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A PRC2-independent function for EZH2 in regulating rRNA 2′-O methylation and IRES-dependent translation

Abstract

Dysregulated translation is a common feature of cancer. Uncovering its governing factors and underlying mechanism are important for cancer therapy. Here, we report that enhancer of zeste homologue 2 (EZH2), previously known as a transcription repressor and lysine methyltransferase, can directly interact with fibrillarin (FBL) to exert its role in translational regulation. We demonstrate that EZH2 enhances rRNA 2′-O methylation via its direct interaction with FBL. Mechanistically, EZH2 strengthens the FBL–NOP56 interaction and facilitates the assembly of box C/D small nucleolar ribonucleoprotein. Strikingly, EZH2 deficiency impairs the translation process globally and reduces internal ribosome entry site (IRES)-dependent translation initiation in cancer cells. Our findings reveal a previously unrecognized role of EZH2 in cancer-related translational regulation.

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Fig. 1: EZH2 directly interacts with FBL in PCa cells and tissues.
Fig. 2: EZH2 modulates rRNA 2′-O-Me by interacting with FBL.
Fig. 3: EZH2 bridges the FBL–NOP56 interaction by binding to both proteins.
Fig. 4: Formation of a EZH2–FBL–NOP56 trimer facilitates box C/D snoRNP assembly.
Fig. 5: EZH2 regulates the translation process in a positive manner.
Fig. 6: EZH2 promotes XIAP IRES-dependent translation.
Fig. 7: Contributions of FBL and NOP56 in EZH2-driven PCa tumorigenesis.

Data availability

The next-generation sequencing data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession codes GSE159004 (RiboMeth-seq) and GSE143975 (Ribo-seq/RNA-seq). Previously published EZH2 RIP-seq data that were re-analysed here are available under accession code GSE63230. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank the Center for Advanced Microscopy/Nikon Imaging Center of Northwestern University for assistance with confocal microscopy. We thank the Vancouver Prostate Centre for assistance with IHC and PLA assays. We appreciate the Metabolomics Core Facility at Robert H. Lurie Comprehensive Cancer Center of Northwestern University for assistance with LC–MS/MS to detect the 2′-O-Me status of RNA. We thank J. C. Trinidad and the Laboratory for Biological Mass Spectrometry Facility at Indiana University Bloomington for conducting the MS analysis of crosslinked samples. This work was supported by a start-up fund provided by the Northwestern University. Q.C. was supported by Northwestern University, the US Department of Defense (W81XWH-17-1-0357, W81XWH-19-1-0563 and W81XWH-20-1-0504), the American Cancer Society (RSG-15-192-01), the NIH/NCI (R01CA208257 and Prostate SPORE P50CA180995 Development Research Program) and the Polsky Urologic Cancer Institute of the Robert H. Lurie Comprehensive Cancer Center of Northwestern University at Northwestern Memorial Hospital. K.C. was supported by start-up funding provided by The Houston Methodist Research Institute and Boston Children’s Hospital. W.Z. was supported by the National Natural Science Foundation of China (81972651, 31771630, 81802974 and 81702784), the Guangdong Innovative and Entrepreneurial Research Team Program (2016ZT06S029), the Natural Science Foundation of Guangdong Province (2017A030310228, 2017A030312009 and 2016A030313238), the Medical Scientific Research Foundation of Guangdong Province of China (A2017110), and the Special funds for Dapeng New District Industry Development (KY20160309). H.N. was supported by National Institutes of Health grant R35GM124765. Z.J. was supported by R00 CA 207865 from National Cancer Institute. E.M.S. was supported in part by the National Institutes of Health grant 5U01CA196390, the Prostate Cancer Foundation, Department of Defense grant W81XWH-15-1-0661 (E.M.S. and T.L.L.). A.B.W. was supported by the 2019 Urology Care Foundation Residency Research Award Program and the Russell Scott, Jr, MD Urology Research Fund.

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Authors

Contributions

Y.Y. and Q.C. conceived and designed the research, and W.Z. helped. Y.Y. performed the majority of the experiments, and Q.M., Qiaqia L., F.L., B.L., A.S., Qianru L., Z.J. and L.W. assisted. C.L., W.J., R.W. and Qipeng L. performed the mouse xenograft studies. L.F. and X.D. performed the IHC and PLA assays. J.S. performed the BS3 crosslinking assay, and H.N. supervised. W.Q. conducted the TEM assay. S.K. provided control and Ezh1/Ezh2 double-knockout XEN cells. Y.Y. and Q.M. performed the next-generation sequencing. A.B.W. conducted the gene expression and survival analyses using the JHMI cohort, and T.L.L. and E.M.S. supervised. Y.L. and D.Z. conceived, designed and performed the bioinformatics analysis, and K.C. supervised. Y.Y. and Q.C. wrote the paper. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Kaifu Chen or Qi Cao.

