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Cited by 80RNA and protein synthesis mechanismsRNA modifications and cancerPI3K/AKT/mTOR signaling in cancer

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eIF5B and eIF1A reorient initiator tRNA to allow ribosomal subunit joining

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Lapointe, Christopher P., Grosely, Rosslyn, Sokabe, Masaaki, Alvarado, Carlos, Wang, Jinfan, Montabana, Elizabeth, Villa, Nancy, Shin, Byung-Sik, Dever, Thomas E., Fraser, Christopher S., Fernández, Israel S., Puglisi, Joseph D.·2022·Nature
DOI 10.1038/s41586-022-04858-zPMC PMC9728550

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Abstract

Translation initiation defines the identity and quantity of a synthesized protein. The process is dysregulated in many human diseases 1,2. A key commitment step is when the ribosomal subunits join at a translation start site on a messenger RNA to form a functional ribosome. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when the universally conserved eukaryotic initiation factors eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we visualized initiation complexes that contained both eIF1A and eIF5B using single-particle cryo-electron microscopy. The resulting structure revealed how eukaryote-specific contacts between the two proteins remodel the initiation complex to orient the initiator aminoacyl-tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during translation initiation in humans.

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  1. Tahmasebi, S., Khoutorsky, A., Mathews, M. B. & Sonenberg, N. Translation deregulation in human disease. Nat. Rev. Mol. Cell Biol. 19, 791–807 (2018). · DOI
  2. Robichaud, N., Sonenberg, N., Ruggero, D. & Schneider, R. J. Translational control in cancer. Cold Spring Harb. Perspect. Biol. 11, a032896 (2019). · DOI
  3. Pain, V. M. Initiation of protein synthesis in eukaryotic cells. Eur. J. Biochem. 236, 747–771 (1996). · DOI
  4. Merrick, W. C. & Pavitt, G. D. Protein synthesis initiation in eukaryotic cells. Cold Spring Harb. Perspect. Biol. 10, a033092 (2018). · DOI
  5. Hershey, J. W. B., Sonenberg, N. & Mathews, M. B. Principles of translational control. Cold Spring Harb. Perspect. Biol. 11, a032607 (2019). · DOI
  6. Aitken, C. E. & Lorsch, J. R. A mechanistic overview of translation initiation in eukaryotes. Nat. Struct. Mol. Biol. 19, 568–576 (2012). · DOI
  7. Sokabe, M. & Fraser, C. S. Toward a kinetic understanding of eukaryotic translation. Cold Spring Harb. Perspect. Biol. 11, a032706 (2019). · DOI
  8. Hinnebusch, A. G. The scanning mechanism of eukaryotic translation initiation. Annu. Rev. Biochem. 83, 779–812 (2014). · DOI · Paper page
  9. Hinnebusch, A. G. Structural Insights into the mechanism of scanning and start codon recognition in eukaryotic translation initiation. Trends Biochem. Sci. 42, 589–611 (2017). · DOI
  10. Hashem, Y. & Frank, J. The jigsaw puzzle of mRNA translation initiation in eukaryotes: a decade of structures unraveling the mechanics of the process. Annu. Rev. Biophys. 47, 125–151 (2018). · DOI
  11. Pestova, T. V. et al. The joining of ribosomal subunits in eukaryotes requires eIF5B. Nature 403, 332–335 (2000). · DOI
  12. Lee, J. H. et al. Initiation factor eIF5B catalyzes second GTP-dependent step in eukaryotic translation initiation. Proc. Natl Acad. Sci. USA 99, 16689–16694 (2002). · DOI
  13. Shin, B. S. et al. Uncoupling of initiation factor eIF5B/IF2 GTPase and translational activities by mutations that lower ribosome affinity. Cell 111, 1015–1025 (2002). · DOI
  14. Wang, J. et al. eIF5B gates the transition from translation initiation to elongation. Nature 573, 605–608 (2019). · DOI
  15. Fernández, I. S. et al. Molecular architecture of a eukaryotic translational initiation complex. Science 342, 1240585 (2013). · DOI
  16. Wang, J. et al. Structural basis for the transition from translation initiation to elongation by an 80S–eIF5B complex. Nat. Commun. 11, 5003 (2020). · DOI
  17. Yamamoto, H. et al. Structure of the mammalian 80S initiation complex with initiation factor 5B on HCV-IRES RNA. Nat. Struct. Mol. Biol. 21, 721–727 (2014). · DOI
  18. Choi, S. K. et al. Physical and functional interaction between the eukaryotic orthologs of prokaryotic translation initiation factors IF1 and IF2. Mol. Cell. Biol. 20, 7183–7191 (2000). · DOI
