Abstract

Synchronization of mitochondrial and cytoplasmic translation rates is critical for the maintenance of cellular fitness, with cancer cells being especially vulnerable to translational uncoupling. Although alterations of cytosolic protein synthesis are common in human cancer, compensating mechanisms in mitochondrial translation remain elusive. Here we show that the malignant long non-coding RNA (lncRNA) SAMMSON promotes a balanced increase in ribosomal RNA (rRNA) maturation and protein synthesis in the cytosol and mitochondria by modulating the localization of CARF, an RNA-binding protein that sequesters the exo-ribonuclease XRN2 in the nucleoplasm, which under normal circumstances limits nucleolar rRNA maturation. SAMMSON interferes with XRN2 binding to CARF in the nucleus by favoring the formation of an aberrant cytoplasmic RNA–protein complex containing CARF and p32, a mitochondrial protein required for the processing of the mitochondrial rRNAs. These data highlight how a single oncogenic lncRNA can simultaneously modulate RNA–protein complex formation in two distinct cellular compartments to promote cell growth.

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Data availability

Small RNA sequencing data have been submitted to the Sequence Read Archive under accession code SRP151840. Source data and statistical analysis for Figs. 1a,b,d,e, 2a,c,e,f, 3a,d,f,h, 4c,e, 5c, 6d,g,h and 7b–g are available in the in the Supplementary Information online. All other data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

GapmeRs were designed by J. Lai (Exiqon, Copenhagen Denmark). We would like to thank M. Leucci for reading and editing the manuscript. This study was supported by the Fund Emile Carpentier—Fund André Vander Stricht—Fund Van Damme 2017-J1810830-207301. The authors wish to thank H. Brems for providing NHEM cultures, A. Sablina (VIB-KULeuven) for providing the SV40 LTA plasmid, M. Spinazzi (VIB-KULeuven) for the technical assistance and for sharing some antibodies, G. Ghanem (Jules Bordet Institute) for the patient-derived melanoma cell line and somersault18:24 (http://www.somersault1824.com/) for providing some graphical illustrations. R.V. is a recipient of the FWO PhD fellowship 1S08316N. D.L. is supported by Fonds National de la Recherche (FRS/FNRS). G.G.T.’s research is supported by the European Research Council (grant no. RIBOMYLOME_309545) and the Spanish Ministry of Economy and Competitiveness (grant nos. BFU2014-55054-P and BFU2017-86970-P).

Author information

Affiliations

  1. Laboratory for RNA Cancer Biology, Department of Oncology, LKI, KU Leuven, Leuven, Belgium

    • Roberto Vendramin
    • , Yvessa Verheyden
    • , Lucas Goedert
    •  & Eleonora Leucci
  2. Laboratory for Molecular Cancer Biology, Department of Oncology, LKI, KU Leuven, Leuven, Belgium

    • Roberto Vendramin
    •  & Jean-Christophe Marine
  3. Laboratory For Molecular Cancer Biology, Center for Cancer Biology, VIB, Leuven, Belgium

    • Roberto Vendramin
    •  & Jean-Christophe Marine
  4. Department of Applied Biological Science, Global Innovation Research Organizations, Tokyo University of Agriculture and Technology, Tokyo, Japan

    • Hideaki Ishikawa
    • , Keichi Izumikawa
    •  & Nobuhiro Takahashi
  5. RNA Molecular Biology, Center for Microscopy and Molecular Imaging, Université Libre de Bruxelles, Charleroi, Belgium

    • Emilien Nicolas
    • , Kritika Saraf
    •  & Denis L. J. Lafontaine
  6. Centre for Genomic Regulation, University Pompeu Fabra and Catalan Institution for Research and Advanced Studies, Barcelona, Spain

    • Alexandros Armaos
    • , Riccardo Delli Ponti
    •  & Gian Gaetano Tartaglia
  7. Center for Medical Genetics, Gent University, Gent, Belgium

    • Pieter Mestdagh
  8. Cancer Research Institute Gent, Gent University, Gent, Belgium

    • Pieter Mestdagh

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Contributions

E.L. and R.V. performed most experiments. Y.V. performed experiments described in Fig. 2f. E.N. and K.S. performed the nuclear rRNA northern blot (Fig. 5b and Supplementary Fig. 4a). R.D.P. and A.A. ran the RNA secondary structure prediction and in silico binding (Fig. 4f). P.M. performed the small RNA-seq (Fig. 5d and Supplementary Fig. 4d). H.I. and K.I. performed the experiments in Fig. 4g–i and Supplementary Fig. 4c. L.G. helped with Figs. 5a and 6a and in additional experiments provided to the reviewers and not included in the final manuscript. D.L., G.G.T. and N.T. helped in the interpretation of the data. E.L. and R.V. designed the study. J.C.M. and E.L. wrote the manuscript with input from all the authors.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Eleonora Leucci.

