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Structural snapshots of actively translating human ribosomes.

ABSTRACT: Macromolecular machines, such as the ribosome, undergo large-scale conformational changes during their functional cycles. Although their mode of action is often compared to that of mechanical machines, a crucial difference is that, at the molecular dimension, thermodynamic effects dominate functional cycles, with proteins fluctuating stochastically between functional states defined by energetic minima on an energy landscape. Here, we have used cryo-electron microscopy to image ex-vivo-derived human polysomes as a source of actively translating ribosomes. Multiparticle refinement and 3D variability analysis allowed us to visualize a variety of native translation intermediates. Significantly populated states include not only elongation cycle intermediates in pre- and post-translocational states, but also eEF1A-containing decoding and termination/recycling complexes. Focusing on the post-translocational state, we extended this assessment to the single-residue level, uncovering striking details of ribosome-ligand interactions and identifying both static and functionally important dynamic elements.

SUBMITTER: Behrmann E

PROVIDER: S-EPMC4432480 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Structural snapshots of actively translating human ribosomes.

Behrmann Elmar E Loerke Justus J Budkevich Tatyana V TV Yamamoto Kaori K Schmidt Andrea A Penczek Pawel A PA Vos Matthijn R MR Bürger Jörg J Mielke Thorsten T Scheerer Patrick P Spahn Christian M T CM

Cell 20150501 4

Macromolecular machines, such as the ribosome, undergo large-scale conformational changes during their functional cycles. Although their mode of action is often compared to that of mechanical machines, a crucial difference is that, at the molecular dimension, thermodynamic effects dominate functional cycles, with proteins fluctuating stochastically between functional states defined by energetic minima on an energy landscape. Here, we have used cryo-electron microscopy to image ex-vivo-derived hu ...[more]

PMID: 25957688

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Project description:BackgroundEukaryotic Initiation Factor 5A (eIF-5A), an essential translation factor, is post-translationally activated by the polyamine spermidine. Two human genes encode eIF-5A, being eIF5-A1 constitutively expressed whereas eIF5-A2 is frequently found overexpressed in human tumours. The contribution of both isoforms with regard to cellular proliferation and invasion in non-small cell lung cancer remains to be characterized.MethodsWe have evaluated the use of eIF-5A2 gene as prognosis marker in lung adenocarcinoma (LUAD) patients and validated in immunocompromised mice. We have used cell migration and cell proliferation assays in LUAD lines after silencing each eIF-5A isoform to monitor their contribution to both phenotypes. Cytoskeleton alterations were analysed in the same cells by rhodamine-phalloidin staining and fluorescence microscopy. Polysome profiles were used to monitor the effect of eIF-5A2 overexpression on translation. Western blotting was used to study the levels of eIF-5A2 client proteins involved in migration upon TGFB1 stimulation. Finally, we have co-localized eIF-5A2 with puromycin to visualize the subcellular pattern of actively translating ribosomes.ResultsWe describe the differential functions of both eIF-5A isoforms, to show that eIF5-A2 properties on cell proliferation and migration are coincident with its features as a poor prognosis marker. Silencing of eIF-5A2 leads to more dramatic consequences of cellular proliferation and migration compared to eIF-5A1. Overexpression of eIF-5A2 leads to enhanced global translation. We also show that TGFβ signalling enhances the expression and activity of eIF-5A2 which promotes the translation of polyproline rich proteins involved in cytoskeleton and motility features as it is the case of Fibronectin, SNAI1, Ezrin and FHOD1. With the use of puromycin labelling we have co-localized active ribosomes with eIF-5A2 not only in cytosol but also in areas of cellular protrusion. We have shown the bulk invasive capacity of cells overexpressing eIF-5A2 in mice.ConclusionsWe propose the existence of a coordinated temporal and positional interaction between TFGB and eIF-5A2 pathways to promote cell migration in NSCLC. We suggest that the co-localization of actively translating ribosomes with hypusinated eIF-5A2 facilitates the translation of key proteins not only in the cytosol but also in areas of cellular protrusion. Video Abstract.

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Structural snapshots of actively translating human ribosomes.

Publications

Structural snapshots of actively translating human ribosomes.

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