  19. Olsen, D. S. Domains of eIF1A that mediate binding to eIF2, eIF3 and eIF5B and promote ternary complex recruitment in vivo. EMBO J. 22, 193–204 (2003). · DOI
  20. Acker, M. G., Shin, B.-S., Dever, T. E. & Lorsch, J. R. Interaction between eukaryotic initiation factors 1A and 5B is required for efficient ribosomal subunit joining. J. Biol. Chem. 281, 8469–8475 (2006). · DOI
  21. Fringer, J. M., Acker, M. G., Fekete, C. A., Lorsch, J. R. & Dever, T. E. Coupled release of eukaryotic translation initiation factors 5B and 1A from 80S ribosomes following subunit joining. Mol. Cell. Biol. 27, 2384–2397 (2007). · DOI
  22. Pisareva, V. P. & Pisarev, A. V. eIF5 and eIF5B together stimulate 48S initiation complex formation during ribosomal scanning. Nucleic Acids Res. 42, 12052–12069 (2014). · DOI
  23. Chen, J. et al. High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence. Proc. Natl Acad. Sci. USA 111, 664–669 (2014). · DOI
  24. Lapointe, C. P. et al. Dynamic competition between SARS-CoV-2 NSP1 and mRNA on the human ribosome inhibits translation initiation. Proc. Natl Acad. Sci. USA 118, e2017715118 (2021). · DOI
  25. Sokabe, M. & Fraser, C. S. Human eukaryotic initiation factor 2 (eIF2)–GTP–Met–tRNAi ternary complex and eIF3 stabilize the 43 S preinitiation complex. J. Biol. Chem. 289, 31827–31836 (2014). · DOI
  26. Koch, A., Aguilera, L., Morisaki, T., Munsky, B. & Stasevich, T. J. Quantifying the dynamics of IRES and cap translation with single-molecule resolution in live cells. Nat. Struct. Mol. Biol. 27, 1095–1104 (2020). · DOI
  27. Acker, M. G. et al. Kinetic analysis of late steps of eukaryotic translation initiation. J. Mol. Biol. 385, 491–506 (2009). · DOI
  28. Huang, B. Y. & Fernández, I. S. Long-range interdomain communications in eIF5B regulate GTP hydrolysis and translation initiation. Proc. Natl Acad. Sci. USA 117, 1429–1437 (2020). · DOI
  29. Passmore, L. A. et al. The eukaryotic translation initiation factors eIF1 and eIF1A induce an open conformation of the 40S ribosome. Mol. Cell 26, 41–50 (2007). · DOI
  30. Hashem, Y. et al. Structure of the mammalian ribosomal 43S preinitiation complex bound to the scanning factor DHX29. Cell 153, 1108–1119 (2013). · DOI
  31. Lomakin, I. B. & Steitz, T. A. The initiation of mammalian protein synthesis and mRNA scanning mechanism. Nature 500, 307–311 (2013). · DOI
  32. Weisser, M., Voigts-Hoffmann, F., Rabl, J., Leibundgut, M. & Ban, N. The crystal structure of the eukaryotic 40S ribosomal subunit in complex with eIF1 and eIF1A. Nat. Struct. Mol. Biol. 20, 1015–1017 (2013). · DOI
  33. Hussain, T. et al. Structural changes enable start codon recognition by the eukaryotic translation initiation complex. Cell 159, 597–607 (2014). · DOI
  34. Des Georges, A. et al. Structure of mammalian eIF3 in the context of the 43S preinitiation complex. Nature 525, 491–495 (2015). · DOI
  35. Simonetti, A. et al. eIF3 peripheral subunits rearrangement after mRNA binding and start-codon recognition. Mol. Cell 63, 206–217 (2016). · DOI
  36. Llácer, J. L. et al. Translational initiation factor eIF5 replaces eIF1 on the 40S ribosomal subunit to promote start-codon recognition. eLife 7, e39273 (2018). · DOI
  37. Brito Querido, J. et al. Structure of a human 48S translational initiation complex. Science 369, 1220–1227 (2020). · DOI
  38. Simonetti, A., Guca, E., Bochler, A., Kuhn, L. & Hashem, Y. Structural insights into the mammalian late-stage initiation complexes. Cell Rep. 31, 107497–107497 (2020). · DOI
  39. Kratzat, H. et al. A structural inventory of native ribosomal ABCE1–43S pre-initiation complexes. EMBO J. 40, e105179 (2021). · DOI
  40. Nag, N. et al. eIF1A/eIF5B interaction network and its functions in translation initiation complex assembly and remodeling. Nucleic Acids Res. 44, 7441–7456 (2016) .
  41. Marintchev, A., Kolupaeva, V. G., Pestova, T. V. & Wagner, G. Mapping the binding interface between human eukaryotic initiation factors 1A and 5B: A new interaction between old partners. Proc. Natl Acad. Sci. USA 100, 1535–1540 (2003). · DOI
  42. Shao, S. et al. Decoding mammalian ribosome-mrna states by translational GTPase complexes. Cell 167, 1229–1240.e1215 (2016). · DOI
  43. Pisarev, A. V. et al. Specific functional interactions of nucleotides at key −3 and +4 positions flanking the initiation codon with components of the mammalian 48S translation initiation complex. Genes Dev. 20, 624–636 (2006). · DOI
  44. Suresh, S. et al. eIF5B drives integrated stress response-dependent translation of PD-L1 in lung cancer. Nat. Cancer 1, 533–545 (2020). · DOI
