Uncoupling of GTP hydrolysis from eIF6 release on the ribosome causes Shwachman-Diamond syndrome

Abstract

Removal of the assembly factor eukaryotic initiation factor 6 (eIF6) is critical for late cytoplasmic maturation of 60S ribosomal subunits. In mammalian cells, the current model posits that eIF6 release is triggered following phosphorylation of Ser 235 by activated protein kinase C. In contrast, genetic studies in yeast indicate a requirement for the ortholog of the SBDS (Shwachman-Bodian-Diamond syndrome) gene that is mutated in the inherited leukemia predisposition disorder Shwachman-Diamond syndrome (SDS). Here, by isolating late cytoplasmic 60S ribosomal subunits from Sbds-deleted mice, we show that SBDS and the GTPase elongation factor-like 1 (EFL1) directly catalyze eIF6 removal in mammalian cells by a mechanism that requires GTP binding and hydrolysis by EFL1 but not phosphorylation of eIF6 Ser 235. Functional analysis of disease-associated missense variants reveals that the essential role of SBDS is to tightly couple GTP hydrolysis by EFL1 on the ribosome to eIF6 release. Furthermore, complementary NMR spectroscopic studies suggest unanticipated mechanistic parallels between this late step in 60S maturation and aspects of bacterial ribosome disassembly. Our findings establish a direct role for SBDS and EFL1 in catalyzing the translational activation of ribosomes in all eukaryotes, and define SDS as a ribosomopathy caused by uncoupling GTP hydrolysis from eIF6 release.

Figure 1.

Histopathological abnormalities in Sbds-deleted mouse livers. (A) Schematic of the targeted Sbds allele on mouse chromosome 5. Exons I–V are shown in red, loxP sites are shown in yellow, and the neomycin cassette is shown in green. (B) Representative filter hybridization analysis of neomycin-resistant embryonic stem cell clones. Genomic DNA was digested with BamH1 and hybridized to the 5′ probe (Supplemental Fig. S1). The 15-kb- and 11-kb bands represent wild-type Sbds⁺ (lanes 1,3) and targeted Sbds^fl (lane 2) alleles, respectively. (C) Representative filter hybridization for genotyping (Geno) of genomic tail DNA from mice carrying wild-type (+) and targeted (fl) Sbds alleles. DNA was digested with BamH1 and hybridized with the 5′ probe. (D) PCR genotyping of tail DNA to detect wild-type (+) and floxed (fl) (panel I) or exon 2 deleted (−) (panel II) Sbds alleles. The sizes of the PCR products are indicated. (E) Sbds protein is absent in Sbds-deleted mouse livers. The Sbds genotype and the absence (−Cre) or presence (+Cre) of the pMx1-cre transgene are indicated. Liver extracts were immunoblotted to visualize Sbds and Rps14. (F) Histopathological abnormalities in Sbds-deleted mouse livers. (Panels I–IX) H&E sections of livers from representative Sbds^fl/- mice after 4 wk of treatment with poly(I:C) in the absence (−Cre) or presence (+Cre) of the pMx1-cre transgene demonstrating normal zone 2 (50×) (panel I); disordered architecture in zone 2 between the portal triads and central veins (50×) (panel II); hydropic cytoplasmic swelling of hepatocytes in zone 2 (400×) (panel III); hepatocytes showing apparent cavitation of the nucleus (thin arrow), degenerative nuclear change (thick arrow), and apoptotic cells (asterisks) (400×) (panel IV); a swollen hepatocyte containing an enlarged abnormal nucleus (arrow) (400×) (panel V); an enlarged, ring-shaped nucleolus with an eosinophilic center (arrow) (400×) (panel VI); multiple enlarged eosinophilic nucleoli (arrow) (400×) (panel VII); the area of necrosis adjacent to the liver capsule with surrounding neutrophil infiltrate (asterisk) (100×) (panel VIII); and the necrotic area showing karyolysis of hepatocytes (400×) (panel IX). See also Supplemental Figure S1.

Figure 2.

60S subunit maturation defect in Sbds-deleted mouse livers. (A) Polysome profiles of liver cell extracts from undeleted (−Cre) or Sbds-deleted (+Cre) mice. Halfmer ribosomes are indicated with arrows. (B) Cytoplasmic accumulation of late pre-60S assembly factors in Sbds-deleted cells. Subcellular fractions from undeleted (−Cre) or Sbds-deleted (+Cre) liver cells were immunoblotted to visualize the indicated factors. Rps14 and Rpl28 reveal the 40S and 60S ribosomal subunits, respectively. Gapdh is a cytoplasmic marker, Npm is a nuclear marker, and histone H3 is a chromatin marker. (C) Cytoplasmic fraction; (N) soluble nuclear fraction; (I) insoluble nuclear fraction containing nucleoli and chromatin. (C) Cosedimentation of eIF6, Nmd3, and Lsg1 with pre-60S subunits in Sbds-deleted cells. Extracts from undeleted (−Cre) or Sbds-deleted (+Cre) liver cells as above were fractionated by sucrose gradient sedimentation and immunoblotted to visualize the indicated factors. P0 reveals the 60S ribosomal stalk, and the sedimentation positions of the 40S, 60S, and 80S ribosomal particles are indicated. See also Supplemental Figure S2.

Figure 3.

