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Multi-color single molecule imaging uncovers extensive heterogeneity in mRNA decoding

Sanne Boersma, Deepak Khuperkar, Bram M.P. Verhagen, Stijn Sonneveld, Jonathan B. Grimm, Luke D. Lavis, Marvin E. Tanenbaum
doi: https://doi.org/10.1101/477661
Sanne Boersma
1Oncode Institute, Hubrecht Institute–KNAW and University Medical Center Utrecht, Utrecht, the Netherlands
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Deepak Khuperkar
1Oncode Institute, Hubrecht Institute–KNAW and University Medical Center Utrecht, Utrecht, the Netherlands
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Bram M.P. Verhagen
1Oncode Institute, Hubrecht Institute–KNAW and University Medical Center Utrecht, Utrecht, the Netherlands
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Stijn Sonneveld
1Oncode Institute, Hubrecht Institute–KNAW and University Medical Center Utrecht, Utrecht, the Netherlands
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Jonathan B. Grimm
2Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA
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Luke D. Lavis
2Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA
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Marvin E. Tanenbaum
1Oncode Institute, Hubrecht Institute–KNAW and University Medical Center Utrecht, Utrecht, the Netherlands
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  • For correspondence: m.tanenbaum@hubrecht.eu
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Abstract

mRNA translation is a key step in decoding genetic information. Genetic decoding is surprisingly heterogeneous, as multiple distinct polypeptides can be synthesized from a single mRNA sequence. To study translational heterogeneity, we developed the MoonTag, a new fluorescence labeling system to visualize translation of single mRNAs. When combined with the orthogonal SunTag system, the MoonTag enables dual readouts of translation, greatly expanding the possibilities to interrogate complex translational heterogeneity. By placing MoonTag and SunTag sequences in different translation reading frames, each driven by distinct translation start sites, start site selection of individual ribosomes can be visualized in real-time. We find that start site selection is largely stochastic, but that the probability of using a particular start site differs among mRNA molecules, and can be dynamically regulated over time. Together, this study provides key insights into translation start site selection heterogeneity, and provides a powerful toolbox to visualize complex translation dynamics.

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Posted November 24, 2018.
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Multi-color single molecule imaging uncovers extensive heterogeneity in mRNA decoding
Sanne Boersma, Deepak Khuperkar, Bram M.P. Verhagen, Stijn Sonneveld, Jonathan B. Grimm, Luke D. Lavis, Marvin E. Tanenbaum
bioRxiv 477661; doi: https://doi.org/10.1101/477661
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Multi-color single molecule imaging uncovers extensive heterogeneity in mRNA decoding
Sanne Boersma, Deepak Khuperkar, Bram M.P. Verhagen, Stijn Sonneveld, Jonathan B. Grimm, Luke D. Lavis, Marvin E. Tanenbaum
bioRxiv 477661; doi: https://doi.org/10.1101/477661

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