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Quantifying the spatiotemporal dynamics of IRES versus Cap translation with single-molecule resolution in living cells

Amanda Koch, Luis Aguilera, View ORCID ProfileTatsuya Morisaki, Brian Munsky, View ORCID ProfileTimothy J. Stasevich
doi: https://doi.org/10.1101/2020.01.09.900829
Amanda Koch
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA
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Luis Aguilera
Keck Scholars, Department of Chemical and Biological Engineering and School of Biomedical Engineering, Colorado State University, Fort Collins, CO 80523, USA
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Tatsuya Morisaki
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA
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Brian Munsky
Keck Scholars, Department of Chemical and Biological Engineering and School of Biomedical Engineering, Colorado State University, Fort Collins, CO 80523, USA
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  • For correspondence: Brian.Munsky@colostate.edu Tim.Stasevich@colostate.edu
Timothy J. Stasevich
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USAWorld Research Hub Initiative, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Kanagawa 226-8503, Japan
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  • ORCID record for Timothy J. Stasevich
  • For correspondence: Brian.Munsky@colostate.edu Tim.Stasevich@colostate.edu
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ABSTRACT

Viruses use IRES sequences within their RNA to hijack translation machinery and thereby rapidly replicate in host cells. While this process has been extensively studied in bulk assays, the dynamics of hijacking at the single-molecule level remain unexplored in living cells. To achieve this, we developed a bicistronic biosensor encoding complementary repeat epitopes in two ORFs, one translated in a Cap-dependent manner and the other translated in an IRES-mediated manner. Using a pair of complementary probes that bind the epitopes co-translationally, our biosensor lights up in different colors depending on which ORF is being translated. In combination with single-molecule tracking and computational modeling, we measured the relative kinetics of Cap versus IRES translation and show: (1) Two non-overlapping ORFs can be simultaneously translated within a single mRNA; (2) EMCV IRES-mediated translation sites recruit ribosomes less efficiently than Cap-dependent translation sites but are otherwise nearly indistinguishable, having similar mobilities, sizes, spatial distributions, and ribosomal initiation and elongation rates; (3) Both Cap-dependent and IRES-mediated ribosomes tend to stretch out translation sites; (4) Although the IRES recruits two to three times fewer ribosomes than the Cap in normal conditions, the balance shifts dramatically in favor of the IRES during oxidative and ER stresses that mimic viral infection; and (5) Translation of the IRES is enhanced by translation of the Cap, demonstrating upstream translation can positively impact the downstream translation of a non-overlapping ORF. With the ability to simultaneously quantify two distinct translation mechanisms in physiologically relevant live-cell environments, we anticipate bicistronic biosensors like the one we developed here will become powerful new tools to dissect both canonical and non-canonical translation dynamics with single-molecule precision.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license.
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Posted January 09, 2020.
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Quantifying the spatiotemporal dynamics of IRES versus Cap translation with single-molecule resolution in living cells
Amanda Koch, Luis Aguilera, Tatsuya Morisaki, Brian Munsky, Timothy J. Stasevich
bioRxiv 2020.01.09.900829; doi: https://doi.org/10.1101/2020.01.09.900829
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Quantifying the spatiotemporal dynamics of IRES versus Cap translation with single-molecule resolution in living cells
Amanda Koch, Luis Aguilera, Tatsuya Morisaki, Brian Munsky, Timothy J. Stasevich
bioRxiv 2020.01.09.900829; doi: https://doi.org/10.1101/2020.01.09.900829

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