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Mechanism of eIF6 release from the nascent 60S ribosomal subunit

Nature Structural & Molecular Biology volume 22, pages 914–919 (2015)Cite this article

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Abstract

SBDS protein (deficient in the inherited leukemia-predisposition disorder Shwachman-Diamond syndrome) and the GTPase EFL1 (an EF-G homolog) activate nascent 60S ribosomal subunits for translation by catalyzing eviction of the antiassociation factor eIF6 from nascent 60S ribosomal subunits. However, the mechanism is completely unknown. Here, we present cryo-EM structures of human SBDS and SBDS–EFL1 bound to Dictyostelium discoideum 60S ribosomal subunits with and without endogenous eIF6. SBDS assesses the integrity of the peptidyl (P) site, bridging uL16 (mutated in T-cell acute lymphoblastic leukemia) with uL11 at the P-stalk base and the sarcin-ricin loop. Upon EFL1 binding, SBDS is repositioned around helix 69, thus facilitating a conformational switch in EFL1 that displaces eIF6 by competing for an overlapping binding site on the 60S ribosomal subunit. Our data reveal the conserved mechanism of eIF6 release, which is corrupted in both inherited and sporadic leukemias.

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Figure 1: SBDS shields the active sites of the 60S subunit.
Figure 2: EFL1 and eIF6 compete for an overlapping binding site.
Figure 3: Rotational displacement of SBDS upon EFL1 binding.
Figure 4: Disease-related SBDS variants disrupt critical interactions with the 60S rRNA.
Figure 5: Mechanism of eIF6 release by SBDS and EFL1.

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Acknowledgements

We thank A. Johnson (University of Texas) and A. Newman (Medical Research Council Laboratory of Molecular Biology) for yeast strains; B. Trumpower (Dartmouth Medical School) for providing uL16 antiserum; S. Chen, C. Savva, S. De Carlo, S. Welsch, F. De Haas, M. Vos and K. Sader for technical support with cryo-EM; G. McMullan for help with movie data acquisition; T. Darling and J. Grimmett for help with computing; A. Brown for help with refinement; and S. Scheres for discussion. This work was supported by a Federation of European Biochemical Societies Long Term Fellowship (to F.W.), the Specialist Programme from Bloodwise (12048 to A.J.W.), the UK Medical Research Council (MRC) (MC_U105161083 to A.J.W. and U105115237 to R.R.K.), a Wellcome Trust strategic award to the Cambridge Institute for Medical Research (100140), a core support grant from the Wellcome Trust and MRC to the Wellcome Trust–Medical Research Council Cambridge Stem Cell Institute, the Tesni Parry Trust (to A.J.W.), Ted's Gang (to A.J.W.) and the Cambridge National Institute for Health Research Biomedical Research Centre.

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Authors and Affiliations

  1. Cambridge Institute for Medical Research, Cambridge, UK

    Félix Weis, Christine Hilcenko & Alan J Warren

  2. Medical Research Council Laboratory of Molecular Biology, University of Cambridge Research Unit, Cambridge, UK

    Félix Weis, Mark Churcher, Li Jin, Christine Hilcenko & Alan J Warren

  3. Department of Haematology, University of Cambridge, Cambridge, UK

    Félix Weis, Mark Churcher, Li Jin, Christine Hilcenko & Alan J Warren

  4. Wellcome Trust–Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, UK

    Félix Weis, Mark Churcher, Li Jin, Christine Hilcenko & Alan J Warren

  5. Université de Rennes 1, Centre Nationale de la Recherche Scientifique, Unité Mixte de Recherche 6290, Institut de Génétique et Développement de Rennes, Rennes, France

    Emmanuel Giudice

  6. Medical Research Council Laboratory of Molecular Biology, Cambridge, UK

    Li Jin, David Traynor & Robert R Kay

  7. Experimental Cancer Genetics, Wellcome Trust Sanger Institute, Cambridge, UK

    Chi C Wong

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Contributions

F.W. performed sample preparation, EM data collection, image processing and model refinement. E.G. performed model building and fitting; M.C., L.J. and A.J.W. performed genetic and biochemical experiments; C.H. performed protein expression and purification; C.C.W. generated mutant Dictyostelium strains with advice from D.T. and R.R.K.; and D.T. and F.W. cultured Dictyostelium cells. F.W. and A.J.W. designed experiments and wrote the manuscript with input from all authors.

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Integrated supplementary information

Supplementary Figure 1 Overview of cryo-EM image-processing procedure.

Maximum likelihood classification scheme and masks used to obtain the complexes of eIF6, SBDS and EFL1 bound to the 60S ribosomal subunit. Insets show the position of the spherical masks (grey) on the 60S subunit (cyan).

Supplementary Figure 2 Resolution of the 60S-ribosomal-subunit complexes and validation of the atomic model.

