eIF6 rebinding dynamically couples ribosome maturation and translation

Abstract

Protein synthesis is a cyclical process consisting of translation initiation, elongation, termination and ribosome recycling. The release factors SBDS and EFL1-both mutated in the leukemia predisposition disorder Shwachman-Diamond syndrome - license entry of nascent 60S ribosomal subunits into active translation by evicting the anti-association factor eIF6 from the 60S intersubunit face. We find that in mammalian cells, eIF6 holds all free cytoplasmic 60S subunits in a translationally inactive state and that SBDS and EFL1 are the minimal components required to recycle these 60S subunits back into additional rounds of translation by evicting eIF6. Increasing the dose of eIF6 in mice in vivo impairs terminal erythropoiesis by sequestering post-termination 60S subunits in the cytoplasm, disrupting subunit joining and attenuating global protein synthesis. These data reveal that ribosome maturation and recycling are dynamically coupled by a mechanism that is disrupted in an inherited leukemia predisposition disorder.

Fig. 1. eIF6 maintains free mammalian 60S subunits in a translationally inactive state.

a Sucrose gradient sedimentation of eIF6 in cell extracts prepared from murine c-kit+ bone marrow cells. The distribution of eIF6, uL5, and eS19 was visualized by immunoblotting. Data are representative of at least five independent experiments. b Cryo-EM classification scheme to quantify the frequency of eIF6-bound 60S subunits in the cytoplasm. See “Methods” for further details. c eIF6 binds the intersubunit face of free cytoplasmic 60S subunits. Crown views of the cryo-EM maps of native 60S–eIF6 complexes isolated from murine c-kit+ bone marrow cells. eIF6 is highlighted by the red color. d Atomic model for the murine 60S ribosomal subunit bound to human eIF6.

Fig. 2. SBDS and EFL1 catalyze GTP-dependent release of rebound eIF6 from mature cytoplasmic 60S ribosomal subunits.

a Sucrose gradient sedimentation of c-Kit+ bone marrow cell extracts (without cycloheximide) lysed in 20 mM Hepes pH 7.5, 2 mM Mg(CH₃COO)₂, 500 mM KCl and incubated for 10 min at 37 °C to allow eIF6 rebinding. eIF6 was detected by immunoblotting. Data are representative of two independent experiments. b Schematic overview of in vitro eIF6 release assay. See “Methods” section for further details. c Sucrose gradient sedimentation of reconstituted eIF6 release reaction mixes. Immunoblotting was used to detect eIF6. The ratio of 80S monosomes to 60S subunits is indicated. Shown is a representative experiment out of a total of two independent experiments.

Fig. 3. Genetic interactions between Sbds, Efl1, and eIF6.

a Increased eIF6 dosage enhances the growth defects of Sbds-deficient Drosophila. Flies were photographed at 1, 3, 5, and 11 days after egg laid. Scale bar, 1 mm. b Genetic interactions between Sbds, Efl1, and eIF6 in the Drosophila eye. Representative photomicrographs of adult eyes from flies with the indicated genotypes. EES, abbreviation of (eIF6/+, Efl1^RNAi/+, Sbds^RNAi/+). Scale bar, 100 μm. c Overexpression of eIF6 suppresses global protein synthesis in Drosophila wing disc cells. Third instar larval wing disc cells with the indicated genotypes were immunostained to reveal OP-Puro incorporation (red, gray). Posterior wing disc cells are marked with GFP; nucleus is blue (DAPI), scale bar: 100 μm. Data are representative of three independent experiments including ten wing discs each.

