ZNF692 organizes a hub specialized in 40S ribosomal subunit maturation enhancing translation in rapidly proliferating cells

Abstract

Increased nucleolar size and activity correlate with aberrant ribosome biogenesis and enhanced translation in cancer cells. One of the first and rate-limiting steps in translation is the interaction of the 40S small ribosome subunit with mRNAs. Here, we report the identification of the zinc finger protein 692 (ZNF692), a MYC-induced nucleolar scaffold that coordinates the final steps in the biogenesis of the small ribosome subunit. ZNF692 forms a hub containing the exosome complex and ribosome biogenesis factors specialized in the final steps of 18S rRNA processing and 40S ribosome maturation in the granular component of the nucleolus. Highly proliferative cells are more reliant on ZNF692 than normal cells; thus, we conclude that effective production of small ribosome subunits is critical for translation efficiency in cancer cells.

Keywords: 40S; CP: Molecular biology; EXOSC7; EXOSC8; KRR1; MYC; ZNF692; exosome; nucleolus; rRNA; ribosome biogenesis.

Figure 1.. MYC promotes the transcription of ZNF692, an evolutionarily conserved nucleolar factor

(A) Zfp692 mRNA expression in Rat1 myc−/− cells expressing empty vector or human MYC upon control siRNA (siCtrl) or MYC siRNA transfection. Biological replicates n = 3 with two technical replicates. (B) WB for Zfp692 in Rat1 myc−/− cells expressing empty vector or human MYC. (C) ZNF692 mRNA expression in ARPE cells expressing empty vector or MYC. Biological replicates n = 4. (D) WB for ZNF692 in ARPE cells expressing empty vector or MYC. (E) WB for ZNF692 in control siRNA (siCtrl) or MYC siRNA-transfected HCT116 cells. (F) IF for ZNF692 in ARPE cells expressing empty vector or MYC. (G) Predicted nucleolar localization sequence (NoLS) in the N terminus of ZNF692. (H) Identification of a NoLS in ZNF692 using nucleolar localization sequence detector (NoD). (I) IF of endogenous ZNF692 colocalizing with NPM1. (J) IF of NPM1 in WT and ΔNoLS GFP-ZNF692-transfected cells. (k) IF of RPA40 in WT and ΔNoLS GFP-ZNF692-transfected cells. Deletion of the NoLS prevented ZNF692 colocalization with NPM1 or RPA40. *p < 0.05, two-tailed unpaired Student’s t test statistical analysis. Graphs with mean ± SD. Scale bars represent 10 μm.

Figure 2.. Knockdown of ZNF692 reduced the viability of proliferative cells without affecting the expression of cell-cycle regulators

(A) WB of HCT116 expressing empty vector (E.V.) or ZNF692 4, 6, 24 or 48 h after FBS stimulation. (B) Heatmap showing quantification of (A) related to tubulin. (C) Relative proliferation of HCT116 cells 3 days after transfection with control or ZNF692 siRNAs. Biological replicates n = 4. (D) WB of HCT116 cells transfected with control or ZNF692 siRNA. (E) Relative proliferation of ARPE cells expressing empty vector or MYC 3 days after transfection with control or ZNF692 siRNA. Biological replicates n = 3. (F) WB of ARPE cells expressing empty vector (E.V.) or MYC 3 days after transfection with control or ZNF692 siRNA. (G) Relative proliferation of HCT116 cells stably expressing ZNF692 or shRNA for ZNF692. Biological replicates n = 3.(H) WB of HCT116 cells stably expressing ZNF692 or shRNA for ZNF692. (I) Relative proliferation of DLD1 cells CRISPR KO for ZNF692 or control. Biological replicates n = 4. (J) WB of DLD1 cells CRISPR KO for ZNF692 or control. (K) Schematic representation of xenograft experiment. (L) Representative pictures of tumors collected at endpoint of the xenografts experiment. (See also Figure S3H.) (M) Tumor weight from xenograft experiment. SgCtrl n = 9, sgZNF692–3 n = 8. (N) Previously reported ZNF692 target genes in our RNA-seq. Biological replicates n = 3. (O) ZNF692 expressing levels in normal and tumor tissues of TCGA COAD samples. (P) Patient survival correlation with 25% highest and lowest ZNF692 mRNA levels of TCGA COAD samples. (Q) ZNF692 mRNA expression in tumor vs normal tissues from patients of different TCGA tumor types. *p < 0.05, two-tailed unpaired Student’s t test statistical analysis. Graphs with mean ± SD.