(a) Gold-standard Fourier shell correlation (FSC) curves for the three 60S ribosome complexes after 3D refinement and statistical movie processing in RELION (Bai, X. C. et al., Elife 2, e00461, 2013; Scheres, S. H. J Struct Biol 180, 519-530, 2012).

(b) Surface (top panel) and cross-sectional (bottom panel) views of the unsharpened final maps colored according to local resolution calculated with the ResMap software package (Kucukelbir, A. et al., Nat Methods, 11, 63-65, 2014).

(c, d, e, f) Examples of densities with the model fitted showing helix 72 of the 26S rRNA from the 60S-eIF6-SBDS complex with the density filtered at 3.3 Å (c), α-helix of the uL6 protein from the 60S-eIF6-SBDS complex with the density filtered at 3.3 Å (d), SBDS from the 60S-eIF6-SBDS complex with the density is filtered at 4 Å (e), EFL1 from the 60S-eIF6-SBDS-EFL1 complex with the density is filtered at 8 Å (f).

(g, h, i) Cross-validation against over-fitting. FSC curves are shown for the 60S-eIF6-SBDS (g), 60S-eIF6-SBDS-EFL1 (h) and 60S-SBDS-EFL1 (i) complexes between the final refined atomic model and the reconstructions from all particles (black); between the model refined in the reconstruction from only half of the particles and the reconstruction from that same half (FSCwork, red); and between that same model and the reconstruction from the other half of the particles (FSCtest, green).

Supplementary Figure 3 Atomic models of the 60S–SBDS–eIF6 complex.

(a) Secondary structure diagram of modeled Dictyostelium 26S rRNA. The diagram was modified from S. cerevisiae 25S rRNA (Comparative RNA Web Site, www.rna.ccbb.utexas.edu). The PTC is shown in bold. Base pairs involved in tertiary interactions are indicated with lines connecting the circles or boxes around the bases involved in the interaction. (-) corresponds to canonical base pairs, while (•, ○ and ●) correspond to non-canonical base pairs.

(b, c) Back (b) and front (c) views of the atomic model of the 60S-eIF6-SBDS complex. SRL, sarcin-ricin loop.

Supplementary Figure 4 Molecular environment of eIF6, SBDS and EFL1.

(a, b) Schematic representations of Homo sapiens EF-2 (a) and EFL1 (b).

(c) Ribbon representation of the atomic model of human EFL1 with the subdomain structure highlighted using the same color scheme as (b).

(d-g) Back (d, f) and front (e, g) views of the atomic models of the 60S-eIF6-SBDS-EFL1 and 60S-SBDS-EFL1 complexes, respectively. SRL, sarcin-ricin loop.

Supplementary Figure 5 uL16 is required to recruit Sdo1 to the 60S subunit in vivo.

(a) The EFL1 domain II insertion is dispensable in vivo. Random sporulation assay showing efl1Δ cells transformed with empty vector (pRS316) control or plasmids expressing wild type EFL1 or the EFL1Δ420-580 mutant (left).

(b) Tenfold serial dilutions (left to right) of the indicated yeast cells spotted onto selective –URA medium (left). efl1Δ cells transformed with empty vector (pRS316) are non-viable and are therefore not shown.

(c) S174 and S175 are dispensable for Tif6 function in vivo. Random sporulation assay showing tif6Δ cells transformed with empty vector (pRS316) control or plasmids expressing wild type TIF6 or the indicated TIF6 mutants.

(d) Tenfold serial dilutions (left to right) of the indicated yeast strains spotted onto selective –URA medium (left). tif6Δ cells transformed with empty vector (pRS316) are non-viable and are therefore not shown.

(e) Severe fitness defect of the uL16-H123P allele. Indicated yeast cells with (galactose) or without (glucose) endogenous uL16 spotted in tenfold serial dilutions (left to right) onto solid media.

(f) The uL16-H123P allele markedly reduces uL16 expression. uL16 and uL14 (control) were visualized by immunoblotting of extracts from the indicated strains with (galactose) or without (glucose) endogenous uL16. As controls, uL16 and uL14 were visualized by immunoblotting of extracts from strain C375 (wild type).

(g) The T-ALL associated alleles uL16-H123P, uL16-R98S and uL16-R98C impair Sdo1 binding to the 60S subunit in vivo. Sdo1-FLAG or uL14 were visualized by immunoblotting of supernatant (S) or pellet (P) from cell extracts with (galactose) or without (glucose) endogenous uL16 across the indicated range of NaCl concentrations.

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Weis, F., Giudice, E., Churcher, M. et al. Mechanism of eIF6 release from the nascent 60S ribosomal subunit. Nat Struct Mol Biol 22, 914–919 (2015). https://doi.org/10.1038/nsmb.3112

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