SAMMSON fosters cancer cell fitness by concertedly enhancing mitochondrial and cytosolic translation

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.

Figure 1. SAMMSON induces a malignant phenotype outside of the melanocytic lineage.

(a) SAMMSON relative expression measured by RT-qPCR in LCL cells infected with an empty (Ctrl) or a SAMMSON-encoding (SAM O/E) expression vector and/or with SV40 Large T Antigen (LTA); n=6. (b) Cell proliferation assays in LCL cells described in a. Error bars represent mean ± s.e.m.; n=4. (c) Soft agar assay in LCL cells described in a. Representative image of three independent experiments. (d) Quantification of number of colonies formed in soft agar as described in c. Error bars represent mean ± s.e.m.; n=3. (e) Quantification of colony size of soft agar colonies as described in c; Error bars represent mean ± s.e.m.; n=3. Box boundaries represent 25th and 75th percentiles; center line represents the median; whiskers, last data point within ±1.5 interquartile range. P values were calculated by paired two-tailed Student’s t-test. * P<0.05; ** P<0.01; **** P<0.0001.

Figure 2. Desynchronization of cytosolic and mitochondrial translation machinery decreases melanoma cell fitness and can be therefore exploited therapeutically.

(a) SAMMSON relative expression measured by RT-qPCR in SK-MEL-28 cells transfected with a non-targeting GapmeR(Ctrl) or GapmeR3. Error bars represent mean ± s.e.m.; n=3. (b) Western blotting after a 10-minute pulse with puromycin and subsequent cytosol(Cyto)-mitochondria (Mito)-mitoplast(Mitopl) fractionation in SK-MEL-28 cells described in a. Representative images of three independent experiments. (c) Quantification of relative protein synthesis (%) in SK-MEL-28 cells as described in a and b. Error bars represent mean ± s.e.m.; n=3. (d) Colony formation assays 5 days after seeding 1′ 10³ SK-MEL-28 cells and treating them with increasing amounts of chloramphenicol or vehicle (EtOH). The violet colour is given by crystal violet, a compound that binds intracellular DNA and protein thus highlighting the cells in the plate. Representative image of three independent experiments. (e) Caspase 3/7 activity 72 hours after treating SK-MEL-28 cells with increasing amounts of chloramphenicol or vehicle (EtOH). Error bars represent mean ± s.e.m.; n=3. P values was calculated with two-way ANOVA. (f) Relative cell viability of MM099 cells measured 48 hours after treatment with either Tigecycline or a combination of Tigecycline, BRAFi (Dabrafenib) and MEKi (Trametinib). Error bars represent mean ± s.e.m.; n=3. P values were calculated by paired two-tailed Student’s t-test. * P<0.05; ** P<0.01; *** P<0.001; NS, not significant. Uncropped gel images are shown in Supplementary Data Set 1.

Figure 3. SAMMSON interacts with XRN2 and CARF.

(a) SAMMSON (but not TBP or UBC) is specifically recovered by RAP after UV crosslinking SK-MEL-28 cells. Representative image of three independent experiments. (b) SAMMSON and HRPT pulldown in native 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. (c) SAMMSON and HRPT pulldown in native conditions (using biotinylated probes, p) and western blotting in mouse p38 cells. Representative image of three independent experiments. (d) MALAT1 (but not SAMMSON or HPRT) is recovered by RIP using an SRSF1- specific antibody as measured by RT-qPCR in SK-MEL-28 cells. Error bars represent mean ± s.e.m.; n=3. (e) Western blot confirming enrichment of SRSF1 following RIP with an anti- SRSF1 antibody in SK-MEL-28 cells. Representative image of three independent experiments. (f) SAMMSON and TERRA are recovered by RIP using an XRN2-specific antibody as measured by RT-qPCR in SK-MEL-28 cells. Error bars represent mean ± s.e.m.; n=3. (g) Western blot confirming enrichment of XRN2 (and CARF) following RIP with an anti-XRN2 antibody in SK-MEL-28 cells. Representative image of three independent experiments. (h) SAMMSON and LINC00698 are recovered by RIP using a CARF-specific antibody as measured by RT-qPCR in SK-MEL-28 cells. Error bars represent mean ± s.e.m.; n=3. (i) Western blot confirming enrichment of CARF (and XRN2) following RIP with an anti-CARF antibody in SK-MEL-28 cells. Representative image of three independent experiments. Uncropped gel images are shown in Supplementary Data Set 1.

