ZNF692 regulates nucleolar morphology by interacting with NPM1 and modifying its self-assembly properties

Abstract

The nucleolus, a membrane-less organelle, is responsible for ribosomal RNA transcription, ribosomal RNA processing, and ribosome assembly. Nucleolar size and number are indicative of a cell's protein synthesis rate and proliferative capacity, and abnormalities in the nucleolus have been linked to neurodegenerative diseases and cancer. In this study, we demonstrated that the nucleolar protein ZNF692 directly interacts with nucleophosmin 1 (NPM1). Knocking down ZNF692 resulted in the nucleolar redistribution of NPM1 in ring-like structures and reduced protein synthesis. Purified NPM1 forms spherical condensates in vitro but mixing it with ZNF692 produces irregular condensates more closely resembling living cell nucleoli. Our findings indicate that ZNF692, by interacting with NPM1, plays a critical role in regulating nucleolar architecture and function in living cells.

Keywords: NPM1; ZNF692; condensates; nucleolus; protein assembly.

Figure 1

ZNF692 directly interacts with NPM1 in the nucleolus.A, schematic of ZNF692 pulldown proteomics. B, R-motif sequences of NPM1 interactors compared to the ZNF692 Nt domain (NoLS). C, immunoprecipitation of HCT116 cells transfected with GFP, GFP-ZNF692 or GFP-ZNF692 ΔNt and immunoblotted for ZNF692, NPM1, FBL, and RPA40. D, ZNF692 and NPM1 immunoprecipitation in HCT116 ZNF692-Flag. E, ZNF692 and NPM1 immunoprecipitation in HCT116 ZNF692-Flag (with and without RNase A). F, schematic of recombinant GFP-ZNF692. GFP-ZNF692 (ZNF692 amino acids 1–519), GFP-ZNF692 ΔNt (lacking ZNF692 amino acids 3–33), GFP-ZNF692 ΔZNF2-4 lacking zinc fingers 2 to 4 (lacking ZNF692 amino acids 360–439), and GFP-ZNF692 ΔNt-Ct (lacking amino acids 1–97 and 320–519). G, immunoprecipitation of the recombinant ZNF692 in (F) with DLD1 nuclear extracts followed by immunoblot for NPM1, FBL, and RPA40. H, Immunofluorescence of ZNF692, NPM1, and FBL in DLD1 cells expressing ZNF692 WT or mutants. Scale bar = 20 μm.

Figure 2

ZNF692 depends on NPM1 to promote protein synthesis.A, puromycylation: Puromycing incorportates into translating peptides. Puromycin-labeled peptides can be detected by Western blot using an anti-puromycin antibody. B, puromycylation in ARPE cells 72 h after transfection with control, NPM1 or ZNF692 siRNA. Immunoblot for indicated antibodies. C, puromycylation in HCT116 cells expressing ZNF692 or empty vector (EV) after 72 h of control, ZNF692 and NPM1 siRNA transfection. Immunoblot for indicated antibodies.

Figure 3

ZNF692 alters the morphology of NPM1 droplets.A, NPM1 predicted disordered region identified with Protein DisOrder prediction System (PrDOS). B, NPM1 predicted protein structure using AlphaFold. C, ZNF692 predicted disordered regions identified with Protein DisOrder prediction System (PrDOS). D, ZNF692 predicted protein structure using AlphaFold. E, coomassie-stained gel of purified mCherry-NPM1 and GFP-ZNF692. F, schematic of the in vitro complex formation experiments using GFP-ZNF692 and mCherry-NPM1 alone or in combination. G, self-assembly of 5 μM GFP-ZNF692 or mCherry-NPM1 in the presence of increasing PEG amounts. H, self-assembly of 5 μM purified mCherry-NPM1 in 10% PEG and increasing GFP-ZNF692 amounts. I, self-assembly of 5 μM purified GFP-ZNF692 WT or mutants and mCherry-NPM1 in 10% PEG. Scale bar = 10 μm. Scale bar insert picture = 5 μm.

Figure 4

NPM1 N-terminal domain is necessary to colocalize and interact with ZNF692 in the nucleolus.A, NPM1-GFP: wild type NPM1 (amino acids 1–305), and NPM1 mutants: NPM1-GFP ΔCt (lacking amino acids 245–305), NPM1-GFP ΔNt (lacking amino acids 1–117), and NPM1-GFP ΔNt-ΔCt (lacking amino acids 1–117 and 245–305). B, WB showing NPM1-GFP expression in HCT116 ZNF692-Flag. C, immunofluorescence of NPM1-GFP mutants and ZNF692 in HCT116 ZNF692-Flag cells. Max intensity from z-stack images is shown. D, ZNF692-Flag immunoprecipitation with Flag-trap beads or agarose beads (control) in HCT116 ZNF692-Flag cells stably expressing NPM1-GFP or GFP constructs or control (no construct). E, orthogonal views from z-stack images in (C) showing partial colocalization of WT and ΔCt NPM1 and ZNF692 (yellow area). Scale bar = 2 μm.

Figure 5

NPM1 distribution within the nucleolus is affected by the presence of ZNF692.A, immunofluorescence of NPM1 in ARPE and ARPE-MYC 72 h after transfection with control or ZNF692 siRNAs. Scale bar = 20 μm. B, example of intensity profile of nucleolar NPM1 indicated by white arrow in Figure 5A. Scale bar = 4 μm. C, immunofluorescence of NPM1 in DLD1 and DLD1 ZNF692 cells transfected with control or ZNF692 siRNAs. Scale bar = 20 μm. D, example of the intensity profile of nucleolar NPM1 indicated by white arrow in Figure 5C. Scale bar = 4 μm. E, percentage of cells with homogenous or redistributed NPM1 from (A). Each data point represents NPM1 distribution percentage within an image. N = 100 cells from three experiments. F, percentage of cells with homogenous or redistributed NPM1 from (C). Each data point represents NPM1 distribution percentage within an image. N = 100 cells from 3 experiments. G, working model. NPM1 self-assemblies into spherical structures. In the presence of ZNF692, ZNF692 interacts with NPM1 restructuring NPM1 self-assembly and allowing for a more interconnected and active nucleoli.