Integrated supplementary information

  1. Supplementary Figure 1 SAMMSON actively participates to malignant transformation.

    a, SAMMSON relative expression measured by RT–qPCR in Mel-ST cells infected with an empty (Ctrl) or a SAMMSON-encoding (SAMMSON O/E) expression vector; n = 7. b, Cell proliferation assays in Mel-ST cells described in a. Error bars represent mean +/− s.e.m.; n = 3. c, Colony formation assays 5 days after seeding 1 × 103 Mel-ST cells as described in a. The violet color is given by crystal violet, a compound that binds intracellular DNA and protein thus highlighting the cells in the plate. Representative image of five independent experiments. d, Quantification of colony formation assays of Mel-ST cells as described in a and c presented as the mean density (% of area occupancy); n = 5. e, Representative picture of xenograft tumors (encircled by the white dashed line) grown in nude mice derived from subcutaneous injection of 5 × 103 Mel-ST cells as described in a. f, Representative picture of resected xenograft tumors as described in e. Scale bar, 1 cm. g, Tumor volume of xenografts as described in e. Error bars represent mean +/− s.e.m.; n = 6. Box boundaries represent 25th and 75th percentiles; center line represents the median; whiskers, last data point within a ±1.5 interquartile range. P values were calculated by paired two-tailed Student’s t-test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

  2. Supplementary Figure 2 XRN2, CARF and p32 levels are increased in melanoma.

    a, Western blot for XRN2, CARF and p32 in NHEM, in SK-MEL-28 WT or BRAFi resistant (SK-MEL-28-R) and in a panel of short-term melanoma cultures (MM-lines, with different mutational backgrounds and phenotype). b, SAMMSON and HRPT pulldown in native (−) and ultraviolet crosslinking (+) conditions (using two sets of 48 biotinylated probes recognizing mature transcripts, p) and western blotting in SK-MEL-28 cells. Representative image of three independent experiments. Uncropped gel images are shown in Supplementary Data Set 1.

  3. Supplementary Figure 3 CARF and XRN2 localization are specifically affected by SAMMSON knockdown and not by other stressors.

    a, XRN2 (cyan) and CARF (yellow) IF in SK-MEL-28 cells 30 h after transfection with a non-targeting GapmeR (Ctrl), GapmeR3 or GapmeR11. Scale bar low magnification, 10 μm; high magnification, 2 μm. Representative image of three independent experiments. b, Puromycin (Puro, white) or p32 (magenta) IF in SK-MEL-28 cells treated for 48 h with 200 μg ml−1 chloramphenicol or vehicle (EtOH). Cell nuclei are stained with DAPI (cyan). Scale bar low magnification, 10 μm; high magnification, 2 μm. Representative image of three independent experiments. c, XRN2 (cyan) and fibrillarin (yellow) IF in SK-MEL-28 cells treated as in b. Scale bar low magnification, 10 μm; high magnification, 7 μm. Representative image of three independent experiments. d, NEAT1 (red) RNA FISH in untreated SK-MEL-28 cells (Mock) or in cells 72 h after transfection with a control siRNA pool (siCtrl) or pools targeting NEAT1 (siNEAT1) or siNEAT1 long form only (siNEAT1 long). Cell nuclei are stained with DAPI (blue). Scale bar low magnification, 10 μm; high magnification, 2 μm. Representative image of three independent experiments. e, XRN2 (cyan) and fibrillarin (yellow) IF in SK-MEL-28 cells treated as described in d. Scale bar low magnification, 10 μm; high magnification, 2 μm. Representative image of three independent experiments.

  4. Supplementary Figure 4 XRN2 functions are specifically affected by SAMMSON knockdown.

    a, Left, schematic representation of the pre-rRNAs and the mature rRNAs detected by northern blotting, the orange arrows indicate the sites of pre-rRNA-processing inhibition in the 5'-ETS (01, A0, and 1). The probe used is highlighted in grey. ETS: external transcribed spacers; ITS: internal transcribed spacers. The aberrant 34S RNA is produced when cleavage occurs in ITS1 prior to 5’-ETS. *, truncated form of the 34S RNA. Right, northern blot hybridization analysis of pre-rRNA isolated from three melanoma cell lines (with different mutational backgrounds and phenotype) transfected with a non-targeting GapmeR (Ctrl) or GapmeR11 (G11) or of SK-MEL-28 transfected with a XRN2-targeting (siXRN2) or control (siCtrl) siRNA. Knockdown efficiency is shown for both SAMMSON knockdown and XRN2 knockdown. Representative image of three independent experiments. b, SAMMSON (SAM), 18S, 16S, 12S, Cyclooxygenase 1 (COX1) and NADH-ubiquinone oxidoreductase chain 1 (ND1) relative expression measured by RT–qPCR in SK-MEL-28 cells 30 h after transfection with a non-targeting GapmeR (Ctrl) or with GapmeR11; n = 5. c, Northern blot hybridization analysis of mitochondrial pre-rRNA (mt_pre-rRNA) isolated from SK-MEL-28 cells transfected with a non-targeting GapmeR (Ctrl), GapmeR3 (G3) or a GapmeR11 (G11). Efficiency of SAMMSON knockdown and ratios of mt_pre-rRNA over 28S (the mature 28S is visualized by methylene blue staining of the denaturing agarose gel) rRNA are shown below the gel. Representative image of three independent experiments. d, tRNA61-MetCAT expression levels in SK-MEL-28 cells treated with a non-targeting GapmeR (Ctrl) and GapmeR3; n = 3. Box boundaries represent 25th and 75th percentiles; center line represents the median; whiskers, last data point within a ±1.5 interquartile range. P values were calculated by paired two-tailed Student’s t-test. * P < 0.05; ** P < 0.01; NS, not significant. Uncropped gel images are shown in Supplementary Data Set 1.