Figure 4. Identification of SAMMSON-binding region on XRN2 and of SAMMSON protein-binding domain.

(a) Schematic representation of the different HA-XRN2-constructs. (b) Western blot confirming enrichment of XRN2 (and CARF) in SK-MEL-28 cells transfected with different XRN2 constructs or an untransfected control (Ctrl) followed by RIP with an anti- HA-tag antibody. Representative images of four independent experiments. (c) SAMMSON and 18S are recovered by RIP using a HA-tag-specific antibody only in one out of the three XRN2 deletion constructs (N1 and C respectively) as measured by RT-qPCR in SK-MEL-28 cells. All deletion mutants were normalised on the positive control (WT) sample. Red asterisks refer to SAMMSON significance (paired two-tailed Student’s t-test WT vs the deletion mutants), yellow asterisks refer to 18S significance (paired two-tailed Student’s t-test WT vs the deletion mutants). Error bars represent mean ± s.e.m.; SAMMSON, n=4; 18S, n=3. (d) Western blotting of SK-MEL-28 cells 72 hours after transfection with a control siRNA (siCtrl) or two different siRNAs targeting XRN2 (siXRN2#1 and siXRN2#2). Representative images of six independent experiments. (e) SAMMSON and TERRA relative expression as measured by RT- qPCR in SK-MEL-28 cells treated as described in d. Error bars represent mean ± s.e.m.; SAMMSON, n=6; TERRA, n=3. (f) Binding of SAMMSON to XRN2, CARF and p32 were predicted using the catRAPID,. Background correction was obtained by subtracting the profile of a bona fide negative control. (g) Schematic representation of the different SAMMSON_RAT-tag constructs (h) FLAG-tag RIP in HEK-293T cells transfected either with the different SAMMSON constructs or control plasmid (Ctrl) followed by northern blot using the probes for the RAT-tag and SAMMSON indicated in g. Representative images of three independent experiments. (i) Western blot with the protein fraction recovered from the RIP described in h. Top panel long exposure, bottom panel short exposure (for 0.5% Input only). Representative images of three independent experiments. 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.

Figure 5. SAMMSON modulates XRN2 localization and function.

(a) Representative XRN2 (cyan) and fibrillarin (yellow) IF in SK-MEL-28 cells 30 hours after transfection with a non-targeting GapmeR(Ctrl), GapmeR3 or GapmeR11 or untransfected (Mock). Scale bar low magnification, 10 µm; high magnification, 2 µm. Representative images of three independent experiments. (b) 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 four melanoma cell lines (with different mutational backgrounds and phenotype) transfected with a non- targeting GapmeR (Ctrl) or a GapmeR3 (G3) or of SK-MEL-28 transfected with a XRN2- targeting (siXRN2) or control (siCtrl) siRNA. KD efficiency is shown for both SAMMSON KD and XRN2 KD. Representative image of three independent experiments. (c) 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 hours after transfection with a non-targeting GapmeR (Ctrl) or with GapmeR3 (G3); n=8 for SAMMSON, 18S, 16S and 12S; n=4 for COX1 and ND1. (d) Mean coverage ratio between SAMMSON KD and a non-targeting GapmeR across all expressed snoRNAs. Data are displayed for a snoRNA meta-gene, demonstrating increased coverage beyond the snoRNA boundaries. Box boundaries represent 25th and 75th percentiles; center line represents the median; whiskers, last data point within ±1.5 interquartile range. P values were calculated by paired two-tailed Student’s t-test. ** P<0.01; *** P<0.001; **** P<0.0001; NS, not significant. Uncropped gel images are shown in Supplementary Data Set 1.

Figure 6. SAMMSON promotes CARF localization to the cytoplasm and its binding to p32.

