PMC

(A-B) We speculate that SBDS mutation poises cells at a low energy level due to impaired global translation. Cells have a complex transcriptional rewiring and adapt by increasing the proteolytic flux. In this condition, they are less responsive to growth stimuli (example ), but more sensitive to stressors respect to wt cells. Interventions to increase translational efficiency are predicted to increase their fitness. (B) Summar table indicating all phenotypes observed in this study in Sbds mutant cells.

(A-B) In Vitro Translation Assay shows a 2- to 3-fold impairment in translation of SBDS-mutant extracts. Equal amounts of translation competent extracts prepared from indicated cells programmed with equal amounts of capped mRNAs. All graphs represent mean ± s.d. Statistic applied was T-test, paired, two-tailed, n≥4. (C-D) SUnSET single cell assay indicates a reduction in the number of cells incorporating mid-high puromycin both in Sbds^R126T/R126T (C) and in Sbds^MOCK fibroblasts (D) respect to their controls. Negative cells are low-expressing cells. Graphs represent mean ± s.d. Statistic applied was T-test, paired, two-tailed, n≥3.

(A) 100 molecules drug screening. Wild type, Sbds^R126T/R126T_, Sbds^RESCUE and Sbds^MOCK MEFs were treated for 48h with 100 molecules selected from oral commercial drugs in UK. The heatmap shows a similar trend in all genotypes. However, the hierarchical clustering underlies that Sbds^R126T/R126T and Sbds^MOCK cells are more similar among them, than the wild type and Sbds^RESCUE cells. (B) Dose-response sensitivity to Chlorambucil a DNA alkylating drug. After the first large screening, molecules showing different activity in Sbds^R126T/R126T were selected for a second dose-response assay. The chemotherapic Chlorambucil is more toxic to Sbds^R126T/R126T and Sbds^MOCK MEFs (1,6 μM and 3,2 μM, 48h). Two-tailed t-test, paired (***P value P≤0.001).

(A-B) UV exposure damages more SBDS mutant cells than wt or Sbds^RESCUE. (A) Experiment on Sbds^R126T/R126T versus wt. There is an increase in cell death in Sbds^R126T/R126T versus wild type cells. In basal conditions no differences are observed among the genotypes (first two bar triplets, left). Upon UV damage cell death affects more Sbds^R126T/R126T (second two bar triplets, right). Cells were stressed with UV irradiation (9999 μJ/cm², three times), recovered for 4 hours and stained for annexin with 7-AAD. (B) Observed ratios in all genotypes. Ratio ≥ 1 indicates higher sensitivity, the rescue with SBDS only partly restores the ratios. (C-D) COMET assay. Representative images of indicated cells treated as indicated in manufacturer protocol (Comet assay kit, Trevigen) in (C). Scale bars indicate 20 μm. (D) Absolute quantitation of the tail moment unveils a minor but significative difference.

(A) Changes in the steady-state levels (total, bona fide for transcriptional) of genes with peptidase activity as detected both on polysomes and total. Heat maps representing absolute gene expression levels in Sbds^MOCK and Sbds^RESCUE samples for the subset of gene sets with peptidase activity by Gene Ontology analysis. (B) qPCR validation of selected genes associated to lysosome activity. Real time analysis of selected genes associated to lysosome trafficking and activity. Data shown for n≥3; mean±s.d., T-test, paired, two-tailed. (C) Sbds^MOCK cells have a decrease in intracellular pH value. Representative graph showing increased lysotracker fluorescence intensitity in Sbds^MOCK cells respect to Sbds^RESCUE cells. This result suggests more lysosome trafficking and activity in Sbds^R126T/R126T cells. Data shown for n≥3; mean±s.d., T-test, paired, two-tailed. (D-E). Lamp1 immunostaining (representative cells, D) and quantitation (E). Scale bar indicates 8.3μm. Globally, A-E show increase in the proteolitic potential of Sbds^MOCK respect to Sbds^RESCUE cells. (F-G) SBDS levels positively regulate ATP accumulation (F) and lactate/pyruvate production, an index of glycolysis (G). All graphs represent mean ± s.d. Statistic applied was T-test, paired, two-tailed, n≥4.