Fig. 4. eIF6 binds post-termination 60S subunits to prevent ribosomal subunit joining.

a Schematic overview of the transgenic Dox-inducible eIF6 overexpression system. b Breeding strategy for graded overexpression of eIF6, with color coding of indicated genotypes. c Quantitative real-time PCR of EIF6 transcript levels (n = 4, 3, and 3 biologically independent samples per genotype). d eIF6 protein immunoblotting analysis in extracts from cultured c-Kit+ bone marrow cells derived from the indicated mouse strains after 24 h of Dox induction. e, f. Sucrose gradient sedimentation of extracts (including cycloheximide) from cultured c-Kit+ bone marrow cells derived from the indicated mouse strains. Dox induction, 24 h. Buffers in (e) and (f) contain 50 mM or 200 mM KCl, respectively. Shown is representative of two independent experiments. g Sucrose gradient sedimentation of extracts prepared in absence of magnesium to dissociate 80S ribosomes and polysomes. h Quantification of the 60S:40S subunit ratios shown in (g) (n = 3 per genotype). Student’s t test was used to determine statistical significance. Two-tailed P values are shown. All graphs show mean ± standard deviation.

Fig. 5. Increased eIF6 dosage impairs erythroblast enucleation in mice.

a Increased eIF6 dosage causes macrocytic anemia. Hematological parameters including hemoglobin concentration, erythrocyte count and mean corpuscular volume (MCV) are shown over the indicated time-course of Dox induction for eIF6^hi mice versus control. 2 and 20 weeks, n = 13 and 15 animals per genotype; 1 year, n = 12 and 13 animals per genotype. Hemoglobin at 2 weeks, P = 0.00000000002. b Reticulocyte counts (n = 3 animals per genotype). c Representative flow cytometry analysis of erythroid precursors in control versus eIF6^hi bone marrow. Gated populations are designated 1–6 in red. d Frequency of erythroid precursors in the bone marrow (n = 7 biologically independent samples per genotype), corresponding to gated populations 1–6 in the flow cytometry analysis. Pro, proerythroblast; Baso, basophilic erythroblast; Poly, polychromatic erythroblast; Ortho, orthochromatic erythroblast; Retic, reticulocyte. e Morphology of erythroid precursors, corresponding to populations 4–6 by flow cytometry. f Representative images of enucleating erythroblasts, defined by Amnis ImageStream IDEAS gating strategy, shown in Supplementary Fig. 11. g Frequencies of enucleating erythroblasts within the late erythroblast population (corresponding to gate 6 in IDEAS gating strategy), in the bone marrow after 2 weeks of Dox administration (n = 4 biologically independent samples per genotype). All graphs show mean ± standard deviation. Student’s t test was used to determine statistical significance. Two-tailed P values are shown.

Fig. 6. Increased eIF6 dosage impairs erythroblast enucleation by attenuating protein synthesis.

a OP-Puro incorporation in the indicated bone marrow cells in vivo after two weeks of Dox administration (n = 3 and 4 biologically independent samples per genotype). Median fluorescence intensities were normalized against the respective control cell populations. b Expression of eIF6 in CFU-E erythroid progenitor cells and erythroid precursors in vivo after two weeks of Dox treatment. Immunoblots are shown for eIF6 and eS19 using extracts generated from identical numbers of the indicated bone marrow cells. Shown is representative of two independent experiments. CFU-E progenitor cells are defined as CD71+ TER-119- bone marrow cells. c Total cellular nucleic acid content in vivo during terminal erythroid differentiation. Freshly isolated bone marrow cells (n = 4 biologically independent samples per genotype) were stained with thiazole orange. Thiazole orange intensities are shown relative to CD44+ TER-119- non-erythroid bone marrow cells. d Enucleation of FACS-purified wild type orthochromatic erythroblasts in culture after 3, 5, or 24 hr treatment with homoharringtonine (n = 3 independent experiments). Enucleation efficiency is expressed as the ratio of reticulocytes to orthochromatic erythroblasts. All graphs show mean ± standard deviation. Student’s t test was used to determine statistical significance. Two-tailed P values are shown.

Fig. 7. Model illustrating how dynamic rebinding of eIF6 couples ribosome maturation and translation.

eIF6 functions as a ribosome anti-association factor to hold nascent pre-60S and mature post-termination 60S subunits in a translationally inactive state. SBDS and EFL1 couple nascent 60S subunit maturation and ribosome recycling by acting as general eIF6 release factors.