trace, where n = 5, 4, 6, 6, 8, 6, 6, 6, 6, 6, 6 images). Whiskers extend from the box to the furthest point within 1.5x the inter-quartile range. The black arrow highlights the increased NPM1 saturation concentration, ΔC_sat, upon substitution of p14^ARF hydrophobic residues to Gly/Ser spacer residues. The gray arrow highlights the reentrant phase transition, which occurs at elevated p14^ARF concentrations. D ΔG^tr for NPM1 in condensates with p14^ARF (gray boxes, whiskers and trace, where n = 696, 42, 61, 159, 225, 285, 333, 276, 306, 227, 773 condensates) and p14^ARFΔH1-3 (blue boxes, whiskers and trace, where n = 2561, 1787, 29, 31, 82, 92, 134, 153, 166, 145, 162 condensates). Whiskers extend from the box to the furthest point within 1.5x the inter-quartile range. The C_sat for NPM1 increases when p14^ARF hydrophobic residues are substituted. The gray arrow highlights the destabilization of NPM1 during the reentrant phase transition. E CV-SANS curves for p14^ARFΔH1-3-[²H]-NPM1 condensates collected at 50% D₂O, where p14^ARFΔH1-3 is contrast matched ([²H]-NPM1 detected; green trace), at 85% D₂O where [²H]-NPM1 is contrast matched (p14^ARFΔH1-3 detected; blue trace), and p14^ARFΔH1-3-NPM1 condensate at 100% D₂O for full scattering intensity (NPM1 and p14^ARFΔH1-3 detected; gray trace). All curves are offset for clarity. Scatter points represent the average, the error bars represent the uncertainty derived from the counting statistics of the SANS instrument, as described and cited in the Methods. F Schematic describing condensed NPM1 with extended IDR conformations. G Schematic describing condensed p14^ARFΔH1-3 in an extended conformation. H FRAP of NPM1-AF488 within condensates shows that substitution of p14^ARF hydrophobic residues to Gly/Ser spacer residues restores NPM1 mobility. Scale bars = 1 µm. I FRAP recovery curves for p14^ARF-NPM1 and p14^ARFΔH1-3-NPM1 condensates with fits, as described in Methods (statistical significance was assessed by two-sided Wilcoxon rank-sum test, n = 10 curves for each condition, the p-value is shown in the figure). J NPM1-AF488 D_App values extracted from the FRAP recovery curves in panel I (statistical significance was assessed by two-sided Wilcoxon rank-sum test, n = 10 D_App values for each condition, the p-value is shown in the figure).

Fig. 5. p14^ARF reduces nucleolar NPM1 diffusion in a concentration dependent manner.

A Zoomed in regions from fluorescence microscopy images of live DLD-1^NPM1-G (clone B11) cells, before and after 48 h of doxycycline induced p14^ARF-iRFP expression. Scale bars = 2 µm. B 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 (statistical significance was assessed by two-sided Mann–Whitney U-test, n = 2272, 122, 54 cells, p-values are shown in the figure) C FRAP curves with fits, as described in Methods, for cells sorted from the DLD-1^NPM1-G population shown in B. The curves on the left are from a cell expressing a high level of nucleolar NPM1 (clone F6; green trace) and a low level of nucleolar p14^ARF (clone F6; blue trace). The curves on the right are from a cell expressing a low level of nucleolar NPM1 (clone G2; green trace) and a high level of nucleolar p14^ARF (clone G2; blue traces). In unsorted DLD-1^NPM1-G cells, D The D_App and E mobility for nucleolar NPM1-GFP and p14^ARF-iRFP (small green and blue transparent markers, respectively, n = 45 cells) are reduced as nucleolar p14^ARF-iRFP levels increase. Reductions also occur as the duration of p14^ARF-iRFP expression is extended (large opaque markers; scatter points represent the mean and error bars represent the standard deviation, where n = 20 cells). F A schematic describing the correlated reductions in p14^ARF and NPM1 dynamics and their assembly into large molecular weight complexes within the granular component (GC) of the nucleolus.