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The transcription factor ERG recruits CCR4–NOT to control mRNA decay and mitotic progression

Nature Structural & Molecular Biology volume 23, pages 663–672 (2016)Cite this article

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Abstract

Control of mRNA levels, a fundamental aspect in the regulation of gene expression, is achieved through a balance between mRNA synthesis and decay. E26-related gene (Erg) proteins are canonical transcription factors whose previously described functions are confined to the control of mRNA synthesis. Here, we report that ERG also regulates gene expression by affecting mRNA stability and identify the molecular mechanisms underlying this function in human cells. ERG is recruited to mRNAs via interaction with the RNA-binding protein RBPMS, and it promotes mRNA decay by binding CNOT2, a component of the CCR4–NOT deadenylation complex. Transcriptome-wide mRNA stability analysis revealed that ERG controls the degradation of a subset of mRNAs highly connected to Aurora signaling, whose decay during S phase is necessary for mitotic progression. Our data indicate that control of gene expression by mammalian transcription factors may follow a more complex scheme than previously anticipated, integrating mRNA synthesis and degradation.

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Figure 1: ERG localizes in PBs and promotes decay of tethered mRNAs.
Figure 2: ERG interacts with the CNOT2 component of the CCR4–NOT deadenylation complex in inducing mRNA decay.
Figure 3: ERG is recruited to mRNA through its interaction with RBPMS.
Figure 4: ERG, RBPMS and CNOT2 promote decay of mitotic mRNAs.
Figure 5: ERG depletion induces blockage in early metaphase.
Figure 6: siERG-induced mitotic defects are due to overactivation of Aurora signaling.
Figure 7: ERG controls decay of Aurora-related mRNA transcripts in the cytoplasm via RBPMS and CNOT2.

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Acknowledgements

We thank the Vidal laboratory at the Center for Cancer Systems Biology at the Dana-Farber Cancer Institute (CCSB, DFCI) for supplying reagents, advice and technical support for the HT-Y2H screen. We thank the Tuschl laboratory (Howard Hughes Medical Institute, Rockefeller University) for sharing reagents. We are grateful to E. Izaurralde (European Molecular Biology Laboratory) for kindly providing HA-tagged CNOT2, CNOT3, CNOT6 and CNOT. We would like to thank the laboratories of the Molecular Biology in Diseases Unit (GIGA-R, ULg) for helpful discussions and C. Vindry from the Kruys laboratory for technical assistance. We thank the GIGA interactomics, imaging and flow cytometry and genotranscriptomics platforms for their technical support. This work was supported by grants from the University of Liège, the Fonds Léon Frédéricq, the Belgian Foundation against Cancer (Special Equipment Fund), the Belgian National Fund for Scientific Research (FNRS), Télévie, the Interuniversity Attraction Poles Program–Belgian Science Policy (IUAP-BELSPO PVI/28 and PVII/13 to F.D. and M. Bollen) and the National Human Genome research Institute (grant R01HG001715 to M.V. and D.E.H.). X.R., J-C.T. and N.S. are supported as an FNRS Research Fellow, Research Associate and Postdoctoral Fellow, respectively. C.D. and K.G. are supported as Télévie-FNRS PhD students.

Author information

Authors and Affiliations

  1. Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), University of Liège (ULg), Liège, Belgium

    Xavier Rambout, Cécile Detiffe, Jonathan Bruyr, Emeline Mariavelle, Majid Cherkaoui, Pauline Demoitié, Marielle Lebrun, Katia Guedri, Richard Kettmann, Ingrid Struman, Jean-Claude Twizere & Franck Dequiedt

  2. GIGA-Molecular Biology in Diseases, ULg, Liège, Belgium

    Xavier Rambout, Cécile Detiffe, Jonathan Bruyr, Emeline Mariavelle, Majid Cherkaoui, Pauline Demoitié, Katia Guedri, Richard Kettmann, Jean-Claude Twizere & Franck Dequiedt

