Quantifying the dynamics of IRES and cap translation with single-molecule resolution in live cells

Koch, Amanda; Aguilera, Luis U.; Morisaki, Tatsuya; Stasevich, Timothy J.

doi:10.1038/s41594-020-0504-7

Cited by 45 publications

(35 citation statements)

References 59 publications

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“…4B, C ). This range is consistent with the range we recently measured using a different reporter without BoxB stem loops 23 . These data therefore demonstrate Ago2 tethering does not impede translation elongation compared to the β-gal control.…”

Section: Resultssupporting

confidence: 92%

Imaging translational control by Argonaute with single-molecule resolution in live cells

et al. 2022

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A major challenge to our understanding of translational control has been deconvolving the individual impact specific regulatory factors have on the complex dynamics of mRNA translation. MicroRNAs (miRNAs), for example, guide Argonaute and associated proteins to target mRNAs, where they direct gene silencing in multiple ways that are not well understood. To better deconvolve these dynamics, we have developed technology to directly visualize and quantify the impact of human Argonaute2 (Ago2) on the translation and subcellular localization of individual reporter mRNAs in living cells. We show that our combined translation and Ago2 tethering sensor reflects endogenous miRNA-mediated gene silencing. Using the sensor, we find that Ago2 association leads to progressive silencing of translation at individual mRNA. Silencing was occasionally interrupted by brief bursts of translational activity and took 3–4 times longer than a single round of translation, consistent with a gradual increase in the inhibition of translation initiation. At later time points, Ago2-tethered mRNAs cluster and coalesce with P-bodies, where a translationally silent state is maintained. These results provide a framework for exploring miRNA-mediated gene regulation in live cells at the single-molecule level. Furthermore, our tethering-based, single-molecule reporter system will likely have wide-ranging application in studying RNA-protein interactions.

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Section: Resultssupporting

confidence: 92%

Imaging translational control by Argonaute with single-molecule resolution in live cells

et al. 2022

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“…The interaction between eIF4G and PABPC is well characterized and provides a physical link connecting both ends of the mRNA, and some studies are able to catch a glimpse of possible circular structures of mRNAs in fixed tissues ( Christensen et al, 1987 ) or in vitro ( Wells et al, 1998 ). However, recent single-molecule imaging studies in mammalian cells question the widespread existence of mRNA closed-loop structures ( Adivarahan et al, 2018 ; Koch et al, 2020 ). Our finding that depletion of PABPC had minimal impact on TE in mammalian cell lines supports a model in which pervasive eIF4G–PABPC-associated looping of mRNAs is generally lacking in uncoupled systems.…”

Section: Discussionmentioning

confidence: 99%

The molecular basis of coupling between poly(A)-tail length and translational efficiency

Xiang

Bartel

2021

eLife

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In animal oocytes and early embryos, mRNA poly(A)-tail length strongly influences translational efficiency (TE), but later in development this coupling between tail length and TE disappears. Here, we elucidate how this coupling is first established and why it disappears. Overexpressing cytoplasmic poly(A)-binding protein (PABPC) in Xenopus oocytes specifically improved translation of short-tailed mRNAs, thereby diminishing coupling between tail length and TE. Thus, strong coupling requires limiting PABPC, implying that in coupled systems longer-tail mRNAs better compete for limiting PABPC. In addition to expressing excess PABPC, post-embryonic mammalian cell lines had two other properties that prevented strong coupling: terminal-uridylation-dependent destabilization of mRNAs lacking bound PABPC, and a regulatory regime wherein PABPC contributes minimally to TE. Thus, these results revealed three fundamental mechanistic requirements for coupling and defined the context-dependent functions for PABPC, which promotes TE but not mRNA stability in coupled systems and mRNA stability but not TE in uncoupled systems.

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“…[ 91 ] Similarly, recent single‐molecule experiments revealed that translation of a dORF that was initiated by an IRES tended to enhance cap‐dependent initiation at a further upstream ORF, presumably due to changing the structure of the mRNP under translation. [ 92 ]…”

Section: Reinitiation and Regulation Of Gene Expression By Uorfs And ...mentioning

confidence: 99%

“…[91] Similarly, recent single-molecule experiments revealed that translation of a dORF that was initiated by an IRES tended to enhance cap-dependent initiation at a further upstream ORF, presumably due to changing the structure of the mRNP under translation. [92] Since uORFs can control translation of coding sequences due to the tendency for reinitiation to occur after their translation terminates [93] ,…”

Section: Reinitiation and Regulation Of Gene Expression By Uorfs And ...mentioning

confidence: 99%

Rebirth of the translational machinery: The importance of recycling ribosomes

Young

Guydosh

2022

BioEssays

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Translation of the genetic code occurs in a cycle where ribosomes engage mRNAs, synthesize protein, and then disengage in order to repeat the process again. The final part of this process-ribosome recycling, where ribosomes dissociate from mRNAsinvolves a complex molecular choreography of specific protein factors to remove the large and small subunits of the ribosome in a coordinated fashion. Errors in this process can lead to the accumulation of ribosomes at stop codons or translation of downstream open reading frames (ORFs). Ribosome recycling is also critical when a ribosome stalls during the elongation phase of translation and must be rescued to allow continued translation of the mRNA. Here we discuss the molecular interactions that drive ribosome recycling, and their regulation in the cell. We also examine the consequences of inefficient recycling with regards to disease, and its functional roles in synthesis of novel peptides, regulation of gene expression, and control of mRNA-associated proteins. Alterations in ribosome recycling efficiency have the potential to impact many cellular functions but additional work is needed to understand how this regulatory power is utilized.

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Quantifying the dynamics of IRES and cap translation with single-molecule resolution in live cells

Cited by 45 publications

References 59 publications

Imaging translational control by Argonaute with single-molecule resolution in live cells

Imaging translational control by Argonaute with single-molecule resolution in live cells

The molecular basis of coupling between poly(A)-tail length and translational efficiency

Rebirth of the translational machinery: The importance of recycling ribosomes

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