  45. Roll-Mecak, A., Cao, C., Dever, T. E. & Burley, S. K. X-ray structures of the universal translation initiation factor IF2/eIF5B. Cell 103, 781–792 (2000). · DOI
  46. Kuhle, B. & Ficner, R. eIF5B employs a novel domain release mechanism to catalyze ribosomal subunit joining. EMBO J. 33, 1177–1191 (2014). · DOI
  47. Zheng, A. et al. X-ray structures of eIF5B and the eIF5B–eIF1A complex: the conformational flexibility of eIF5B is restricted on the ribosome by interaction with eIF1A. Acta Crystallogr. D 70, 3090–3098 (2014). · DOI
  48. Kaledhonkar, S. et al. Late steps in bacterial translation initiation visualized using time-resolved cryo-EM. Nature 570, 400–404 (2019). · DOI
  49. Marshall, R. A., Aitken, C. E. & Puglisi, J. D. GTP hydrolysis by IF2 guides progression of the ribosome into elongation. Mol. Cell 35, 37–47 (2009). · DOI
  50. Ling, C. & Ermolenko, D. N. Initiation factor 2 stabilizes the ribosome in a semirotated conformation. Proc. Natl Acad. Sci. USA 112, 15874–15879 (2015). · DOI
  51. Sprink, T. et al. Structures of ribosome-bound initiation factor 2 reveal the mechanism of subunit association. Sci. Adv. 2, e1501502 (2016). · DOI
  52. Johnson, A. G. et al. RACK1 on and off the ribosome. RNA 25, 881–895 (2019). · DOI
  53. Yin, J., Lin, A. J., Golan, D. E. & Walsh, C. T. Site-specific protein labeling by Sfp phosphopantetheinyl transferase. Nat. Protoc. 1, 280–285 (2006). · DOI
  54. Fraser, C. S., Berry, K. E., Hershey, J. W. B. & Doudna, J. A. eIF3j is located in the decoding center of the human 40S ribosomal subunit. Mol. Cell 26, 811–819 (2007). · DOI
  55. Özeş, A. R., Feoktistova, K., Avanzino, B. C. & Fraser, C. S. Duplex unwinding and ATPase activities of the DEAD-box helicase eIF4A are coupled by eIF4G and eIF4B. J. Mol. Biol. 412, 674–687 (2011). · DOI
  56. Feoktistova, K., Tuvshintogs, E., Do, A. & Fraser, C. S. Human eIF4E promotes mRNA restructuring by stimulating eIF4A helicase activity. Proc. Natl Acad. Sci. USA 110, 13339–13344 (2013). · DOI
  57. Sokabe, M., Fraser, C. S. & Hershey, J. W. The human translation initiation multi-factor complex promotes methionyl-tRNAi binding to the 40S ribosomal subunit. Nucleic Acids Res. 40, 905–913 (2012). · DOI
  58. Damoc, E. et al. Structural characterization of the human eukaryotic initiation factor 3 protein complex by mass spectrometry. Mol. Cell. Proteomics 6, 1135–1146 (2007). · DOI
  59. Korlach, J. et al. Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures. Proc. Natl Acad. Sci. USA 105, 1176–1181 (2008). · DOI
  60. Aitken, C. E., Marshall, R. A. & Puglisi, J. D. An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys. J. 94, 1826–1835 (2008). · DOI
  61. Erickson, F. L. & Hannig, E. M. Ligand interactions with eukaryotic translation initiation factor 2: role of the gamma-subunit. EMBO J. 15, 6311–6320 (1996). · DOI
  62. Kapp, L. D. & Lorsch, J. R. GTP-dependent recognition of the methionine moiety on initiator tRNA by translation factor eIF2. J. Mol. Biol. 335, 923–936 (2004). · DOI
  63. Juette, M. F. et al. Single-molecule imaging of non-equilibrium molecular ensembles on the millisecond timescale. Nat. Methods 13, 341–344 (2016). · DOI
  64. Bronson, J. E., Fei, J., Hofman, J. M., Gonzalez, R. L. & Wiggins, C. H. Learning rates and states from biophysical time series: a Bayesian approach to model selection and single-molecule FRET data. Biophys. J. 97, 3196–3205 (2009). · DOI
  65. Lawson, M. R. et al. Mechanisms that ensure speed and fidelity in eukaryotic translation termination. Science 373, 876–882 (2021). · DOI
  66. Zivanov, J., Nakane, T. & Scheres, S. H. W. Estimation of high-order aberrations and anisotropic magnification from cryo-EM data sets in. IUCrJ 7, 253–267 (2020). · DOI
  67. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015). · DOI
  68. Casañal, A., Lohkamp, B. & Emsley, P. Current developments in Coot for macromolecular model building of electron cryo-microscopy and crystallographic data. Protein Sci. 29, 1069–1078 (2020). · DOI
  69. Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D 74, 531–544 (2018). · DOI
  70. Nicholls, R. A., Long, F. & Murshudov, G. N. Low-resolution refinement tools in REFMAC5. Acta Crystallogr. D 68, 404–417 (2012). · DOI
  71. Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018). · DOI · Paper page
  72. Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018). · DOI · Paper page

