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eIF3d is an mRNA cap-binding protein that is required for specialized translation initiation

Nature volume 536, pages 96–99 (2016)Cite this article

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Abstract

Eukaryotic mRNAs contain a 5′ cap structure that is crucial for recruitment of the translation machinery and initiation of protein synthesis. mRNA recognition is thought to require direct interactions between eukaryotic initiation factor 4E (eIF4E) and the mRNA cap. However, translation of numerous capped mRNAs remains robust during cellular stress, early development, and cell cycle progression1 despite inactivation of eIF4E. Here we describe a cap-dependent pathway of translation initiation in human cells that relies on a previously unknown cap-binding activity of eIF3d, a subunit of the 800-kilodalton eIF3 complex. A 1.4 Å crystal structure of the eIF3d cap-binding domain reveals unexpected homology to endonucleases involved in RNA turnover, and allows modelling of cap recognition by eIF3d. eIF3d makes specific contacts with the cap, as exemplified by cap analogue competition, and these interactions are essential for assembly of translation initiation complexes on eIF3-specialized mRNAs2 such as the cell proliferation regulator c-Jun (also known as JUN). The c-Jun mRNA further encodes an inhibitory RNA element that blocks eIF4E recruitment, thus enforcing alternative cap recognition by eIF3d. Our results reveal a mechanism of cap-dependent translation that is independent of eIF4E, and illustrate how modular RNA elements work together to direct specialized forms of translation initiation.

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Figure 1: 5′ end recognition of c-Jun mRNA is eIF4F-independent.
Figure 2: Structure of eIF3d reveals a conserved cap-binding domain.
Figure 3: eIF3d cap-binding activity is required for efficient 48S initiation complex formation on specific mRNAs.
Figure 4: An RNA element inhibits eIF4F recruitment and directs mRNAs to use an eIF3-specialized translation pathway.

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Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported crystal structures are deposited at the Protein Data Bank (PDB) under accession codes 5K4B (crystal form 1), 5K4C (crystal form 2), and 5K4D (crystal form 3).

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Acknowledgements

The authors thank J. Berger and K. Chat for discussions. X-ray data were collected at Beamline 8.3.1 of the Lawrence Berkeley National Laboratory Advanced Light Source (ALS), supported in part by the UC Office of the President, Multicampus Research Programs and Initiatives grant MR-15-328599 and the Program for Breakthrough Biomedical Research, which is partially funded by the Sandler Foundation. The authors are grateful to J. Holton, G. Meigs (ALS), and T. Doukov (SSRL) for help with S-SAD data collection. This work used the Vincent J. Proteomics/Mass Spectrometry Laboratory at UC Berkeley, supported in part by NIH S10 Instrumentation Grant S10RR025622. This work was funded by the NIGMS Center for RNA Systems Biology (P50-GM201706). J.A.D. is an HHMI Investigator. A.S.Y.L. is supported as an American Cancer Society Postdoctoral Fellow (PF-14-108-01-RMC) and P.J.K. is supported as an HHMI Fellow of the Life Sciences Research Foundation.

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Author notes

  1. Amy S. Y. Lee & Philip J. Kranzusch

    Present address: † Present addresses: Department of Biology, Brandeis University, Waltham, Massachusetts 02454, USA (A.S.Y.L.); Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA (P.J.K.); Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts 02115, USA (P.J.K.).,

Authors and Affiliations

  1. Department of Molecular & Cell Biology, University of California, Berkeley, Berkeley, 94720, California, USA

    Amy S. Y. Lee, Philip J. Kranzusch, Jennifer A. Doudna & Jamie H. D. Cate

  2. Center for RNA Systems Biology, University of California, Berkeley, Berkeley, 94720, California, USA

    Amy S. Y. Lee, Philip J. Kranzusch, Jennifer A. Doudna & Jamie H. D. Cate

  3. Howard Hughes Medical Institute (HHMI), University of California, Berkeley, Berkeley, 94720, California, USA

    Philip J. Kranzusch & Jennifer A. Doudna

  4. Department of Chemistry, University of California, Berkeley, Berkeley, 94720, California, USA

    Jennifer A. Doudna & Jamie H. D. Cate

  5. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, 94720, California, USA

    Jennifer A. Doudna & Jamie H. D. Cate

  6. Innovative Genomics Initiative, University of California, Berkeley, 94720, California, USA

    Jennifer A. Doudna

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Contributions

Project and experiments were conceived and designed by A.S.Y.L. with consultation with J.H.D.C. Protein purification, biochemistry, and cell biology experiments and analyses were performed by A.S.Y.L. Crystallography and structure determination was performed by P.J.K. The manuscript was written by A.S.Y.L., P.J.K. and J.H.D.C, with editing by J.A.D.

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Correspondence to Jamie H. D. Cate.

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The authors declare no competing financial interests.

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Reviewer Information

Nature thanks I. Topisirovic and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 c-Jun expression is unaffected by 4E-BP1 activation.