  3. BiGRe, Université Libre de Bruxelles (ULB), Bruxelles, Belgium

    Sylvain Brohée & Nicolas Simonis

  4. Computer Science Department, ULB, Bruxelles, Belgium

    Sylvain Brohée

  5. GIGA-Inflammation, Infection & Immunity, ULg, Liège, Belgium

    Marielle Lebrun

  6. Faculté des Sciences, ULB, Gosselies, Belgium

    Romuald Soin & Véronique Kruys

  7. Department of Cellular and Molecular Medicine, University of Leuven (KUL), Leuven, Belgium

    Bart Lesage, Monique Beullens & Mathieu Bollen

  8. Howard Hughes Medical Institute, Rockefeller University, New York, New York, USA

    Thalia A Farazi

  9. GIGA-Cancer, ULg, Liège, Belgium

    Ingrid Struman

  10. Center for Cancer Systems Biology (CCSB),

    David E Hill & Marc Vidal

  11. Department of Cancer Biology, Dana-Farber Cancer Institute (DFCI), Boston, Massachusetts, USA

    David E Hill & Marc Vidal

  12. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA

    David E Hill & Marc Vidal

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  1. Xavier Rambout
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Contributions

F.D. and X.R. conceived the project and designed the experiments. X.R. performed most experiments and processed and analyzed the data. C.D., J.B., E.M., M.C., P.D., M.L., R.S., K.G., R.K., I.S. and V.K. assisted with experiments. D.E.H., M.V., N.S. and J.-C.T. helped perform and analyze the HT-Y2H screen. S.B. performed bioinformatic analysis for the transcriptome-wide decay experiments and contributed to the corresponding manuscript sections. M. Bollen, M. Beullens and B.L. contributed to analysis of ERG in mitotic progression and Aurora kinase regulation and participated in writing the corresponding manuscript sections. T.A.F. assisted in analyzing RBPMS CLIP data. F.D. and X.R. wrote the manuscript, which was edited by all authors.

Corresponding author

Correspondence to Franck Dequiedt.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 ERG localizes in PBs and promotes decay of tethered mRNAs.

Related to Figure 1.

(a) Immunofluorescence of ERG (green), DPC1A (PBs marker, red), EDC4 (PBs marker, white) and Hoechst (nuclear stain, blue) in HeLa cells. Scale bars, 10 µm.

(b) Immunofluorescence of YFP-tagged Erg members FLI1 and FEV (green), RFP-tagged DCP1A (PB marker, in red) and Hoechst (nuclear stain, blue) in HeLa cells. Scale bars, 10 µm.

(c) Immunofluorescence of ERG (green), DPC1A (PBs marker, red), TIA1 (upper panel) or TIAR (lower panel) (SGs markers, white) and Hoechst (nuclear stain, blue) in arsenite-treated HeLa cells. Scale bars, 10 µm.

(d,e) RT-qPCR analysis of ERG mRNA levels (d) and western blot against ERG and control GAPDH (e). Samples are HeLa cells transfected with control (siCTL) or anti-ERG siRNA (siERG). (d) Results are means ± s.d. (n = 4 independent experiments). ***P < 0.001 compared to siCTL condition by two-tailed unpaired Student’s t test.

(f) Luciferase assays in HeLa, U2OS and MRC5 cells transfected with MS2-CP or ERG-MS2-CP together with R-Luc-8MS2 or R-Luc-0MS2 reporters. Results are means of normalized R-Luc activities (white bars) and relative mRNA levels (grey bars) ± s.d. (see Online Methods) (n = 3 independent experiments). *P < 0.05; **P < 0.01; ***P < 0.001 compared to MS2-CP conditions by two-tailed unpaired Student’s t test.

(g,h) RT-qPCR analysis of the stability of the R-Luc-8MS2 mRNA reporter (g) and luciferase assays (h). Samples are RNA (g) or lysates (h) from HeLa cells ActD-treated (g) or not (h) and transfected with MS2-CP (blue, diamond), ERG-MS2-CP (red, squares), or ERG-W334R-MS2-CP (green, triangles) together with R-Luc-8MS2 (or R-Luc-0MS2 (h)) reporters. Results are means ± s.d. (n = 3 independent experiments). *P < 0.05; **P < 0.01; NS, not significant compared to MS2-CP condition by two-tailed unpaired Student’s t test.

(i) Luciferase assays of HeLa cells transfected as in Fig. 1g. Results are means of normalized R-Luc activities ± s.d. (see Online Methods) (n = 3 independent experiments). **P < 0.01; NS, not significant compared to MS2-CP condition by two-tailed unpaired Student’s t test.

(j) Anti-FLAG, -GW182, and -GAPDH western blots. Samples are lysates from HeLa cells transfected as in Fig. 1g.

Supplementary Figure 2 ERG interacts with the CNOT2 component of the CCR4–NOT deadenylation complex and induces mRNA decay.

Related to Figure 2.

