Multi-Color Single-Molecule Imaging Uncovers Extensive Heterogeneity in mRNA Decoding
Sanne Boersma3 Deepak Khuperkar3 Bram M.P. Verhagen Jonathan B. Grimm Luke D. Lavis Marvin E. Tanenbaum4
Published:June 06, 2019 DOI: https://doi.org/10.1016/j.cell.2019.05.001
Highlights
• Development of MoonTag, a fluorescence labeling system to visualize translation
• Development of MoonTag, a fluorescence labeling system to visualize translation
• Combining MoonTag and SunTag enables visualization of translational heterogeneity
• mRNAs from a single gene vary in initiation frequency at different start sites
• Ribosomes take many different “paths” along the 5′ UTR of a single mRNA molecule
Summary
mRNA translation is a key step in decoding genetic information. Genetic decoding is surprisingly heterogeneous because multiple distinct polypeptides can be synthesized from a single mRNA sequence. To study translational heterogeneity, we developed the MoonTag, a 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. 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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