SBDS and EFL1 cooperate to directly catalyze eIF6 release. (A) Schematic of eIF6 release assay. Pre-60S subunits were isolated from Sbds-deleted liver cell extracts by sucrose gradient sedimentation, incubated with recombinant release factors, and pelleted through a 30% (w/v) sucrose cushion. Immunoblotting revealed the distribution of eIF6 in the supernatant (“free”) and pellet (“bound”). (B) SBDS and EFL1 are jointly required for GTP-dependent release of eIF6 from purified pre-60S subunits. Indicated combinations of recombinant human SBDS and EFL1 were incubated with pre-60S subunits in the presence of GTP. eIF6, Nmd3, and Ebp1 were visualized by immunoblotting. A minus sign (−) indicates no added factor. (C) Nucleotide dependence of eIF6 release by SBDS and EFL1. Recombinant human SBDS and EFL1 were incubated with pre-60S subunits in the presence of the indicated guanine nucleotides. eIF6 and Ebp1 were visualized by immunoblotting. (D) Ser 235 is not required for GTP-dependent eIF6 release by SBDS and EFL1 in vitro. (Left panel) Purified RRL 60S subunits (preloaded with eIF6^1-225) were incubated with the indicated combinations of recombinant human SBDS and EFL1 and GTP. (Right panel) RRL 60S subunits were incubated with SBDS and EFL1 and the indicated nucleotides. EDTA was used as a positive control for eIF6 release. eIF6 and Rpl28 were visualized by immunoblotting. See also Supplemental Figures S3 and S4 and Supplemental Tables S1 and S2.

Figure 4.

Solution structure of human SBDS and impact of SDS-associated mutations. (A) Ribbon representation of the lowest-energy human SBDS NMR structure surrounded by the solvent-accessible surface (radius probe 1.4 Å), prepared using the program PyMOL (http://www.pymol.org). Domain I is colored red, domain II is colored yellow, domain III is colored green, and loops are colored gray. The indicated SDS-associated mutations modify surface epitopes (blue spheres) or protein stability (pink spheres). (B–D) Overlay of the backbone atoms of the 20 lowest-energy structures from domain I (A16–T89) (A), domain II (D97–K164) (B), and domain III (H171–L237) (C). The 20 conformers were overlaid using Clusterpose (Diamond 1995). (E) Representation of the electrostatic surface potential of the human SBDS protein, calculated by the program APBS (Baker et al. 2001) and colored using a linear color ramp from −15 kT (red) to +15 kT (blue). The SDS-associated mutation K151N is indicated. (F) Overlays of the ¹H,¹⁵N HSQC spectra for wild-type (blue) and three SDS-associated mutants (C84R, R126T, and K151N) (red). Arrows indicate peaks visible in the wild-type spectrum but not in the C84R mutant. See also Supplemental Figures S5 and S6 and Supplemental Tables S3–S5.

Figure 5.

Conserved interdomain motion of the SBDS protein. (A) Quasi-independent mobility of domain I relative to domains II and III. The experimentally determined longitudinal relaxation rate ¹⁵N R₁ (sec⁻¹) for human (blue circles) and yeast (red triangles) SBDS is plotted against human SBDS residue number. Black circles represent ¹⁵N R₁ (sec⁻¹) values calculated for a simulated fully rigid SBDS model, and gray triangles represent ¹⁵N R₁ (sec⁻¹) values calculated for a simulated model in which the motion of domain I is completely independent of domains II and III. (B) Schematic representation of the quasi-independent mobility of domain I (red) relative to domains II and III (blue). Arrow indicates the hinge region at the N terminus of helix α5. (C) {¹H}¹⁵N-heteronuclear NOE values for human (blue circles) and yeast (red triangles) SBDS proteins plotted against human SBDS residue number. Residues for which no data are shown correspond to prolines or overlapped cross-peaks. (D) {¹H}¹⁵N-heteronuclear NOE values mapped onto a ribbon representation of the human SBDS structure. Residues are colored from most (red) to least (blue) flexible.

Figure 6.

SBDS tightly couples activation of EFL1 GTP hydrolysis to eIF6 release. (A) SBDS and EFL1 bind independently to 60S subunits. SBDS and EFL1 were bound to RRL 60S subunits over the indicated range of KCl concentrations and pelleted through 30% (w/v) sucrose cushions. Bound SBDS, EFL1, and Rpl28 were visualized by immunoblotting. (B) SBDS stimulates 60S-dependent GTP hydrolysis by EFL1. 60S-dependent GTPase activity of human wild-type EFL1 or a catalytically inactive mutant (H96A) was measured in the presence of wild-type or variant (R126T and K151N) SBDS. The experiment was repeated three times and the average values with SD are presented. (Inset) Dose response curve for 60S-dependent EFL1 catalytic activity as a function of GTP concentration in the presence (black line) or absence (red line) of human SBDS. Measurements were performed in duplicate. (C) SDS-associated SBDS variants are defective in eIF6 release. Pre-60S subunits were incubated with GTP, EFL1, and either wild-type or variant (R126T or K151N) SBDS proteins. Following sucrose pelleting, “bound” and “free” fractions were immunoblotted to visualize eIF6 and Ebp1. See also Supplemental Figure S7.

Figure 7.

Model of eIF6 release by SBDS and EFL1. (1) SBDS stimulates 60S-dependent GTP hydrolysis by EFL1, generating EFL1.GDP.Pi. (2) Following release of inorganic Pi, EFL1 adopts its GDP-bound conformation and domain I of SBDS is rotated relative to domains II and III, directly or indirectly disrupting the intersubunit bridge B6. (3) Binding of eIF6 is destabilized, release of eIF6 is triggered, and EFL1.GDP and SBDS dissociate from the ribosome. Release of eIF6 allows the formation of actively translating 80S ribosomes. The putative conformations of EFL1 are not indicated.