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

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.

[1]  J. Bujnicki,et al.  Structure of a multipartite protein-protein interaction domain in splicing factor prp8 and its link to retinitis pigmentosa. , 2007, Molecular cell.

[2]  Joachim Frank,et al.  Preparation of macromolecular complexes for cryo-electron microscopy , 2007, Nature Protocols.

[3]  Friedrich Förster,et al.  Near-atomic resolution structural model of the yeast 26S proteasome , 2012, Proceedings of the National Academy of Sciences.

[4]  Spectrin Domain of Eukaryotic Initiation Factor 3a Is the Docking Site for Formation of the a:b:i:g Subcomplex* , 2013, The Journal of Biological Chemistry.

[5]  M. Martin-Magniette,et al.  The RNA Helicases AtMTR4 and HEN2 Target Specific Subsets of Nuclear Transcripts for Degradation by the Nuclear Exosome in Arabidopsis thaliana , 2014, PLoS genetics.

[6]  U. Hassiepen,et al.  Crystal structure of the human COP9 signalosome , 2014, Nature.

[7]  Fabienne Beuron,et al.  Structural insights into the COP9 signalosome and its common architecture with the 26S proteasome lid and eIF3. , 2010, Structure.

[8]  K Schulten,et al.  VMD: visual molecular dynamics. , 1996, Journal of molecular graphics.

[9]  Christian R S Reis,et al.  The translation initiation complex eIF3 in trypanosomatids and other pathogenic excavates – identification of conserved and divergent features based on orthologue analysis , 2014, BMC Genomics.

[10]  J. Frank,et al.  Automated particle picking for low-contrast macromolecules in cryo-electron microscopy. , 2014, Journal of structural biology.

[11]  A. Tzakos,et al.  Structure of eIF3b RNA Recognition Motif and Its Interaction with eIF3j , 2007, Journal of Biological Chemistry.

[12]  J. Frank,et al.  High-resolution cryo-electron microscopy structure of the Trypanosoma brucei ribosome , 2013, Nature.

[13]  M. Stewart,et al.  Structural biology of the PCI-protein fold , 2012, Bioarchitecture.

[14]  Gabriel C. Lander,et al.  Complete subunit architecture of the proteasome regulatory particle , 2011, Nature.

[15]  Herbert Thiele,et al.  Bioinformatics Strategies in Life Sciences: from Data Processing and Data Warehousing to Biological Knowledge Extraction , 2022 .

[16]  Thomas D. Goddard,et al.  Quantitative analysis of cryo-EM density map segmentation by watershed and scale-space filtering, and fitting of structures by alignment to regions. , 2010, Journal of structural biology.

[17]  Leonardo G. Trabuco,et al.  Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics. , 2008, Structure.

[18]  M. Yusupov,et al.  Ribosomal position and contacts of mRNA in eukaryotic translation initiation complexes , 2008, The EMBO journal.

[19]  E. Nogales,et al.  Architecture of human translation initiation factor 3. , 2013, Structure.

[20]  J. Dubochet,et al.  Cryo-electron microscopy of vitrified specimens , 1988, Quarterly Reviews of Biophysics.

[21]  Ji-Chun Yang,et al.  Structural analysis of an eIF3 subcomplex reveals conserved interactions required for a stable and proper translation pre-initiation complex assembly , 2011, Nucleic acids research.

[22]  Nancy Y. Villa,et al.  Human Eukaryotic Initiation Factor 4G (eIF4G) Protein Binds to eIF3c, -d, and -e to Promote mRNA Recruitment to the Ribosome* , 2013, The Journal of Biological Chemistry.

[23]  A. Hinnebusch,et al.  The C-Terminal Region of Eukaryotic Translation Initiation Factor 3a (eIF3a) Promotes mRNA Recruitment, Scanning, and, Together with eIF3j and the eIF3b RNA Recognition Motif, Selection of AUG Start Codons , 2010, Molecular and Cellular Biology.

[24]  Alexander R Ivanov,et al.  PCI proteins eIF3e and eIF3m define distinct translation initiation factor 3 complexes , 2005, BMC Biology.

[25]  N. Sonenberg,et al.  Reconstitution reveals the functional core of mammalian eIF3 , 2007, The EMBO journal.

[26]  J. Frank,et al.  Direct localization of the tRNA--anticodon interaction site on the Escherichia coli 30 S ribosomal subunit by electron microscopy and computerized image averaging. , 1988, Journal of molecular biology.

[27]  L. Valášek,et al.  Small Ribosomal Protein RPS0 Stimulates Translation Initiation by Mediating 40S-Binding of eIF3 via Its Direct Contact with the eIF3a/TIF32 Subunit , 2012, PloS one.

[28]  Jordi Querol-Audí,et al.  Functional reconstitution of human eukaryotic translation initiation factor 3 (eIF3) , 2011, Proceedings of the National Academy of Sciences.

[29]  Daniel Barsky,et al.  Mass spectrometry reveals modularity and a complete subunit interaction map of the eukaryotic translation factor eIF3 , 2008, Proceedings of the National Academy of Sciences.

[30]  P. Neumann,et al.  Translation initiation factor eIF3b contains a nine-bladed β-propeller and interacts with the 40S ribosomal subunit. , 2014, Structure.

[31]  P. Chacón,et al.  Formation of an intricate helical bundle dictates the assembly of the 26S proteasome lid. , 2013, Structure.

[32]  Anchi Cheng,et al.  Automated molecular microscopy: the new Leginon system. , 2005, Journal of structural biology.

[33]  D. Boehringer,et al.  Structure of a Yeast 40S–eIF1–eIF1A–eIF3–eIF3j initiation complex , 2015, Nature Structural &Molecular Biology.

