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 language | English |
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Pages (from-to) | 1078-1089.e4 |
Journal | Molecular Cell |
Volume | 84 |
Issue number | 6 |
Early online date | 2 Feb 2024 |
DOIs | |
Publication status | Published - 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.
Funders | Funder number |
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European Union's Horizon 2020 Program | |
Medical Research Council , as part of United Kingdom Research and Innovation | MC_UP_1201/32 |
Medical Research Council, as part of United Kingdom Research and Innovation | |
National Institutes of Health | GM133598 |
National Institutes of Health | |
European Research Council | 724425 - BENDER |
European Research Council | |
Nederlandse Organisatie voor Wetenschappelijk Onderzoek | 212.152, 724.016.001 |
Nederlandse Organisatie voor Wetenschappelijk Onderzoek | |
Horizon 2020 |
Keywords
- cryoelectron tomography
- initiation
- polysome
- ribosome collision
- ribosome quality control
- translation regulation