American Society of Hematology

Figure 3.

$Figure 3. SDS-associated EFL1 mutations impair eIF6 release. (A) Total cell lysates from fibroblasts and (B) B-LCL from wild-type and 3 individuals (P1, P2, and P3) with SDS were immunoblotted to visualize the indicated proteins. (C) Defective ribosome assembly in EFL1 mutant fibroblasts. Polysome profiles from fibroblast extracts from 3 unrelated individuals with SDS compared with wild-type control. Quantification of the 60S:80S ribosomal subunit ratios is indicated as a bar chart (n ≥ 5) (D). EFL1 genotypes are provided in supplemental Table 1. (E) EFL1 is required for eIF6 recycling in human cells. Indicated proteins were visualized by immunoblotting in cytoplasmic or nuclear fractions from wild-type and EFL1 mutant fibroblasts. Histone H3, nuclear marker; HSP9, cytoplasmic marker. (F) Relative amount of eIF6 in the nucleus of EFL1 mutant cells compared with wild-type (n = 5). (G-H) EFL1 deficiency attenuates protein synthesis. OP-Puro incorporation in fibroblast cells lines from individuals P1, P2, and P3 relative to wild-type control cells quantified 1 hour after OP-Puro administration (n = 6). (I) Complementation of EFL1-deficient fibroblasts with wild-type EFL1. Lysates from EFL1 mutant fibroblasts transduced with a vector expressing GFP alone (+empty) or GFP + EFL1 (+EFL1) and from wild-type fibroblasts as a control. The indicated proteins were visualized by immunoblotting. (J) Wild-type EFL1 rescues global translation in patient fibroblast cell lines from P1 and P3 (n = 4). (K) Complementation of EFL1-deficient fibroblasts with inducible vector allows wild-type EFL1 expression after doxycycline (Dox) treatment. Lysates from EFL1 mutant fibroblasts transduced with an empty vector or EFL1-expressing vector with (+) or without (−) doxycycline. Vinculin is used as a loading control. (L) Comparison of polysome profiles from wild-type and EFL1-mutant fibroblasts transduced with empty vector or inducible EFL1-expression vector treated (+) or not (−) with doxycycline. Arrows indicate increased 80S formation in complemented cells from individuals P1, P2, and P3. (M) Quantification of the 60S:80S ribosomal subunit ratios in cells transduced with inducible EFL1-expression vector treated (+) or not (−) with doxycycline (n = 3). (N) Inducible expression of wild-type EFL1 rescues global protein translation rates in EFL1-deficient fibroblast cell lines (n = 6). (O) Schematic of eIF6 release assay. Pre-60S subunits extracted from P3-derived fibroblasts were incubated with the indicated release factors and pelleted through a 15% sucrose cushion. Immunoblotting reveals the eIF6 distribution in the supernatant (“free”) and pellet (“bound”). (P) Release of eIF6 by SDS-associated EFL1 variants. P3-derived pre-60S subunits were incubated with guanosine triphosphate (GTP), SBDS, and the indicated EFL1 variants. EDTA was added as a positive control for eIF6 release. eIF6 and uL14 were visualized by immunoblotting. (−) indicates the negative control lacking EFL1. All data represent mean ± standard error. Statistical significance between samples was assessed by a 2-tailed Student t test. ns, not significant.$

SDS-associated EFL1 mutations impair eIF6 release. (A) Total cell lysates from fibroblasts and (B) B-LCL from wild-type and 3 individuals (P1, P2, and P3) with SDS were immunoblotted to visualize the indicated proteins. (C) Defective ribosome assembly in EFL1 mutant fibroblasts. Polysome profiles from fibroblast extracts from 3 unrelated individuals with SDS compared with wild-type control. Quantification of the 60S:80S ribosomal subunit ratios is indicated as a bar chart (n ≥ 5) (D). EFL1 genotypes are provided in supplemental Table 1. (E) EFL1 is required for eIF6 recycling in human cells. Indicated proteins were visualized by immunoblotting in cytoplasmic or nuclear fractions from wild-type and EFL1 mutant fibroblasts. Histone H3, nuclear marker; HSP9, cytoplasmic marker. (F) Relative amount of eIF6 in the nucleus of EFL1 mutant cells compared with wild-type (n = 5). (G-H) EFL1 deficiency attenuates protein synthesis. OP-Puro incorporation in fibroblast cells lines from individuals P1, P2, and P3 relative to wild-type control cells quantified 1 hour after OP-Puro administration (n = 6). (I) Complementation of EFL1-deficient fibroblasts with wild-type EFL1. Lysates from EFL1 mutant fibroblasts transduced with a vector expressing GFP alone (+empty) or GFP + EFL1 (+EFL1) and from wild-type fibroblasts as a control. The indicated proteins were visualized by immunoblotting. (J) Wild-type EFL1 rescues global translation in patient fibroblast cell lines from P1 and P3 (n = 4). (K) Complementation of EFL1-deficient fibroblasts with inducible vector allows wild-type EFL1 expression after doxycycline (Dox) treatment. Lysates from EFL1 mutant fibroblasts transduced with an empty vector or EFL1-expressing vector with (+) or without (−) doxycycline. Vinculin is used as a loading control. (L) Comparison of polysome profiles from wild-type and EFL1-mutant fibroblasts transduced with empty vector or inducible EFL1-expression vector treated (+) or not (−) with doxycycline. Arrows indicate increased 80S formation in complemented cells from individuals P1, P2, and P3. (M) Quantification of the 60S:80S ribosomal subunit ratios in cells transduced with inducible EFL1-expression vector treated (+) or not (−) with doxycycline (n = 3). (N) Inducible expression of wild-type EFL1 rescues global protein translation rates in EFL1-deficient fibroblast cell lines (n = 6). (O) Schematic of eIF6 release assay. Pre-60S subunits extracted from P3-derived fibroblasts were incubated with the indicated release factors and pelleted through a 15% sucrose cushion. Immunoblotting reveals the eIF6 distribution in the supernatant (“free”) and pellet (“bound”). (P) Release of eIF6 by SDS-associated EFL1 variants. P3-derived pre-60S subunits were incubated with guanosine triphosphate (GTP), SBDS, and the indicated EFL1 variants. EDTA was added as a positive control for eIF6 release. eIF6 and uL14 were visualized by immunoblotting. (−) indicates the negative control lacking EFL1. All data represent mean ± standard error. Statistical significance between samples was assessed by a 2-tailed Student t test. ns, not significant.

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