Translation dynamics of single mRNAs in live cells and neurons

Wu, Bin; Eliscovich, Carolina; Yoon, Yong‐Dal; Singer, Robert H.

doi:10.1126/science.aaf1084

Cited by 465 publications

(605 citation statements)

References 41 publications

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“…Using the CrPV IRES, we measured the speed of eukaryotic ribosomes and were able to identify two characteristic times: a very long time (40 sec) that corresponds to the first (or the first plus the sec) translocation step, and a short time (1.4 sec) for all remaining translocations. This translocation time is in good agreement with recent in vivo experiments that indicated a ribosome translocation rate around 3 ± 1 amino acids per second (Morisaki et al 2016;Wang et al 2016;Wu et al 2016;Yan et al 2016).…”

Section: Discussionsupporting

confidence: 79%

“…This value is slightly slower than the one obtained by the ribosome profiling approach (0.2 sec per codon) (Ingolia et al 2012). However, with nonpurified ribosomes, the translation rate increases threefold (0.5 ± 0.2 sec per codon), which is in good agreement with the kinetics already obtained in vivo (Morisaki et al 2016;Wang et al 2016;Wu et al 2016;Yan et al 2016). Although the kinetics of initiation has recently been addressed in a heterogeneous cell-free system , the consequences of the IRES-dependent initiation for the elongation cycles have never been evaluated in an unmodified translation system.…”

Section: Discussionsupporting

confidence: 64%

“…Although several clever approaches have been developed to study eukaryotic translation by single-molecule FRET (Ferguson et al 2015), all of them rely on modifying either the ribosome itself or translational factors like tRNA to attach a fluorescent dye. An approach with unmodified translational components is of great interest to analyze eukaryotic translation dynamics, as shown by the recent in vivo quantification of translational speed (Morisaki et al 2016;Murray et al 2016;Wang et al 2016;Wu et al 2016).…”

Section: Introductionmentioning

confidence: 99%

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Kinetics of CrPV and HCV IRES-mediated eukaryotic translation using single-molecule fluorescence microscopy

Bugaud

Barbier

Chommy

et al. 2017

RNA

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Protein synthesis is a complex multistep process involving many factors that need to interact in a coordinated manner to properly translate the messenger RNA. As translating ribosomes cannot be synchronized over many elongation cycles, single-molecule studies have been introduced to bring a deeper understanding of prokaryotic translation dynamics. Extending this approach to eukaryotic translation is very appealing, but initiation and specific labeling of the ribosomes are much more complicated. Here, we use a noncanonical translation initiation based on internal ribosome entry sites (IRES), and we monitor the passage of individual, unmodified mammalian ribosomes at specific fluorescent milestones along mRNA. We explore initiation by two types of IRES, the intergenic IRES of cricket paralysis virus (CrPV) and the hepatitis C (HCV) IRES, and show that they both strongly limit the rate of the first elongation steps compared to the following ones, suggesting that those first elongation cycles do not correspond to a canonical elongation. This new system opens the possibility of studying both IRES-mediated initiation and elongation kinetics of eukaryotic translation and will undoubtedly be a valuable tool to investigate the role of translation machinery modifications in human diseases.

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Section: Discussionsupporting

confidence: 79%

Section: Discussionsupporting

confidence: 64%

Section: Introductionmentioning

confidence: 99%

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Kinetics of CrPV and HCV IRES-mediated eukaryotic translation using single-molecule fluorescence microscopy

Bugaud

Barbier

Chommy

et al. 2017

RNA

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“…The estimated value is of the same order of magnitude and just slightly higher than those recently determined for untranslating polysomes [47]. The diffusion constant for H 2 O 2 [48] was determined for diffusion in buffer.…”

Section: Models and Methodssupporting

confidence: 47%

Localized redox relays as a privileged mode of cytoplasmic hydrogen peroxide signaling

Travasso

Aidos

Abranches

et al. 2017

Free Radical Biology and Medicine

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A B S T R A C THydrogen peroxide (H 2 O 2 ) is a key signaling agent. Its best characterized signaling actions in mammalian cells involve the early oxidation of thiols in cytoplasmic phosphatases, kinases and transcription factors. However, these redox targets are orders of magnitude less H 2 O 2 -reactive and abundant than cytoplasmic peroxiredoxins. How can they be oxidized in a signaling time frame? Here we investigate this question using computational reaction-diffusion models of H 2 O 2 signaling. The results show that at H 2 O 2 supply rates commensurate with mitogenic signaling a H 2 O 2 concentration gradient with a length scale of a few tenths of μm is established. Even near the supply sites H 2 O 2 concentrations are far too low to oxidize typical targets in an early mitogenic signaling time frame. Furthermore, any inhibition of the peroxiredoxin or increase in H 2 O 2 supply able to drastically increase the local H 2 O 2 concentration would collapse the concentration gradient and/or cause an extensive oxidation of the peroxiredoxins I and II, inconsistent with experimental observations. In turn, the local concentrations of peroxiredoxin sulfenate and disulfide forms exceed those of H 2 O 2 by several orders of magnitude. Redox targets reacting with these forms at rate constants much lower than that for, say, thioredoxin could be oxidized within seconds. Moreover, the spatial distribution of the concentrations of these peroxiredoxin forms allows them to reach targets within 1 μm from the H 2 O 2 sites while maintaining signaling localized. The recruitment of peroxiredoxins to specific sites such as caveolae can dramatically increase the local concentrations of the sulfenic and disulfide forms, thus further helping these species to outcompete H 2 O 2 for the oxidation of redox targets. Altogether, these results suggest that H 2 O 2 signaling is mediated by localized redox relays whereby peroxiredoxins are oxidized to sulfenate and disulfide forms at H 2 O 2 supply sites and these forms in turn oxidize the redox targets near these sites.

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“…However, methods to track translation at single molecule level in vivo have been lacking. On May 5th 2016, four independent groups [3][4][5][6] reported a set of novel tools to capture translational dynamics of individual mRNAs in live cells.…”

mentioning

confidence: 99%