Fig 2. 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.

(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.