Figure 3.. ZNF692 regulates nucleolar morphology and protein synthesis

(A) H&E staining from ZNF692 KO or control DLD1 xenograft tumors. White dotted line outlines the nucleolus. (See also Figure S3G.) Scale bars represent 10 μm. (B) Quantification of nucleolar area and circularity (more circular the closer to 1) of ZNF692 KO or control DLD1 xenograft tumors. (See also Figure S3H.) Number of nucleoli sgCtrl n = 108, sgZNF692–1 n = 124, sgZNF692–3 n = 212. (C) Correlation between levels of ZNF692 measured by IF with nucleolar size and circularity (more circular the closer to 1) in HCT116 cells. (See also Figure S3C.) Number of nucleoli top 25% n = 220, bottom 25% n = 218. Representation of one biological replicate out of three with similar results. (D) Electron microscopy of two examples of HCT116 cells after 3 days of infection with empty vector (PLKO) or shRNA for ZNF692 (#2 and #5) containing virus (See also Figure S2B.) Scale bars represent 2 μm. (E) Quantification of nucleolar area and circularity (more circular the closer to 1) from cells in C. ZNF692 KD reduces the perimeter. Number of nucleoli pLKO n = 25, shZNF692–2 n = 34, shZNF692–5 n = 30. (F) Schematic representation of the puromycylation-based assays to measure protein synthesis. (G) Puromycylation of control or ZNF692 siRNA-transfected DLD1 cells. (H) Puromycylation of control or ZNF692 shRNA transiently infected HCT116 cells. (I) Puromycylation of control or ZNF692 shRNA stably infected HCT116 cells. Three replicates are shown. (J) Puromycylation of WT or ZNF692 KO (sg1 and sg3) DLD1 cells. (K) Puromycylation of HCT116 stably expressing ZNF692. Three replicates are shown.(L) Schematic representation of the harringtonine and puromycylation-based assay to measure translation elongation. (M) Inhibition of translation initiation with harringtonine followed by puromycylation chase in DLD1 ZNF692 KO or control. *p < 0.05, two-tailed unpaired Student’s t test statistical analysis. Graphs with mean ± SD.

Figure 4.. ZNF692 resides in the granular component of the nucleolus where it interacts with ribosomal proteins

(A) Pre-rRNA levels of control or ZNF692 shRNA stably infected HCT116 cells. pLKO n = 3, shZNF692–2 n = 1, shZNF692–5 n = 3, with two to three technical replicates each. Graphs with mean ± SD. (B) Pre-rRNA in ZNF692 KO or control DLD1 xenograft tumors from Figures S2J–S2L. SgCtrl n = 6, sgZNF692–1 n = 5, sgZNF692–3 n = 6. Graphs with mean ± SD and p value. (C) rRNA and ZNF692 IF showing equal rRNA levels in control or ZNF692 shRNA stably infected HCT116 cells. Scale bars represent 10 μm. (D) RNA-FISH for 5ʹ-ETS (targeting 47S pre-rRNA) in ZNF692 KO or control DLD1 cells. Scale bars represent 10 μm. (E) GFP, GFP-tagged WT ZNF692, and ΔNt constructs and their localization when expressed in HCT116 cells. Scale bars represent 5 μm. (F) GFP WB of anti-GFP nanotrap bead immunoprecipitants in HCT116 cells transfected with GFP, GFP-ZNF692, or GFP-ZNF692ΔNt. Immunoprecipitants were analyzed by mass spectrometry to identify ZNF692’s interactome. (G) Gene Ontology of ZNF692 partners (see also Figures S4E–S4F). (H) Representation of a nucleolus and its contained subcompartments. (I) Representation of the expected localization of RPA40 in the fibrillary centers (FCs), fibrillarin (FBL) in the dense fibrillar component (DFC), and NPM1 in the granular component (GC) of the nucleolus. (J) IF of ZNF692-transfected DLD1 cells showing the colocalization of ZNF692 with NPM1 in the GC through double staining of ZNF692 (red) with RPA40, FBL, or NPM1 (green). Scale bars represent 5 μm. (k) Structured illumination microscopy (SIM) in ZNF692-transfected DLD1 cells showing that ZNF692 is colocalized with NPM1 but not RPA40 and FBL. (See also Figure S4I.) Scale bars represent 2 μm.