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The authors declare no competing interests.

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Peer review information Nature Cell Biology thanks Ivan Topisirovic and the other, anonymous, reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 EZH2 interacts with FBL, but does not affect H2AQ104me modification and 18S rRNA processing.

a, Purified proteins of GST-tagged EED and Flag-tagged FBL were subjected to GST pull down assay, followed by western blot analysis. b, AlphaLISA cross-titration assay to determine the optimal protein concentration combination of EZH2 and FBL. A hook point is reached at 30 nM GST-tagged EZH2 and 1 nM Myc-tagged FBL. c, Representative fluorescence images of C4-2 cells with antibodies against endogenous EZH2 (Green) and FBL (Red). The nuclei were visualized by DAPI (Scale bar: 20 μm). Arrows indicate the enrichment of EZH2 signals in the nucleolus. d, Graph showing the immunoactivity values based on the PLA results from four PCa types. Data represent Mean ± SD from 38 samples in NHT Naïve group, 24 sample in NHT treated group, 42 samples in CRPC group and 43 samples in NEPC group. Statistical significance was determined by One-way ANOVA. e, Representative confocal images of immunofluorescence staining of C4-2 cells with antibodies against FBL (Green) and H2AQ104me1 (Red). Nuclei were stained with DAPI. Scale bars: 20 μm. f, Co-IP assay using anti-FBL antibody showing binding of endogenous FBL with histone H2A. g, Northern blot of RNA from C4-2 cells upon FBL or EZH2 suppression. The 30S and 18SE processing intermediates were detected using the ITS-1 probe while the 18S rRNA and U1 snRNA were hybridized by their corresponding probes. The assays in a-c and e-g have been performed three times with similar results. Statistical source data and unprocessed blots are provided in Source data Extended data Fig. 1.

Source data

Extended Data Fig. 2 EZH2 modulates rRNA 2′-O-Me by interacting with FBL.

a, RTL-P assay to detect the 2′-O-Me level in 12 areas in rRNA. Total RNAs were extracted and subjected to reverse transcription (RT) with RT primer at low (1 μM) or high (1 mM) concentration of dNTP, respectively. The obtained cDNA was then amplified with primer pairs corresponding to upstream (Um) or downstream (Dm) regions of specific methylation site(s). This assay has been performed three times with similar results. b, Densitometric analysis of data from a were shown as signal intensity ratio of PCR products at low dNTP (1 μM) over high dNTP (1 mM) level. Methylation levels in control cells were set close to 1. Data represent Mean ± SD from n = 3 biologically independent experiments. c-e, For the 87 sites in which the 2′-O-Me level was significantly decreased upon EZH2 inhibition (detailed information provided in Supplementary Table 1), the MethScore obtained in control C4-2 cells was subtracted from the one in EZH2-deficient C4-2 cells. Sites are shown in order of increasing difference in MethScore for the 18S (c), 5.8S (d), and 28S (e) rRNAs. Data represent Mean ± SD from n=4 biologically independent experiments. f,g, Translation efficiency of firefly (f) and renilla luciferase (g) reporters was evaluated as the ratio of luciferase activity over mRNA levels. Luciferase activities were detected by dual-luciferase assay using a bi-cistronic luciferase reporter construct as shown above, while the luciferase mRNA levels were measured by RT-qPCR assay. Data represent Mean ± SD from n=3 biologically independent experiments. h, The Poliovirus (PV) IRES activity was calculated as the ratio of firefly luciferase activity over renilla luciferase activity. Data represent Mean ± SD from n=4 biologically independent experiments. For all relevant panels, unless otherwise stated, statistical significance was determined by two-tailed Student’s t-test. Statistical source data and unprocessed blots are provided in Source data Extended data Fig. 2.