(A) Scheme of the strategy for immortalization from E12.5 MEFs from Sbds^R126T/R126T or wild type mice and summary data relative to Sbds^R126T/R126T cells. (B-C) Generation of immortalized colonies by infection of E12.5 MEFs at early passages with a retrovirus carrying DNp53 + H-ras^V12. Sbds^R126T/R126T have low clonogenic potential. n = 3 per genotype). (B) representaive plates; (C) quantitation (D-E) Soft agar assay for transformed cells unveils that some Sbds^R126T/R126T transformants grow as well as wt (D), but overall they produce less colonies (E). Scale bar 25 μm. (F) In vivo tumor growth underscores an unexpected incapability of Sbds^R126T/R126T transformants to propagate tumors in mice. Kaplan-Meier curves of CD1 nude mice subcutaneously injected with Sbds^R126T/R126T and wild type MEFs (n = 7 per genotype): death time point is defined at 400mm³ tumor volume, when mice were euthanized. Number of colonies expressed as mean ± s.d. Statistic applied for (B-E) was t-test, paired, two-tailed (*P value≤0.05, ***P≤0.001). Log-rank (Mantel-Cox) Test (***P≤0.0001) was applied to (F).

(A-B) Sucrose gradient separations for RNA extraction and RNAseq. In (A) RNA was extracted from polysomes (fractions corresponding to polysomes were collected in one polysome fraction) and from the whole gradient (100 μL from each fraction of the gradient were pulled in one total fraction). In (B) RNA was extracted from 80S and total (not graphed). Colored rectangles correspond to extracted fractions. (C-D) Translational analysis. Sbds^MOCK cells show 844 genes differently expressed genes on polysome fraction, most of them upregulated respect to the Sbds^RESCUE polysome fraction (C), but relative normalization to total (D) indicates that there is not a consistent change in the specific translational efficiency of individual mRNAs. (C) Volcano plot representing the differential expression analysis for polysome fraction. Results obtained comparing Sbds^MOCK versus Sbds^RESCUE conditions are reported for the pool of tested genes as -Log10 false discovery rate / Log2 moderate fold change. Genes selected as significantly changed (FDR < 5%, absolute Log2 fold-change value > 1) are shown in red. (D) Histogram showing the translational efficiency, calculated as delta Log2 fold-change (Polysome/Total) between Sbds^MOCK and Sbds^RESCUE. The normal distribution indicates that very few genes are specifically regulated in the polysomal fraction, once calculated the Polysome/Total ratio. (E) Volcano plot representing the results of the differential expression analysis for the 80S fraction. (F) Sequencing unveils accumulation on ribosomes of Sbds^MOCK versus Sbds^RESCUE of some snoRNAs.

(A-B) Polysome profiles. Sbds^R126T/R126T cells show an increase in free 60S and lower 80S peaks on sucrose gradient compared to wt (A). The phenotype is completely restored in Sbds^RESCUE cells (B). Note, the increase of free 60S is two-fold. A₂₅₄ nm was measured after 15–50% sucrose gradient sedimentation. Representative experiment of n≥5. (C-D) Nucleoli analysis. Sbds^R126T/R126T (C) and Sbds^MOCK cells (D) do not have differences in the number of nucleoli respect to their control cell lines (wild type and Sbds^RESCUE). Distribution of cells containing less or more of six nucleoli per nucleus in wild type, Sbds^R126T/R126T_, Sbds^RESCUE and Sbds^MOCK cells was counted with Volocity Sofwtare, by analyzing cells stained for the nucleolar marker nucleophosmin (NPM) (n≥200, n = number of nuclei analyzed per genotype). (E-F) SBDS and NPM localization. Confocal images wild type and Sbds^R126T/R126T cells indicate a co-localization of SBDS and NPM proteins within the nucleolus, and a cytoplasmic SBDS. There are no visible differences among all genotypes. Scale bar 25 μm (E) and 2 μm (F). (G-H) Measurement of eIF6 binding sites by iRIA technique shows that wt cells have 25% more free 60S as detected by eIF6 binding. (G) iRIA technique outline: Sbds^R126T/R126T and wild type cellular extracts were immobilized on a 96 well and biotinylated eIF6 is added. This assay is able to detect the binding between eIF6 and 60S. (H) Sbds^R126T/R126T fibroblasts have less binding sites for eIF6, respect to wild type cell line, i.e. 0.12 arbitrary units versus 0.16. Representative technical triplicate experiment of n≥4 biological replicates.