  5. Supplementary Figure 5 CARF localization and its interaction with p32 are RNA and SAMMSON-dependent.

    a, p32 (yellow) IF combined with 12S (red) RNA FISH in SK-MEL-28 cells 30 h after transfection with a non-targeting GapmeR (Ctrl), GapmeR3 or GapmeR11. Cell nuclei are stained with DAPI (cyan). Scale bar, 4 μm. Representative image of three independent experiments. b, PLA (cyan) using antibodies against CARF and p32 in SK-MEL-28 cells in normal conditions (Ctrl) or after addition of RNase A. Cell nuclei are stained with DAPI (blue). Scale bar low magnification, 10 μm; high magnification, 2 μm. Representative image of three independent experiments. c, p32 RIP in LCL cells infected with an empty (−) or a SAMMSON-encoding (+) expression vector and western blotting. Representative image of three independent experiments. d, CARF (red) and p32 (yellow) IF in LCL cells as described in c. Cell nuclei are stained with DAPI (blue). Scale bar low magnification, 10 μm, Scale bar high magnification, 2 μm. Representative image of three independent experiments. Uncropped gel images are shown in Supplementary Data Set 1.

  6. Supplementary Figure 6 SAMMSON regulates the interaction between XRN2, CARF and p32.

    a, CARF (magenta) and mitotracker (white) IF in SK-MEL-28 cells 72 h after transfection with a control siRNA pool (siCtrl) or an siRNA pool targeting CARF (siCARF). Cell nuclei are stained with DAPI (cyan). Scale bar, 10 μm. Representative image of three independent experiments. b, PLA (cyan) assay using antibodies against CARF and p32 in Mel-ST cells described in Supplementary Fig. 1a. Cell nuclei are stained with DAPI (blue). Scale bar low magnification, 10 μm; high magnification, 2 μm. c, SAMMSON relative expression measured by RT–qPCR in SK-MEL-28 cells 30 h after transfection with a non-targeting GapmeR (Ctrl) or GapmeR11 (G11). Error bars represent mean +/− s.e.m.; n = 3. d, CARF IP in SK-MEL-28 cells treated as described in c and western blotting. Representative image of three independent experiments. P values were calculated by paired two-tailed Student’s t-test. ** P < 0.01. Uncropped gel images are shown in Supplementary Data Set 1.

  7. Supplementary Figure 7 SAMMSON regulates rRNA biogenesis and protein synthesis.

    a, Pre-rRNA-processing analysis in Mel-ST cells infected with an empty (Ctrl) or a SAMMSON-encoding (SAM O/E) expression vector. Left, structure of the pre-rRNAs detected and probes used. The aberrant 34S RNA observed after SAMMSON depletion (see Fig. 5b) is highlighted in red. Right, northern blot hybridizations. The mature rRNAs are visualized by ethidium-bromide staining of the denaturing agarose gel. Representative image of three independent experiments. b, Western blotting after a 10-min pulse with puromycin and subsequent cytosol (Cyto)/mitochondria (Mito)/mitoplast (Mitopl) fractionation in Mel-ST cells infected with an empty (Ctrl) or a SAMMSON-encoding (SAM O/E). Representative image of four (Total and Cyto) and three (Mito and Mitopl) independent experiments. c, Western blotting after a 10-min pulse with puromycin and subsequent cytosol (Cyto)/mitochondria (Mito)/mitoplast (Mitopl) fractionation in LCL cells described in a. Representative image of five independent experiments. d, Western blotting after cytosol (Cyto)/mitochondria (Mito)/proteinase K-treated mitochondria (Mito+PK) fractionation in LCL cells described in Fig. 1a. Representative image of four independent experiments. e, Western blotting after a 3-h pulse with azidohomoalanine, followed by cytosol (Cyto)/mitochondria (Mito)/proteinase K-treated mitochondria (Mito+PK) fractionation and subsequent Click-iT alkyne reaction in LCL cells described in Fig. 1a. f, Immunohistochemistry Ki67 staining of xenograft tumors as described in Supplementary Fig. 1e–g. Scale bar, 100 μm. Representative image of eight independent experiments. g, Immunohistochemistry puromycin staining of tumors as described in Supplementary Fig. 1e–g. Scale bar, 100 μm. Representative image of eight independent experiments. Uncropped gel images are shown in Supplementary Data Set 1.

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    Supplementary Figures 1–7 and Supplementary Table 1

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  3. Supplementary Dataset 1

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https://doi.org/10.1038/s41594-018-0143-4