Source data

Extended Data Fig. 3 EZH2 bridges FBL-NOP56 interaction by binding to both proteins.

a, Western blot analysis of FBL protein level in PCa cell lines upon EZH2 knockdown. b, Western blot analysis of EZH2 and H3K27me3 levels in PCa cell lines upon FBL knockdown. c, Equal amount of FBL protein in control and EZH2-deficient C4-2 cells were pulled down using anti-FBL antibody followed by western blot to detect its trimethyl-lysine (Kme3) level. d, Co-IP of Nop56, Nop58 and Snu13 with Fbl in control and Ezh1/Ezh2 double-knockout XEN cells, followed by western blot analysis with indicated antibodies. Graph represents the relative Nop56 protein level coimmunoprecipitated with Fbl in each group. Data represent Mean ± SD (n=3 biologically independent measurements). Interaction intensity at control XEN group was set as 1. Statistical significance was determined by two-tailed Student’s t-test. e, AlphaLISA cross-titration assay to determine the optimal protein concentration combination of FBL and NOP56. A hook point is reached at 3 nM His-tagged NOP56 and 30 nM Flag-tagged FBL. To achieve best results, a combination of 3 nM His-tagged NOP56 and 3 nM Flag-tagged FBL was used for the subsequent experiments. f, AlphaLISA displacement assay showing that FBL-NOP56 interaction is unaffected by EED. Data represent Mean ± SD for n=3 biologically independent experiments. g, After nucleolar isolation, proteins in each fraction were separated by SDS-PAGE and visualized by UV (upper panel). Distributions of EZH2, FBL (nucleolar marker), NOP56, FUS/TLS (nucleoplasmic marker) and β-actin (cytoplasmic marker) in each fraction were detected by western blot (lower panel). Wc: whole cells; CN: cytoplasm + nucleoplasm; No: nucleoli. h, AlphaLISA cross-titration assay to determine the optimal protein concentration combination of Flag-tagged-EZH2 and GST-tagged-NOP56. A hook point is reached at 100 nM Flag-tagged-EZH2 and 1 nM GST-tagged-NOP56. i, Co-IP of Myc-tagged FBL with full-length or truncation mutants of Flag-tagged NOP56. The assays in a-c, e and g-i have been performed three times with similar results. Statistical source data and unprocessed blots are provided in Source data Extended data Fig. 3.

Source data

Extended Data Fig. 4 EZH2 alters box C/D snoRNP assembly.

a, Mixtures of recombinant EZH2, FBL and NOP56 proteins with or without BS3 crosslinking were subjected to SDS-PAGE, followed by western blot analysis using their own antibodies to visualize the location of cross-linked species. b, Gel band containing cross-linked proteins was subjected to Mass Spectrometry analyses to detect the presence of all three proteins. c-f, Densitometric analysis of data from Fig. 4a were shown as distribution proportion of FBL (c) /NOP56 (d) /NOP58 (e) /SNU13 (f) protein in each fraction. g, Fractions 17-19 and 27-29 from control C4-2 cell nuclear extracts were subjected to co-IP assay using anti-EZH2 antibody. The assays in a-b and g have been performed three times with similar results. Statistical source data and unprocessed blots are provided in Source data Extended data Fig. 4.

Source data

Extended Data Fig. 5 EZH2 regulates the translation process independently of its mRNA-binding capacities.

a, Representative Immunohistochemistry staining images of PCa TMA slides using the indicated antibodies (Abs). Scale bar, 100 μm. b,c, Graph showing the relative nucleolar area (b) and nucleolar number (c) per cell by counting 60 cells from three TMA cores representing each group. The ends of box are the upper and lower quartiles and box spans the interquartile range. Median is marked by a vertical line inside the box and whiskers represent for the highest and lowest observations. NHT, neoadjuvant hormonal therapy; CRPC, castration-resistant PCa; NEPC, neuroendocrine PCa. d, Global protein synthesis in control and EZH2-overexpressing C4-2 cells were detected by Puromycylation assay followed by western blot. Expression of β-actin was used as reference. e,f, Venn diagram to show overlap between genes from “buffering” mode upon EZH2 or FBL deficiency. P values were calculated by one-tailed Fisher’s exact test. g, KEGG pathway analysis of genes from “buffering” mode upon EZH2 or FBL deficiency. h, GSEA analysis of genes bound by EZH2 to test their enrichment with TE changes after EZH2 knockdown. i, Venn diagram to show overlap between genes from two TE-altered groups after EZH2 knockdown and EZH2 RNA binding targets in C4-2 cells. P values were calculated by one-tailed Fisher’s exact test. j, Heatmap to show EZH2 RIP-seq and Input signals for TE-altered genes in EZH2-deficient C4-2 cells. k, Representative genome browser tracks to show Ribo-seq, RNA-seq and public EZH2 RIP-seq data at the loci of TP53 (a known mRNA binding target of EZH2) and IFT81 (an identified TE-altered gene after EZH2 inhibition). For all relevant panels, unless otherwise stated, statistical significance was determined by two-tailed Student’s t-test. The assays in a and d have been performed three times with similar results. Statistical source data and unprocessed blots are provided in Source data Extended data Fig. 5.

