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Structure of mammalian eIF3 in the context of the 43S preinitiation complex

Nature volume 525, pages 491–495 (2015)Cite this article

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Abstract

During eukaryotic translation initiation, 43S complexes, comprising a 40S ribosomal subunit, initiator transfer RNA and initiation factors (eIF) 2, 3, 1 and 1A, attach to the 5′-terminal region of messenger RNA and scan along it to the initiation codon. Scanning on structured mRNAs also requires the DExH-box protein DHX29. Mammalian eIF3 contains 13 subunits and participates in nearly all steps of translation initiation. Eight subunits having PCI (proteasome, COP9 signalosome, eIF3) or MPN (Mpr1, Pad1, amino-terminal) domains constitute the structural core of eIF3, to which five peripheral subunits are flexibly linked. Here we present a cryo-electron microscopy structure of eIF3 in the context of the DHX29-bound 43S complex, showing the PCI/MPN core at ∼6 Å resolution. It reveals the organization of the individual subunits and their interactions with components of the 43S complex. We were able to build near-complete polyalanine-level models of the eIF3 PCI/MPN core and of two peripheral subunits. The implications for understanding mRNA ribosomal attachment and scanning are discussed.

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Figure 1: Structure of eIF3 core.
Figure 2: Model of the eIF3 core.
Figure 3: Contacts of the eIF3 core with the 40S subunit in the 43S complex.
Figure 4: Peripheral subunits of eIF3.
Figure 5: Schematic representation of the arrangement of initiation factors and their subunits in the DHX29-bound 43S complex.

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Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

Cryo-EM maps and atomic coordinates for the reported structures have been deposited in the Electron Microscopy Data Bank under the accession numbers EMD-3056, EMD-3057 and EMD-3058, and in the Protein Data Bank under the accession numbers 5A5T and 5A5U.

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Acknowledgements

We thank R. A. Grassucci for helping collect the electron microscopy images, H. Kao for help with computations, and the proteomic platform Strasbourg-Esplanade for conducting the nano-LS–MS/MS analysis. This work was supported by both HHMI and NIH R01 GM29169 (to J.F.) and NIH R01 GM59660 (to T.V.P.). This work has been published under the framework of the LABEX: ANR-10-LABX-0036_NETRNA and benefits from funding from the state managed by the French National Research Agency as part of the Investments for the future program (to Y.H.).

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biophysics, HHMI, Columbia University, New York, 10032, New York, USA

    Amedee des Georges & Joachim Frank

  2. Department of Cell Biology, SUNY Downstate Medical Center, Brooklyn, 11203, New York, USA

    Vidya Dhote, Christopher U. T. Hellen & Tatyana V. Pestova

  3. CNRS, Proteomic Platform Strasbourg - Esplanade, Strasbourg, 67084, France

    Lauriane Kuhn

  4. Department of Biological Sciences, Columbia University, New York, 10032, New York, USA

    Joachim Frank

  5. CNRS, Architecture et Réactivité de l’ARN, Université de Strasbourg, Strasbourg, 67084, France

    Yaser Hashem

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  1. Amedee des Georges
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  2. Vidya Dhote
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  4. Christopher U. T. Hellen
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  5. Tatyana V. Pestova
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Contributions

A.d.G., V.D., L.K., C.U.T.H., T.V.P., J.F. and Y.H. interpreted the data and wrote the manuscript. V.D. and T.V.P. prepared the samples. Y.H. and A.d.G. performed the cryo-electron microscopy experiments, data processing and the atomic modelling. L.K. performed the mass-spectrometry experiment. T.V.P., J.F. and Y.H. directed research.

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Correspondence to Tatyana V. Pestova, Joachim Frank or Yaser Hashem.

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Extended data figures and tables

Extended Data Figure 1 Domain organization of the eIF3 subunits and resolution of eIF3 core.

a, Schematic representation of the domain organization of the rabbit eIF3 subunits (see Methods). Domain boundaries are indicated, and based where possible on our polyalanine-level model of eIF3. HD, helical domain; Z, zinc-recognition motif. Dashed line in eIF3l subunit diagram indicates that the helical domain might extend further in the N terminus, but it was not possible to be conclusive based on our density map. b, Fourier shell correlation (FSC) of the different 43S complex reconstructions used during analysis. The resolution estimation followed the ‘gold standard’ protocol ensuring independence of the half-set reconstructions. x axis, resolution in Å; y axis, FSC. Green line denotes 43S complexes including particles that present the eIF3 core subunits; blue line denotes 43S complexes including all particles presenting DHX29 and the eIF3 peripheral subunits b, g and i; and red line denotes 43S complexes including all particles presenting the density attributed to eIF3d. For each reconstruction, a dashed line marks the resolution at which the FSC reaches the value of 0.143. c–f, Qualitative comparison of eIF3 core resolution in the present and in previous structures. c, d, eIF3 core cryo-EM structure from our previous study at 11.6 Å (ref. 7). e, f, eIF3 core cryo-EM structure from the present study at 6 Å after focused refinement. The eIF3 core is labelled according to the anthropomorphic nomenclature. g, Unassigned density region of the eIF3 core cryo-EM structure, coloured in red, seen from three different orientations. The green surface represents most of the core region that was modelled.