[34]  Huaiyu Mi,et al.  The InterPro protein families database: the classification resource after 15 years , 2014, Nucleic Acids Res..

[35]  Vidya Dhote,et al.  Roles of individual domains in the function of DHX29, an essential factor required for translation of structured mammalian mRNAs , 2012, Proceedings of the National Academy of Sciences.

[36]  Hemant D. Tagare,et al.  The Local Resolution of Cryo-EM Density Maps , 2013, Nature Methods.

[37]  L. Valášek,et al.  The Saccharomyces cerevisiae HCR1 Gene Encoding a Homologue of the p35 Subunit of Human Translation Initiation Factor 3 (eIF3) Is a High Copy Suppressor of a Temperature-sensitive Mutation in the Rpg1p Subunit of Yeast eIF3* , 1999, The Journal of Biological Chemistry.

[38]  R. Jackson,et al.  The mechanism of eukaryotic translation initiation and principles of its regulation , 2010, Nature Reviews Molecular Cell Biology.

[39]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[40]  N. Guex,et al.  SWISS‐MODEL and the Swiss‐Pdb Viewer: An environment for comparative protein modeling , 1997, Electrophoresis.

[41]  D. Agard,et al.  Electron counting and beam-induced motion correction enable near atomic resolution single particle cryoEM , 2013, Nature Methods.

[42]  Joshua E. Elias,et al.  Target-Decoy Search Strategy for Mass Spectrometry-Based Proteomics , 2010, Proteome Bioinformatics.

[43]  R. Aebersold,et al.  Molecular Architecture of the 40S⋅eIF1⋅eIF3 Translation Initiation Complex , 2014, Cell.

[44]  R. Henderson,et al.  High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy☆ , 2013, Ultramicroscopy.

[45]  M. Karplus,et al.  CHARMM: A program for macromolecular energy, minimization, and dynamics calculations , 1983 .

[46]  Aaron K. LeFebvre,et al.  Translation Initiation Factor eIF4G-1 Binds to eIF3 through the eIF3e Subunit* , 2006, Journal of Biological Chemistry.

[47]  P. Schleyer.  Encyclopedia of computational chemistry , 1998 .

[48]  J. Frank,et al.  Structure of the Mammalian Ribosomal 43S Preinitiation Complex Bound to the Scanning Factor DHX29 , 2013, Cell.

[49]  Narmada Thanki,et al.  CDD: NCBI's conserved domain database , 2014, Nucleic Acids Res..

[50]  Daniel N. Wilson,et al.  Structures of the human and Drosophila 80S ribosome , 2013, Nature.

[51]  L. Valášek.  ‘Ribozoomin’ – Translation Initiation from the Perspective of the Ribosome-bound Eukaryotic Initiation Factors (eIFs) , 2012, Current protein & peptide science.

[52]  A Leith,et al.  SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. , 1996, Journal of structural biology.

[53]  J. Doudna,et al.  The j-Subunit of Human Translation Initiation Factor eIF3 Is Required for the Stable Binding of eIF3 and Its Subcomplexes to 40 S Ribosomal Subunits in Vitro* , 2004, Journal of Biological Chemistry.

[54]  Joachim Frank,et al.  Hepatitis-C-virus-like internal ribosome entry sites displace eIF3 to gain access to the 40S subunit , 2013, Nature.

[55]  Yingpu Yu,et al.  Common conformational changes induced in type 2 picornavirus IRESs by cognate trans-acting factors , 2011, Nucleic acids research.

[56]  Y. Mechulam,et al.  Structure of the ternary initiation complex aIF2–GDPNP–methionylated initiator tRNA , 2012, Nature Structural &Molecular Biology.

[57]  L. Valášek,et al.  The RNA Recognition Motif of Eukaryotic Translation Initiation Factor 3g (eIF3g) Is Required for Resumption of Scanning of Posttermination Ribosomes for Reinitiation on GCN4 and Together with eIF3i Stimulates Linear Scanning , 2010, Molecular and Cellular Biology.

[58]  Sjors H.W. Scheres,et al.  RELION: Implementation of a Bayesian approach to cryo-EM structure determination , 2012, Journal of structural biology.

[59]  Alan G Hinnebusch,et al.  eIF3: a versatile scaffold for translation initiation complexes. , 2006, Trends in biochemical sciences.

[60]  A. Komar,et al.  Translation Initiation on Mammalian mRNAs with Structured 5′UTRs Requires DExH-Box Protein DHX29 , 2008, Cell.

[61]  D. N. Perkins,et al.  Probability‐based protein identification by searching sequence databases using mass spectrometry data , 1999, Electrophoresis.

[62]  L. Valášek,et al.  Functional and Biochemical Characterization of Human Eukaryotic Translation Initiation Factor 3 in Living Cells , 2014, Molecular and Cellular Biology.

[63]  A. Hinnebusch,et al.  The yeast eIF3 subunits TIF32/a, NIP1/c, and eIF5 make critical connections with the 40S ribosome in vivo. , 2003, Genes & development.

[64]  G. Wagner,et al.  Translation initiation: structures, mechanisms and evolution , 2004, Quarterly Reviews of Biophysics.

[65]  R. Henderson,et al.  Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. , 2003, Journal of molecular biology.

[66]  L. Valášek,et al.  The indispensable N-terminal half of eIF3j/HCR1 co-operates with its structurally conserved binding partner eIF3b/PRT1-RRM and eIF1A in stringent AUG selection , 2010, Journal of molecular biology.

[67]  Zhongjun Cheng,et al.  Crystal Structure of Human eIF3k, the First Structure of eIF3 Subunits* , 2004, Journal of Biological Chemistry.