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Metadata

Authors
Lapointe, C. P., Grosely, R., Sokabe, M., Alvarado, C., Wang, J., Montabana, E., Villa, N., Shin, B.-S., Dever, T. E., Fraser, C. S., Fernández, I. S., & Puglisi, J. D.
Affiliations
  • Stanford Medicine (US)
  • University of California, Davis (US)
  • National Institutes of Health (US)
  • St. Jude Children's Research Hospital (US)
Corresponding author
  • Fernández, Israel S.
  • Puglisi, Joseph D.
ORCID
Venue
Nature
Publisher
Springer Science and Business Media LLC
Published
2022-06-22
Volume
607
Issue
7917
Pages
185-190
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
DOI
10.1038/s41586-022-04858-z
PMC
PMC9728550
OpenAlex
W4283259136
Cited by
80
References
1
Citations by year
2026: 16 · 2025: 21 · 2024: 21 · 2023: 16 · 2022: 6
FWCI
5.22
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
Funders
  • Damon Runyon Cancer Research Foundation (2321-18, DRG#2321-18)
  • Knut och Alice Wallenbergs Stiftelse (KAW 2015.0406, 2015.0406)
  • National Institutes of Health (GM011378, AG064690, AI047365)
  • Stanford Bio-X
  • National Institute on Aging (RF1AG064690, R01AG064690)
  • National Institute of General Medical Sciences (R01GM113078, R00GM144678, R01GM051266, K99GM144678, R01GM092927, R35GM145306)
  • SLAC National Accelerator Laboratory
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA modifications and cancer
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › PI3K/AKT/mTOR signaling in cancer
Keywords
translation initiation, eIF1A, eIF5B, initiator tRNA, initiation complex, eukaryotic initiation factors, ribosome assembly, single-molecule fluorescence, single-particle cryo-electron microscopy
MeSH terms
Single Molecule Imaging, Humans, RNA, Transfer, Met, Eukaryotic Initiation Factor-1, Cryoelectron Microscopy, Eukaryotic Initiation Factors, Ribosome Subunits

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