(a) Upper panel: Domain structure of CNOT2 and description of CNOT2 deletion mutants. Lower panel: Immunoprecipitation of FLAG-tagged ERG and anti-FLAG and -Myc western blots. Samples are lysates from HEK-293 cells transfected with either Myc-tagged CNOT2 deletion mutants alone or with FLAG-ERG.

(b) Immunofluorescence of ERG (green), CNOT2 (red), G3BP (SGs marker, white), and Hoechst (nuclear stain, blue) in arsenite-treated HeLa cells. Scale bars, 10 µm.

(c) Immunoprecipitation of FLAG-tagged ERG and anti-FLAG and –HA western blot. Samples are lysates from HEK-293 cells transfected with either of HA-tagged subunits of the CCR4-NOT complex (CNOT2, CNOT3, CNOT6, CNOT7) alone or with FLAG-ERG.

(d) Luciferase assays of HeLa cells transfected as in Fig. 2f. Results are means of normalized R-Luc activities ± s.d. (see Online Methods) (n = 3 independent experiments). *P < 0.05; **P < 0.01; NS, not significant compared to MS2-CP condition by two-tailed unpaired Student’s t test.

(e) Luciferase assays of HeLa cells transfected as in Fig. 2g. Results are means of normalized R-Luc activities ± s.d. (see Online Methods) (n = 3 independent experiments). *P < 0.05; **P < 0.01; NS, not significant compared to MS2-CP or siCTL condition by two-tailed unpaired Student’s t test.

(f) Anti-FLAG, -CNOT2, and -GAPDH western blots. Samples are lysates from HeLa cells transfected as in Fig. 2g.

Supplementary Figure 3 ERG is recruited on mRNA through its interaction with RBPMS.

Related to Figure 3.

(a) Immunoprecipitation of ERG and anti-ERG and -RBPMS western blots. Samples are control (CTL) or anti-ERG immunoprecipitates from HeLa cells.

(b) Immunoprecipitation of FLAG-tagged ERG deletion mutants and anti-FLAG and -HA western blots. Samples are lysates from HEK-293 cells transfected with HA-RBPMS alone or with either of the FLAG-tagged Erg members.

(c) Immunoprecipitation of FLAG-tagged ERG and anti-FLAG or -HA western blots. Samples are nontreated (NT) or RNase A-treated lysates from HEK-293 cells transfected with HA-RBPMS alone or with FLAG-ERG.

(d) Upper panel: Domain structure of RBPMS and description of RBPMS deletion mutants. Lower panel: Immunoprecipitation of FLAG-tagged ERG and anti-FLAG and -HA western blots. Samples are lysates from HEK-293 cells transfected with HA-RBPMS alone or with FLAG-ERG.

(e) Immunoprecipitation of RBPMS and western blot against RBPMS, CNOT2 and CNOT3. Samples are control (CTL) or anti-RBPMS immunoprecipitates from HeLa cells.

(f) Immunofluorescence of ERG (green), RBPMS (red), Hoechst (nuclear stain, blue) and CNOT2 (upper panel) or CNOT6 (lower panel) (white). Scale bars, 10 µm. White and red arrows indicate ERG cytoplasmic foci positive for CNOT2 or CNOT6, and positive or negative for RBPMS, respectively.

Supplementary Figure 4 ERG promotes decay of mitotic mRNAs.

Related to Figure 4.

(a) List of the 22 mitotic genes (GO:0000279 – M phase) identified in Figure 4a.

(b) RT-qPCR stability analysis of the 22 ERG mitotic targets. Samples are RNA from HeLa cells transfected with control (siCTL: blue, diamonds), anti-ERG (siERG: red, squares), anti-CNOT2 (siCNOT2: green, triangles), or anti-RBPMS siRNA (siRBPMS: purple, circles) (n = 5, 4, 3 and 4 independent experiments respectively) and treated with ActD for 0, 1, 2, or 4 hours. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant; ND, not determined compared to siCTL condition by two-tailed unpaired Student’s t test.

(c) RT-qPCR analysis of RBPMS mRNA levels (upper panel) and anti-RBPMS and -GAPDH western blots (lower panel). Samples are HeLa cells transfected with control or anti-RBPMS siRNA. Upper panel: Results are means ± s.d. (n = 4 independent experiments). ***P < 0.001 compared to siCTL condition by two-tailed unpaired Student’s t test.