Figure 5.. ZNF692 interacts with the components of the exosome complex

(A) ZNF692 interactors and their function in ribosomal subunit assembly. (See also Table S4.). (B) ZNF692 recombinant proteins purified from insect cells to be used for in vitro assays. (C) IP of recombinant ZNF692 using GFP nanotrap beads and DLD1 cells nuclear extracts followed by WB for NOP2, KRR1, EXOSC7, and EXOSC8. (D)IF of NOP2, KRR1, EXOSC7, or EXOSC8 with NPM1 showing their co-colocalization in the granular component of the nucleolus. Scale bars represent 10 μm. (E) IP of EXOSC7, EXOSC8 NOP2, KRR1, and ZNF692 from HCT116 cells stably overexpressing ZNF692 followed by WB for ZNF692, NOP2, KRR1, EXOSC7, and EXOSC8. Left panel: control; right panel: lysates were treated with 20 μg/mL RNase A prior to IP. (F) RNA-IP of ZNF692 in DLD1 cells on rRNA; ZNF692 binds rRNA. n = 3 (two technical replicates each). *p < 0.05, two-tailed unpaired Student’s t test statistical analysis. Graphs with mean ± SD and p value. (G) Puromycylation in control or NOP2, KRR1, EXOSC7, or EXOSC8 shRNA transiently infected DLD1 cells 3 days after infection. (H) WB for NOP2, KRR1, EXOSC7, and EXOSC8 in cell myc−/− fibroblasts expressing empty vector (E.V.) or human MYC. (I) WB for MYC, NOP2, KRR1, EXOSC7, and EXOSC8 in control or MYC siRNA DLD1 cells. (J) Heatmap of ZNF692, KRR1, EXOSC7, EXOSC8, and NOP2 expression in tumor vs normal tissues of TCGA COAD samples.

Figure 6.. ZNF692 enhances the last steps of 18S rRNA processing

(A) Representation of the pre-rRNA processing pathway showing the functions of ZNF692 interactors. (B) Northern blot of ZNF692, EXOSC10, or control siRNA transfected ARPE-MYC cells. (C) Quantification of 5ʹ-ITS1and 5ʹ-ITS2 by ratio analysis of multiple precursors (RAMP) for siZNF692 in B. N = 3. (D) Northern blot of ZNF692 or control siRNA-transfected DLD1 cells. (E) Quantification of 5ʹ-ITS1 by RAMP of D. N = 3. (E) Schematic representation of the role of ZNF692 on 18S rRNA maturation. siCtrl., siRNA control; siZ-3, siRNA for ZNF692 #3; siEX10, siRNA for EXOSC10. *p < 0.05, two-tailed unpaired Student’s t test statistical analysis. Graphs with mean ± SD and p value.

Figure 7.. ZNF692 enhances small ribosomal subunit assembly and translation initiation

(A) Representation of cellular/nucleolar fractionation using the three-step Pre-ribosome Sequential Extraction (PSE) protocol. Adapted from Nieto et al., 2021. (B) WB of the cellular/nucleolar fractionation for ZNF692 and ZNF692 interactors in control or ZNF692 KO DLD1 cells. (C and D) 3D reconstruction of SIM IF images (Figure S7A) for NPM1, ZNF692, and FBL in ZNF692 KO DLD1 cells stably overexpressing ZNF692 showing the surface (C) and the volume (D) of ZNF692, NPM1, and FBL in the nucleolus. (See also Figure S7B.). (E) WB of the cellular/nucleolar fractionation for ZNF692 and EXOSC10 in E.V. or ZNF692-overexpressing HCT116 cells. (F) Northern blot of SN1 and SN2 fractions of ZNF692-overexpressing HCT116 cells. (G) Polysome profiling of control or ZNF692 KO DLD1 cells. (H) RNA bioanalyzer analysis of 40S, 60S, and 80S cytoplasmic fractions collected from (G). (I) Proteomics analysis heatmap showing proteins whose abundance changed in 40S, 60S, and 80S single ribosomes of control or ZNF692 KO DLD1 cells from (G). (J) WB for ribosomal proteins of 40S, 60S, and 80S fractions (G) of control or ZNF692 KO DLD1 cells. Actin was used as loading control. (K) Click-IT AHA experiments in DLD1 ZNF692 KO cells (−/+ ectopic ZNF692) or control cells. Graphical abstract: ZNF692 and SSU processome components promotes 18S rRNA processing and small ribosomal subunit assembly. Model for the function of ZNF692 in driving small ribosome subunit maturation and translation initiation in highly proliferative cells. MYC drives the expression of ZNF692 as well as components of the SSU processome such as KRR1 and the exosome complex promoting small ribosomal subunit maturation.