Visualization of translation reorganization upon persistent ribosome collision stress in mammalian cells

Juliette Fedry*, Joana Silva, Mihajlo Vanevic, Stanley Fronik, Yves Mechulam, Emmanuelle Schmitt, Amédée des Georges, William James Faller, Friedrich Förster

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Aberrantly slow ribosomes incur collisions, a sentinel of stress that triggers quality control, signaling, and translation attenuation. Although each collision response has been studied in isolation, the net consequences of their collective actions in reshaping translation in cells is poorly understood. Here, we apply cryoelectron tomography to visualize the translation machinery in mammalian cells during persistent collision stress. We find that polysomes are compressed, with up to 30% of ribosomes in helical polysomes or collided disomes, some of which are bound to the stress effector GCN1. The native collision interface extends beyond the in vitro-characterized 40S and includes the L1 stalk and eEF2, possibly contributing to translocation inhibition. The accumulation of unresolved tRNA-bound 80S and 60S and aberrant 40S configurations identifies potentially limiting steps in collision responses. Our work provides a global view of the translation machinery in response to persistent collisions and a framework for quantitative analysis of translation dynamics in situ.

Original languageEnglish
Pages (from-to)1078-1089.e4
JournalMolecular Cell
Volume84
Issue number6
Early online date2 Feb 2024
DOIs
Publication statusPublished - 21 Mar 2024

Bibliographical note

Publisher Copyright:
© 2024 MRC Laboratory of Molecular Biology

Funding

We thank Simon Bekker-Jensen for sharing U2OS WT and ZNF598 KO cell lines. We thank Sofia Ramalho, Joyce van Loenhout, and Anjani Parag for help with preliminary experiments. We are grateful to Stuart C. Howes and Menno Bergmeijer for cryo-EM support, as well as to Mariska Gröllers Mulderij for support with cell culture. We thank Jan Dreyer, Francesca Mattiroli, and the Hubrecht Institute FACS facility for help with FACS experiments. We thank Anne Bertolotti and Stefan Pfeffer for insightful discussions. Finally, we are particularly grateful to Ramanujan Hegde for enlightening exchanges and comments on our manuscript. This project benefitted from access to the Netherlands Centre for Electron Microscopy (NeCEN), with support from the operator Dr. W. Noteboorn. NeCEN access is part of the research program National Roadmap for Large-Scale Research Infrastructure 2017–2018 with project number 184.034.014, which is (partly) financed by the Dutch Research Council (NWO). The work was supported by the European Research Council under the European Union’s Horizon 2020 Program (ERC Consolidator grant agreement 724425 - BENDER), the Nederlandse Organisatie voor Wetenschappelijke Onderzoek ( Vici 724.016.001 to F.F. and Veni 212.152 to J.F.), and the Medical Research Council , as part of United Kingdom Research and Innovation ( MC_UP_1201/32 to J.F.). A.d.G was supported by the US National Institutes of Health (NIH) grant GM133598 . We thank Simon Bekker-Jensen for sharing U2OS WT and ZNF598 KO cell lines. We thank Sofia Ramalho, Joyce van Loenhout, and Anjani Parag for help with preliminary experiments. We are grateful to Stuart C. Howes and Menno Bergmeijer for cryo-EM support, as well as to Mariska Gröllers Mulderij for support with cell culture. We thank Jan Dreyer, Francesca Mattiroli, and the Hubrecht Institute FACS facility for help with FACS experiments. We thank Anne Bertolotti and Stefan Pfeffer for insightful discussions. Finally, we are particularly grateful to Ramanujan Hegde for enlightening exchanges and comments on our manuscript. This project benefitted from access to the Netherlands Centre for Electron Microscopy (NeCEN), with support from the operator Dr. W. Noteboorn. NeCEN access is part of the research program National Roadmap for Large-Scale Research Infrastructure 2017–2018 with project number 184.034.014, which is (partly) financed by the Dutch Research Council (NWO). The work was supported by the European Research Council under the European Union's Horizon 2020 Program (ERC Consolidator grant agreement 724425 - BENDER), the Nederlandse Organisatie voor Wetenschappelijke Onderzoek (Vici 724.016.001 to F.F. and Veni 212.152 to J.F.), and the Medical Research Council, as part of United Kingdom Research and Innovation (MC_UP_1201/32 to J.F.). A.d.G was supported by the US National Institutes of Health (NIH) grant GM133598. J.F. designed the project and performed the in situ cryo-ET data acquisition and processing; J.F. and M.V. performed nearest-neighbor analysis; J.S. performed the S35 protein synthesis experiment; S.F. performed preliminary experiments; J.F. M.V. J.S. Y.M. E.S. A.d.G. W.J.F. and F.F. analyzed the data; and J.F. wrote the manuscript with input from all authors. The authors declare no competing interests.

FundersFunder number
European Union's Horizon 2020 Program
Medical Research Council , as part of United Kingdom Research and InnovationMC_UP_1201/32
Medical Research Council, as part of United Kingdom Research and Innovation
National Institutes of HealthGM133598
National Institutes of Health
European Research Council724425 - BENDER
European Research Council
Nederlandse Organisatie voor Wetenschappelijk Onderzoek212.152, 724.016.001
Nederlandse Organisatie voor Wetenschappelijk Onderzoek
Horizon 2020

    Keywords

    • cryoelectron tomography
    • initiation
    • polysome
    • ribosome collision
    • ribosome quality control
    • translation regulation

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