Extended Data Figure 2 Sorting of particle images and focused refinement of the five-lobed core of eIF3.

a, Composition of eIF3 purified from RRL for assembly of 43S complexes, resolved by SDS–PAGE and analysed by nano-LC–MS/MS to characterize truncation of eIF3a and eIF3c due to endoproteolytic cleavage. The intensity of labels in this panel reflects the intensity of bands corresponding to the truncated forms of eIF3a and eIF3c. The sequence of the N-terminal region of rabbit eIF3b has not been conclusively established (see Extended Data Fig. 9c) and numbering therefore refers to human eIF3b. b, Overview of the process of sorting particle images. The population of each class is indicated by the number of particles and the percentage of the total number of particles at the beginning of each of the two classification rounds. The DHX29-bound 43S complex was processed from a total of ∼650,000 particle images, which were first sorted into ten different classes. Class 2 (∼125,000 particles) was sorted into ten subclasses, which are displayed in four different orientations, showing the intersubunit face, front, solvent side and bottom, respectively. Classes 2–7 and 9 from the second classification round were pooled and refined yielding a reconstruction presenting a variable resolution ranging from 4.5 to 15 Å (bottom right). d, f, Cryo-EM reconstruction of the 43S complex, coloured according to the local resolution as measured using ResMap (see Methods). The red and black boxes correspond to close-up views of the eIF3 core viewed from the intersubunit face (d) and the solvent face (f) of the 40S subunit. c, e, CryoEM reconstruction of the eIF3 core after focused refinement, coloured according to the local resolution as measured using ResMap. Maps in c–f are filtered to 6 Å.

Extended Data Figure 3 Comparison of the structure of the mammalian eIF3 core with the structures of the COP9 signalosome and 26S proteasome lid molecules.

a–c, Close-up views of the helical bundles of eIF3, COP9 and 26S lid molecules. d–i, All three molecules shown in two different orientations. Different constitutive subunits are labelled and coloured variably. Homologous subunits among all three molecules are shaded using the same colour in all panels. j, k, Additional quaternary interactions between eIF3a and eIF3c, and between eIF3c and eIF3e, which occur neither in COP9 nor in 26S lid molecules. l, Consequence of these additional quaternary interactions on the structure of eIF3 subunits a, c and e, schematized as rectangles. Black arrows around axes describe the rotation of the helical domains of subunits a, c and e relatively to their respective PCI domains, owing to the existence of these additional quaternary interactions, compared to COP9 and 26S lid molecules.

Extended Data Figure 4 Conservation of quaternary interactions between eIF3a and eIF3c subunits, and between eIF3c and eIF3e subunits.

a, Ribbon representation of eIF3a and eIF3c subunits. b–d, Close-up views of contact regions between eIF3a and eIF3c. e, Ribbon representation of eIF3 c and e subunits. f, g, Close-up views of contact regions between subunits c and e of eIF3. Red spheres represent residues at the interfaces that are conserved in eIF3 from six representative eukaryotic organisms; H. sapiens, C. elegans, A. thaliana, D. melanogaster and X. tropicalis, which are very different multicellular eukaryotic organisms, and N. crassa, a unicellular organism. These organisms all have a full complement of 13 eIF3 subunits. Orange spheres represent residues at the interfaces that are conserved only in the five multicellular eukaryotic organisms. The remaining residues that are suggested by the model and the density map to be involved in quaternary interactions are represented as ribbons in salmon colour. Many of these other residues are conserved in three or four of the compared organisms, but almost all of them have conserved chemical properties.

Extended Data Figure 5 Conservation of quaternary interactions between eIF3 subunits a, m, f and h.

a, In centre, eIF3 a, m, f and h subunits. b–f, Close-up views of contact regions between subunits a, m, f and h of eIF3. Red spheres represent residues at the interfaces that are conserved in six representative eukaryotic organisms; H. sapiens, C. elegans, A. thaliana, D. melanogaster, X. tropicalis and the unicellular N. crassa. Orange spheres represent residues at the interfaces that are conserved only in the five multicellular eukaryotic organisms. The remaining residues that are suggested by the model and the density map to be involved in quaternary interactions are represented as ribbons in salmon colour. Many of these other residues are conserved in three or four of the compared organisms, but almost all of them have conserved chemical properties.

Extended Data Figure 6 Conservation of quaternary interactions between eIF3 subunits e, k and l, and between eIF3 subunits in the region of the helical bundle.

a, Ribbon representation of eIF3 e, k and l subunits. b–f, Close-up views of contact regions between subunits e, k and l of eIF3. g, Ribbon representation of eIF3 octamer core. h, Close-up views of contact regions between subunits a, c, e, m, f, h, k and l of eIF3 in the helical bundle region, seen from the direction of the axis of the latter. i, Region displayed in b rotated by 90°. Red spheres represent residues at the interfaces that are conserved in six representative eukaryotic organisms; H. sapiens, C. elegans, A. thaliana, D. melanogaster, X. tropicalis and the unicellular N. crassa. Orange spheres represent residues at the interfaces that are conserved only in the five multicellular eukaryotic organisms. The remaining residues that are suggested by the model and the density map to be involved in quaternary interactions are represented as ribbons in salmon colour. Many of these other residues are conserved in three or four of the compared organisms, but almost all of them have conserved chemical properties. j, Same view as in i displaying all the hydrophobic residues of the helical bundle region in silver ribbons. The abundance of hydrophobic residues in the helical bundle at the interfaces of different helices suggests the stabilization of the bundle though hydrophobic interactions, hence the low identity conservation of residues at the interfaces.