Supplementary Figure 5 ERG depletion promotes mitotic arrest and impairs decay of mitotic mRNAs during the S phase of the cell cycle.

Related to Figure 5.

(a) Quantification of the western blot analyses, one representative of which is shown in Fig. 5b. Samples are lysates from HeLa cells transfected with control (siCTL), anti-ERG (siERG) or anti-CNOT2 siRNA (siCNOT2) and released from a G2-M block. Cyclin signals were normalized to GAPDH, and set to 1 at the time of release (t = 0 h) for each siRNA. Results are means ± s.d. (n = 3 independent experiments). **P < 0.01; NS, not significant for siERG (higher labels) or siCNOT2 (lower labels) conditions compared to siCTL condition by two-tailed unpaired Student’s t test.

(b) Immunofluorescence of Hoechst (chromosomes, blue), ACA (centromeres, white), α-tubulin (mitotic spindle, green), and γ-tubulin (centrosomes, red) in mitotic HeLa cells. Scale bars, 5 µm.

(c) RT-qPCR stability analysis of the 19 validated ERG mitotic targets presented in Fig. 5d. Samples are RNA from HeLa cells transfected with control (plain, triangles) or anti-ERG siRNA (dashed, squares), synchronized in S-phase (blue), late G2-phase (red), or mitosis (green) (n = 4, 3 and 3 independent experiments respectively) and treated with ActD for 0, 2, or 4 hours. Results are means ± s.d. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant; ND, not determined compared to siCTL condition by two-tailed unpaired Student’s t test.

Supplementary Figure 6 ERG depletion promotes mitotic defects.

Related to Figure 6.

(a-d) Quantification of centrosome defects (a), spindle multipolarity (b), increased length (c) and chromosome congression defects (d) presented in Fig. 6a,b. Results are mean percentages (a,b,d) or length (c) ± s.d. (n = 3 independent experiments, 50-100 (a,b,d) and 5-10 (d) cells in each replicate). *P < 0.05; **P < 0.01; ***P < 0.001 compared to siCTL condition by two-tailed unpaired Student’s t test.

(e) Quantification of mitotic defects. Samples are HeLa cells transfected with control (siCTL), anti-ERG (siERG), anti-CNOT2 (siCNOT2) or anti-RBPMS siRNA (siRBPMS). Results are mean percentages ± s.d. (n = 3 independent experiments). *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant compared to siCTL condition by two-tailed unpaired Student’s t test.

(f) RT-qPCR stability analysis of the 9 Aurora-related ERG mitotic. Samples are RNA from HeLa cells transfected with control (siCTL) or an alternate anti-ERG siRNA (siERG#2), synchronized in S-phase, and treated with ActD for 0, 2, or 4 hours. Results are means ± s.d. (n = 3 independent experiments). Representation is similar to Fig. 4d.

(g) Immunofluorescence of AURKA mRNAs (smFISH, red) and Hoechst (nuclear stain, blue). Samples are HeLa cells transfected with control or anti-ERG siRNA and synchronized in S-phase. Scale bars, 10 µm.

(h) Quantification of the number of AURKA mRNAs in the cytoplasm and nucleus from immunofluorescence images in (g). Results are means ± s.d. (n = 3 independent experiments). *P < 0.05; NS, not significant compared to siCTL condition by two-tailed unpaired Student’s t test.

(i) Quantification of western blot analyses, one representative of which is presented in Fig. 6e. Aurora signals were normalized to GAPDH, and set to 1 in control cells at t = 0 h for AURKA and AURKB, or at t = 14 h for P-Aurora. Results are means ± s.d. (n = 4 independent experiments). *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant compared to siCTL condition by two-tailed unpaired Student’s t test.

(j) Anti-ERG, -phospho-Aurora and -GAPDH western blots. Samples are lysates from HeLa cells transfected with control or anti-ERG siRNAs.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Notes 1–2 (PDF 1999 kb)

Supplementary Data Set 1

Original uncropped western blots (PDF 29563 kb)

Supplementary Table 1

List of ERG-interacting partners identified by HT-Y2H (XLSX 43 kb)

Supplementary Table 2

List of mRNAs whose stability changed after ERG knock-down in HeLa cells (XLSX 72 kb)

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Rambout, X., Detiffe, C., Bruyr, J. et al. The transcription factor ERG recruits CCR4–NOT to control mRNA decay and mitotic progression. Nat Struct Mol Biol 23, 663–672 (2016). https://doi.org/10.1038/nsmb.3243

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