Representative western blot of 293T cells after 24 h treatment with mTOR inhibitor INK128. The results are representative of three independent experiments. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 2 Mapping of a C-terminal region of eIF3d that protects the c-Jun 5′ cap structure.

a, Validation of eIF3d subunit identification. eIF3d-cap crosslinking was validated by immunoprecipitation of eIF3d after crosslinking and denaturing the eIF3 complex by boiling in SDS. The result is representative of biological replicates. b, Limited proteolysis of eIF3 crosslinked to 32P-cap-labelled c-Jun 5′ UTR RNA. Full-length and proteolysis fragments of eIF3d are indicated by black and maroon arrows, respectively, on the phosphorimage and Coomassie-stained SDS gels. c, Mass spectrometry identification of trypsinized peptides from limited proteolysis of cap-crosslinked eIF3d. Identified peptides are highlighted in blue. The results in b and c are representative of three independent experiments.

Extended Data Figure 3 Purification of eIF3d cap-binding domain.

a, Alignment is coloured by phylogenetic conservation of amino acid physiochemical property similarity and a cartoon schematic of the eIF3d secondary structure is depicted below the sequences. Colouring begins at 30% conservation (lightest blue). b, Coomassie-blue-stained SDS gel of recombinant N. vitripennis eIF3d S172–F537 protein expressed in E. coli.

Extended Data Figure 4 Structure-based alignment of eIF3d cap-binding domain and DXO cap-endonuclease domain sequences.

Structure-based alignment of eIF3d and DXO sequences according to superposition of eIF3d and DXO (PDB 4J7L) structures15. Alignment is coloured by phylogenetic conservation as in Extended Data Fig. 2, and cartoon schematics of the secondary structures are depicted below the sequences. eIF3d is coloured in blue and magenta as in Fig. 2, and DXO is coloured in green and magenta.

Extended Data Figure 5 Structural details of eIF3d ‘RNA gate’ stabilizing interactions.

a–c, Structural overview of eIF3d with cut-away sections highlighting charged interactions stabilizing the closed ‘RNA gate’ conformation. No significant van der Waals interactions stabilize the closed gate conformation, supporting likely repositioning of the RNA gate before 5′ mRNA cap recognition. Charged interactions occur in three areas: a, at the beginning of the gate insertion sequence (gate beginning); b, at the tip of the unstructured loop (gate tip); and c, at an ‘arginine anchor’ point stabilizing the return of the loop insertion sequence to the α-helix shared with DXO family endonucleases. Residues are numbered according to the human eIF3d sequence, and all positions are conserved between human and N. vitripennis except S292N. eIF3d RNA gate residues are displayed with blue side chains and the residues making stabilizing contacts are coloured in green. 2Fo − Fc map regions are shown at 1.5σ.

Extended Data Figure 6 Packing interactions observed in alternative eIF3d crystal forms.

Cartoon representation of crystallographic packing in eIF3d crystal form 1, 2 and 3 (a, b and c). Crystal forms 1 and 3 have two copies of eIF3d in the asymmetric unit coloured in blue/magenta and green/magenta, respectively; crystal form 2 has only one copy of eIF3d per asymmetric unit. Symmetry-related molecules are depicted in grey. Cut-away zoom illustrates position of the eIF3d RNA gate (red) relative to the nearest symmetry-related molecule. In crystal form 1, the RNA gate is packed against a neighbouring symmetry molecule, but in crystal forms 2 and 3, the RNA gate is positioned towards a major solvent channel. Relative conformation of the RNA gate remains unchanged in either eIF3d crystal form.

Extended Data Figure 7 eIF3d cap-binding activity requires the eIF3-recruitment stem–loop RNA.

Phosphorimage of SDS gel resolving RNase-protected 32P-cap-labelled c-Jun stem–loop RNA crosslinked to eIF3 subunits. The result is representative of three independent experiments.

Extended Data Figure 8 Incorporation of HA epitope-tagged eIF3d into translation initiation complexes.

a, Coomassie-blue-stained SDS gel of recombinant eIF3 containing wild-type or helix α5- or α11-mutated eIF3d. b, Representative native agarose gel electrophoresis of recombinant wild-type and mutant eIF3 complexes bound to the c-Jun stem–loop. c, Polysome profiles of untransfected 293T cells, plotted as relative absorbance at 254 nm versus elution fractions. d, Western blot analysis of eIF3d and the small (rpS19) and large (rpLP0) ribosomal subunits. The results in b–d are representative of three independent experiments. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 9 eIF4E recognizes the 5′ end of the c-Jun mRNA less efficiently than ACTB mRNA.

a, Coomassie-blue-stained SDS gel of recombinant human eIF4E expressed in E. coli. b, Phosphorimage of SDS gel resolving RNase-protected 32P-cap-labelled ACTB or c-Jun 5′ UTR RNA crosslinked to eIF4E. The result is representative of three independent experiments. For gel source data, see Supplementary Fig. 1.

Extended Data Table 1 Summary of data collection, phasing and refinement statistics
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Lee, A., Kranzusch, P., Doudna, J. et al. eIF3d is an mRNA cap-binding protein that is required for specialized translation initiation. Nature 536, 96–99 (2016). https://doi.org/10.1038/nature18954

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