Visualization of single endogenous polysomes reveals the dynamics of translation in live human cells

Pichon, Xavier; Bastide, Amandine; Safieddine, Adham; Chouaib, Racha; Samacoïts, Aubin; Basyuk, Eugénia; Peter, Matthias; Mueller, Florian; Bertrand, Édouard

doi:10.1083/jcb.201605024

Cited by 172 publications

(286 citation statements)

References 48 publications

(62 reference statements)

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“…(a) Nascent peptide imaging techniques (SINAPS, NTC and others) [74-78] visualize translation in real time. RNA reporter construct includes a labeling tag for the nascent peptide (SunTag or FLAG) in the coding sequence and MS2 or PP7 stem-loops in the 3′-UTR for labeling the mRNA.…”

Section: Figurementioning

confidence: 99%

“…Innovative improvements in reagent design using rapid live fluorescence microscopy, made it possible to determine when and where single molecules of mRNAs translate [73-78]** (Figure 2a,b). It is now possible, using fluorescence fluctuation spectroscopy (FFS, Figure 2c), to ascertain that the association of a specific RBP (ZBP1) with a specific mRNA (β-actin) in living cells is anti-correlated with the assembly of ribosomes, hence validating its role in translational repression and its dissociation at the periphery where actin protein translation then occurs [19]**.…”

Section: Introductionmentioning

confidence: 99%

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RNP transport in cell biology: the long and winding road

Eliscovich

Singer

2017

Current Opinion in Cell Biology

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Regulation of gene expression is key determinant to cell structure and function. RNA localization, where specific mRNAs are transported to subcellular regions and then translated, is highly conserved in eukaryotes ranging from yeast to extremely specialized and polarized cells such as neurons. Messenger RNA and associated proteins (mRNP) move from the site of transcription in the nucleus to their final destination in the cytoplasm both passively through diffusion and actively via directed transport. Dysfunction of RNA localization, transport and translation machinery can lead to pathology. Single-molecule live-cell imaging techniques have revealed unique features of this journey with unprecedented resolution. In this review, we highlight key recent findings that have been made using these approaches and possible implications for spatial control of gene function.

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Section: Figurementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

RNP transport in cell biology: the long and winding road

Eliscovich

Singer

2017

Current Opinion in Cell Biology

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“…Antibody fragments ( Fab ) and single chain variable fragments ( scFv ) fit this criteria and have been successfully used for these purposes [17–21]. Both Fab and scFv can bind short unmodified or modified peptide epitopes with high specificity, like the full antibodies from which they are derived.…”

Section: Fab and Scfv: Useful Antibody-based Probes For Imaging Protementioning

confidence: 99%

“…Recently, five research groups have independently figured out how to address this problem by directly imaging and quantifying the translation process in living cells [17–21]. Remarkably, all solved the problem in essentially the same way: by using antibody-based probes that bind to repeat epitope tags.…”

Section: Probing Translational Gene Regulatory Dynamics In Living Celmentioning

confidence: 99%

Imaging Translational and Post-Translational Gene Regulatory Dynamics in Living Cells with Antibody-Based Probes

Lyon

Stasevich

2017

Trends in Genetics

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Antibody derivatives, such as antibody fragments (Fab) and single-chain variable fragments (scFv), are now being used to image traditionally hard-to-see protein subpopulations, including nascent polypeptides being translated and post-translationally modified proteins. This has allowed researchers to directly image and quantify for the first time (1) translation initiation and elongation kinetics with single-transcript resolution and (2) the temporal ordering and kinetics of post-translational histone and RNA polymerase II modifications. Here we review these developments and discuss the strengths and weaknesses of live-cell imaging with antibody-based probes. Further development of these probes will increase their versatility and open up new avenues of research for dissecting complex gene regulatory dynamics.

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“…If we could track endogenous mRNA while measuring its translational output in response to diverse signaling in live tissues, we would be able to uncover many interesting details regarding the dynamics of gene expression. Further improvements in these imaging techniques (Pichon et al, 2016) will certainly offer new insights into the complex regulation of translation at the single-molecule level in situ .…”

Section: Monitoring Translation From a Single Mrna In Live Neuronsmentioning

confidence: 99%

Imaging Single-mRNA Localization and Translation in Live Neurons

Lee¹,

Bae²,

Shim³

et al. 2016

Molecules and Cells

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Local protein synthesis mediates precise spatio-temporal regulation of gene expression for neuronal functions such as long-term plasticity, axon guidance and regeneration. To reveal the underlying mechanisms of local translation, it is crucial to understand mRNA transport, localization and translation in live neurons. Among various techniques for mRNA analysis, fluorescence microscopy has been widely used as the most direct method to study localization of mRNA. Live-cell imaging of single RNA molecules is particularly advantageous to dissect the highly heterogeneous and dynamic nature of messenger ribonucleoprotein (mRNP) complexes in neurons. Here, we review recent advances in the study of mRNA localization and translation in live neurons using novel techniques for single-RNA imaging.

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Visualization of single endogenous polysomes reveals the dynamics of translation in live human cells

Abstract: Pichon et al. describe a method to visualize translation of single endogenous mRNPs in live cells and provide evidence for specialized translation factories, as well as measurements of translation elongation rate, ribosome loading, and movements of single polysomes.

Cited by 172 publications

References 48 publications

RNP transport in cell biology: the long and winding road

RNP transport in cell biology: the long and winding road

Imaging Translational and Post-Translational Gene Regulatory Dynamics in Living Cells with Antibody-Based Probes

Imaging Single-mRNA Localization and Translation in Live Neurons

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