<i>Sbds</i>^{<i>R126T/R126T</i>} and <i>Sbds</i>^<i>MOCK</i> cells show an increase in cell death in response to UV stress.

<p><b>(A-B)</b> UV exposure damages more SBDS mutant cells than wt or <i>Sbds</i>^{<i>RESCUE</i>}. (A) Experiment on <i>Sbds</i>^{<i>R126T/R126T</i>} versus wt. There is an increase in cell death in <i>Sbds</i>^{<i>R126T/R126T</i>} 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 <i>Sbds</i>^{<i>R126T/R126T</i>} (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>(B)</b> Observed ratios in all genotypes. Ratio ≥ 1 indicates higher sensitivity, the rescue with SBDS only partly restores the ratios. <b>(C-D)</b> 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.</p

SBDS deficiency reduces the maximal translational capability up to 70% due to a defect in 60S maturation that is partly associated with a change in eIF6–free 60S subunits.

<p><b>(A-B) Polysome profiles.</b><i>Sbds</i>^{<i>R126T/R126T</i>} cells show an increase in free 60S and lower 80S peaks on sucrose gradient compared to wt (A). The phenotype is completely restored in <i>Sbds</i>^{<i>RESCUE</i>} 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. (<b>C-D) Nucleoli analysis.</b> <i>Sbds</i>^{<i>R126T/R126T</i>} (C) and <i>Sbds</i>^<i>MOCK</i> cells (D) do not have differences in the number of nucleoli respect to their control cell lines (wild type and <i>Sbds</i>^{<i>RESCUE</i>}). Distribution of cells containing less or more of six nucleoli per nucleus in wild type, <i>Sbds</i>^{<i>R126T/R126T</i>}_, <i>Sbds</i>^{<i>RESCUE</i>} and <i>Sbds</i>^<i>MOCK</i> 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). <b>(E-F) SBDS and NPM localization.</b> Confocal images wild type and <i>Sbds</i>^{<i>R126T/R126T</i>} 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). <b>(G-H)</b> 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: <i>Sbds</i>^{<i>R126T/R126T</i>} 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) <i>Sbds</i>^{<i>R126T/R126T</i>} 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.</p

RNASeq of 80S and polysome associated RNAs shows that <i>SBDS</i> mutation causes limited changes in mRNAs associated to polysomes, accumulation of ACA8 and ACA31 snoRNAs on immature ribosomes.

<p><b>(A-B)</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. <b>(C-D)</b> Translational analysis. <i>Sbds</i>^<i>MOCK</i> cells show 844 genes differently expressed genes on polysome fraction, most of them upregulated respect to the <i>Sbds</i>^{<i>RESCUE</i>} 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 <i>Sbds</i>^<i>MOCK</i> versus <i>Sbds</i>^{<i>RESCUE</i>} 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 <i>Sbds</i>^<i>MOCK</i> and <i>Sbds</i>^{<i>RESCUE</i>}. The normal distribution indicates that very few genes are specifically regulated in the polysomal fraction, once calculated the Polysome/Total ratio. <b>(E)</b> Volcano plot representing the results of the differential expression analysis for the 80S fraction. <b>(F)</b> Sequencing unveils accumulation on ribosomes of <i>Sbds</i>^<i>MOCK</i> versus <i>Sbds</i>^{<i>RESCUE</i>} of some snoRNAs.</p

<i>SBDS</i> mutation results in increased lysomome trafficking and activity, and a decrease in ATP and lactate levels predicted by changes in the steady-state of the mRNAs identified by RNA-seq.

<p><b>(A)</b> Changes in the steady-state levels (total, <i>bona fide</i> for transcriptional) of genes with peptidase activity as detected both on polysomes and total. Heat maps representing absolute gene expression levels in <i>Sbds</i>^<i>MOCK</i> and <i>Sbds</i>^{<i>RESCUE</i>} samples for the subset of gene sets with peptidase activity by Gene Ontology analysis. (<b>B)</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 <i>n</i>≥3; mean±s.d., T-test, paired, two-tailed. <b>(C)</b> <i>Sbds</i>^<i>MOCK</i> cells have a decrease in intracellular pH value. Representative graph showing increased lysotracker fluorescence intensitity in <i>Sbds</i>^<i>MOCK</i> cells respect to <i>Sbds</i>^{<i>RESCUE</i>} cells. This result suggests more lysosome trafficking and activity in <i>Sbds</i>^{<i>R126T/R126T</i>} cells. Data shown for <i>n</i>≥3; mean±s.d., T-test, paired, two-tailed. <b>(D-E)</b>. Lamp1 immunostaining (representative cells, D) and quantitation (E). Scale bar indicates 8.3μm. Globally, A-E show increase in the proteolitic potential of <i>Sbds</i>^<i>MOCK</i> respect to <i>Sbds</i>^{<i>RESCUE</i>} cells. <b>(F-G)</b> 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.</p

Model for SBDS-induced changes.

<p><b>(A-B)</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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006552#pgen.1006552.g001" target="_blank">Fig 1</a>), but more sensitive to stressors respect to wt cells. Interventions to increase translational efficiency are predicted to increase their fitness. <b>(B)</b> Summar table indicating all phenotypes observed in this study in <i>Sbds</i> mutant cells.</p

SBDS deficiency results in less translation capability.

<p><b>(A-B)</b><i>In Vitro</i> 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. <b>(C-D)</b> SUnSET single cell assay indicates a reduction in the number of cells incorporating mid-high puromycin both in <i>Sbds</i>^{<i>R126T/R126T</i>} (C) and in <i>Sbds</i>^<i>MOCK</i> 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.</p

Drugs show a similar sensitivity profile between <i>SBDS</i> mutant or wt cells, but unveil a slight sensibility to selective DNA damage in <i>SBDS</i> mutant cells.

<p><b>(A)</b> 100 molecules drug screening. Wild type, <i>Sbds</i>^{<i>R126T/R126T</i>}_, <i>Sbds</i>^{<i>RESCUE</i>} and <i>Sbds</i>^<i>MOC</i>K 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 <i>Sbds</i>^{<i>R126T/R126T</i>} and <i>Sbds</i>^<i>MOCK</i> cells are more similar among them, than the wild type and <i>Sbds</i>^{<i>RESCUE</i>} cells. <b>(B)</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 <i>Sbds</i>^{<i>R126T/R126T</i>} and <i>Sbds</i>^<i>MOCK</i> MEFs (1,6 μM and 3,2 μM, 48h). Two-tailed t-test, paired (***P value P≤0.001).</p