(a) Representative CARF (red) and p32 (yellow) IF in SK-MEL-28 cells 30 hours after transfection with a non-targeting GapmeR(Ctrl), GapmeR3 or GapmeR11 or untransfected (Mock). Cell nuclei are stained with DAPI (blue). Scale bar low magnification, 10 µm; high magnification, 2 µm. Representative images of three independent experiments. (b) CARF IP in SK-MEL-28 cells in the presence (+) or absence (-) of RNase A and western blotting. Representative image of three independent experiments. (c) CARF IP in LCL cells described in Figure 1a and western blotting, where (-) represents cells infected with an empty control plasmid and (+) the SAMMSON-expressing cells. Representative image of three independent experiments. (d) SAMMSON relative expression measured by RT-qPCR in SK-MEL-28 cells 30 hours after transfection with a non-targeting GapmeR (Ctrl) or GapmeR3 (G3). Error bars represent mean ± s.e.m.; n=3. (e) CARF IP in SK-MEL-28 cells treated as described in d and western blotting. Representative images of three independent experiments. (f) Representative Proximity Ligation Assay (PLA, cyan) assay using antibodies against CARF and p32 in SKMEL-28 cells 30 hours after transfection with a non-targeting (Ctrl), GapmeR3 or GapmeR11. Cell nuclei are stained with DAPI (blue). Scale bar low magnification, 10 µm; high magnification, 2 µm. Representative images of three independent experiments. (g) SAMMSON relative expression measured by RT-qPCR in SK-MEL-28 cells 30 hours after transfection with a non-targeting GapmeR(Ctrl), GapmeR3 (G3) or GapmeR11 (G11). Error bars represent mean ± s.e.m.; n=3. (h) Quantification of PLA assay described in f and g. Error bars represent mean ± s.e.m.; n=3. P values were calculated by paired two-tailed Student’s t-test. * P<0.05. Uncropped gel images are shown in Supplementary Data Set 1.

Figure 7. SAMMSON expression increases rRNA processing thus promoting protein synthesis.

(a) Heatmap representation of pre-rRNA analysis in Mel-ST cells overexpressing SAMMSON (SAM O/E) relative to cells transfected with an empty (Ctrl). Each pre-RNA intermediates detected was quantified with a PhosphorImager; n=3. (b) Quantification of relative protein synthesis (%) after a 10-minute pulse with puromycin and subsequent cytosol(Cyto)-mitochondria(Mito)-mitoplast(Mitopl) fractionation in Mel-ST cells described in a. Error bars represent mean ± s.e.m. Total and Cyto, n=4; Mito and Mitopl, n=3. (c) Quantification of relative protein synthesis (%) after a 10-minute pulse with puromycin and subsequent cytosol(Cyto)-mitochondria(Mito)-mitoplast(Mitopl) fractionation in LCL cells as described in Figure 1a; n=5. (d) p32 relative expression measured by RT-qPCR in LCL cells as described in c; n=6. (e) Quantification of p32 protein levels relative to the control fraction in LCL cells described in c after cytosol(Cyto)-mitochondria(Mito)-mitoplast(Mitopl) fractionation; n=6. (f) Quantification of p32 protein levels relative to the control fraction after cytosol(Cyto)-mitochondria(Mito)-proteinase K-treated mitochondria(Mito+PK) fractionation in LCL cells as described in Figure 1a; n=4. (g) Puromycin quantification (each point represents the average of the quantification of three different fields) of tumors as described in Figure 1e-g; n=8. Box boundaries represent 25th and 75th percentiles; center line represents the median; whiskers, last data point within ±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.

Figure 8. Schematic representation of SAMMSON mechanism of action.

In normal (SAMMSON-negative) cells, CARF controls the nuclear localization of XRN2 by sequestering a pool of it in the nucleoplasm. In the context of melanoma, SAMMSON expression promotes the interaction of CARF with p32 at the expense of the CARF-XRN2 interaction, thus favoring p32 mitochondrial localization and XRN2 nucleolar localization. By modulating these interactions SAMMSON determines a balanced increase in rRNA maturation and protein synthesis in the cytosol and mitochondria. As a result, SAMMSON promotes an increase in cell growth.