Extended Data Figure 7 Comparison of the mammalian eIF3 core model built from the 6 Å cryo-EM reconstruction with a model based on low-resolution cryo-EM studies.

In the centre, our polyalanine-level model of the mammalian eIF3 octamer core (represented in red ribbons) fitted on the atomic model proposed previously18 (represented in dark grey ribbons), shown in two different orientations. The surrounding panels are close-up views of different constitutive subunits, highlighting notable structural differences between the previously proposed model18 and the model proposed in this study. Gold- and cyan-dashed ovals highlight the misassignment of several helices of the helical bundle belonging to the C termini of subunits h and f, respectively, in the previously proposed model. Red-, green-dashed circles and black arrowhead highlight the absence in the previously proposed model of important structural features involved in quaternary interactions. In each panel, the remaining subunits of the eIF3 core octamer are faded out as transparent ribbons.

Extended Data Figure 8 Sorting of particle images and focused classification of eIF3 peripheral subunits near the mRNA channel entrance and exit.

a, Overview of the process of sorting particle images. The population of each class is indicated by the number of particles and the percentage of the total number of particles at the beginning of each of the classification rounds. After the first round of classification, class 2 stands out as the class displaying eIF3. Focused classification of eIF3 peripheral subunits, near the mRNA channel entrance, was performed by applying a smooth-edge mask corresponding to the shape of the concerned subunits of eIF3 and to a region of the 43S complex encompassing DHX29 and h16 of the 40S subunit. The mask is displayed as pink mesh. The resulting classes from the focused sorting of class 2 of the first classification round are displayed in two different orientations, front and solvent side, respectively. Class 6 from the second classification round presents the most solid and complete density of the peripheral subunits of eIF3 at this region of the complex, and it was therefore refined yielding a reconstruction presenting an average resolution of 7.1 Å. Cryo-EM reconstruction of eIF3b and eIF3i along with DHX29, coloured by local resolution. b, After the first round of classification, on class 2, focused classification of an eIF3 peripheral subunit, identified as eIF3d, near the mRNA channel exit behind ribosomal protein RACK1, was performed by applying a smooth-edge mask corresponding to the shape of the concerned subunit of eIF3 and to a region of the head of the 40S subunit encompassing RACK1. The mask is displayed as pink mesh. The resulting classes from the focused sorting of class 2 of the first classification round are displayed in two different orientations, intersubunit face and top, respectively. Class 2 from the second round of classification presents the most solid and complete density of the peripheral subunit of eIF3 at this region of the complex, but due to some apparent heterogeneity in eIF3d, a third classification round was required, yielding four classes displaying a solid eIF3d subunit in slightly different conformations (other classes obtained in this third round of classification were completely empty and therefore not shown). The major class (39% of the particles in this round) yielded a reconstruction presenting an average resolution of 7.7 Å. Cryo-EM reconstruction of eIF3d, coloured by local resolution.

Extended Data Figure 9 Shape and ribosomal binding site of eIF3d, and eIF3b sequence.

a, Segmented cryo-EM reconstruction of the peripheral eIF3d subunit localized on the head of the 40S subunit, behind ribosomal protein RACK1, displayed at a high density threshold to show its most solid features, in four different orientations. b, eIF3d subunit in the context of the 43S preinitiation complex, seen from the back, showing the ribosomal proteins that contact it directly. This figure is complementary to Fig. 4c as it displays the same complex in a different orientation. This panel shows contacts between eIF3d subunit and ribosomal proteins eS28, uS7 and uS9. Contacts with RACK1 cannot be seen from this orientation. c, eIF3b sequence. The amino acid sequences of human eIF3b (GenBank NP_003742.2) and rabbit eIF3b (UniProt G1SZ03_RABIT) aligned using T_COFFEE (http://www.ebi.ac.uk/Tools/msa/tcoffee/) and annotated to show identity with tryptic and chymotryptic peptides derived from purified rabbit eIF3 and identified by nano-LC–MS/MS analysis. The complete sequence of rabbit eIF3b has not been determined, but clearly extends beyond the N terminus of G1SZ03_RABIT, and we therefore used the numbering of residues in human eIF3b when referring in the text to elements of rabbit eIF3b.

Extended Data Table 1 Peptide analysis of eIF3 a and c subunits after digestion and details of interactions between eIF3 and the 40S small ribosomal subunit
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des Georges, A., Dhote, V., Kuhn, L. et al. Structure of mammalian eIF3 in the context of the 43S preinitiation complex. Nature 525, 491–495 (2015). https://doi.org/10.1038/nature14891

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