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Extended Data Fig. 6 EZH2 promotes XIAP IRES-dependent translation.

a, Heatmap to show TE changes for the top 30 IRES genes which underwent TE down-regulation after EZH2 inhibition, as revealed from gene set enrichment analysis (GSEA). b, Western blot analysis of XIAP protein level upon FBL or EZH2 depletion in LNCaP, DU145, 22RV1 and VCaP cells. c, RT-qPCR analysis of XIAP mRNA level upon FBL or EZH2 depletion in LNCaP, DU145, 22RV1 and VCaP cells. Data represent Mean ± SD from n=3 biologically independent experiments. d, RT-qPCR analysis of XIAP mRNA level in C4-2 cells after treatment of various EZH2 inhibitors as indicated. Data represent Mean ± SD from n=3 biologically independent experiments. e, CHX treatment assay was performed to monitor the degradation of XIAP protein in control, FBL-deficient and EZH2-deficient cells. The relative protein level is shown under the bands. f, RT-qPCR analysis of XIAP mRNA level upon serum starvation in control, FBL-deficient and EZH2-deficient C4-2 cells. Data represent Mean ± SD from n=3 biologically independent experiments. For all relevant panels, unless otherwise stated, statistical significance was determined by two-tailed Student’s t-test. The assays in b and e have been performed three times with similar results. Statistical source data and unprocessed blots are provided in Source data Extended data Fig. 6.

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Extended Data Fig. 7 Contributions of FBL and NOP56 in PCa tumorigenesis.

a, Wound healing assay was conducted to evaluate the migration potential of PC-3 cells after FBL depletion. The healing of wounded cell layer was monitored under a microscope every 24 h. Graph showing the rate of filling of the scratched area by cells. Data represent Mean ± SD from n=3 biologically independent experiments. The knockdown efficiency of FBL was validated by western blot. b, Wound healing assay was conducted to evaluate the migration potential of PC-3 cells after NOP56 depletion. The healing of wounded cell layer was monitored under a microscope every 24 h. Graph showing the rate of filling of the scratched area by cells. Data represent Mean ± SD from n=3 biologically independent experiments. The knockdown efficiency of NOP56 was validated by western blot. c,d, Boyden chamber invasion assay was performed to determine the invasive capability of PC-3 cells after FBL depletion (c) or NOP56 depletion (d). Graph showing the number of migrated cells passing through Matrigel at 24 h. Data represent Mean ± SD from n=5 random fields per filter. e,f, Tumor formation in nude mice injected with control, FBL-deficient or NOP56-deficient PC-3 cells. The images of xenograft tumors at the end point of measurement were shown in e. Tumor volume was measured by caliper twice a week and plotted in f. Data represent Mean ± SD from n=8 tumors in each group. Statistical significance was determined by two-way ANOVA. For all relevant panels, unless otherwise stated, statistical significance was determined by two-tailed Student’s t-test. Statistical source data and unprocessed blots are provided in Source data Extended data Fig. 7.

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Extended Data Fig. 8 Model of the implication of EZH2 in control of gene expression.

EZH2 plays a dual-role to regulate gene expression. On one hand, EZH2 inhibits DNA transcription by catalyzing H3K27me3 marks in a PRC2-dependent manner; On the other hand, EZH2 activates mRNA translation by enhancing the functionality of FBL through a non-lysine methyltransferase role. Hence, EZH2 could exert its oncogenic functions by coordination of transcriptional inhibition (that is, tumor suppressors) and promotion of translation (that is, pro-oncogenic, anti-apoptotic, and survival proteins) during cancer progression.

Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Table 1: Detailed information of RiboMeth-seq data. (a) MethScore information for each 2′-O-Me site in 18S rRNA; (b) MethScore information for each 2′-O-Me site in 5.8S rRNA; (c) MethScore information for each 2′-O-Me site in 28S rRNA. Supplementary Table 2: List of translation-affected genes after EZH2 or FBL inhibition. (a) Quantification of TE of the coding genes from the Ribo-seq combined with RNA-seq data after EZH2 inhibition; (b) quantification of TE of the coding genes from the Ribo-seq combined with RNA-seq data after FBL inhibition. Supplementary Table 3: Antibodies used in this study. Supplementary Table 4: Oligonucleotides used for this study. Supplementary Table 5: Recombinant proteins used for this study.

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Yi, Y., Li, Y., Meng, Q. et al. A PRC2-independent function for EZH2 in regulating rRNA 2′-O methylation and IRES-dependent translation. Nat Cell Biol 23, 341–354 (2021). https://doi.org/10.1038/s41556-021-00653-6

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