TY - JOUR
T1 - Multi-Color Single-Molecule Imaging Uncovers Extensive Heterogeneity in mRNA Decoding
AU - Boersma, S.
AU - Khuperkar, Deepak
AU - Verhagen, Bram M.P.
AU - Sonneveld, S.
AU - Grimm, Jonathan B.
AU - Lavis, Luke D.
AU - Tanenbaum, Marvin E.
N1 - Funding Information:
We would like to thank the members of the Tanenbaum lab for helpful discussions. We would also like to thank Merlijn Staps for initial work on the computational pipeline, Ive Logister for help with the northern blot, and Tim Hoek for critical reading of the manuscript. This work was financially supported by an ERC starting grant (ERC-STG 677936-RNAREG), grants from the Netherlands Organization for Scientific Research (NWO) (ALWOP.290 and NWO/016.VIDI.189.005), and by the Howard Hughes Medical Institute through an international research scholar grant to M.E.T. (HHMI/IRS 55008747) and the Janelia Research Campus (to J.B.G. and L.D.L.). M.E.T. was also financially supported by the Oncode Institute. S.B. D.K. and M.E.T. conceived the project. J.B.G. and L.D.L. provided reagents. B.M.P.V. and S.S. developed and optimized the software. S.B. and D.K. performed all experiments. S.B. D.K. B.M.P.V. and M.E.T. analyzed the data. S.B. and D.K. prepared the figures, and M.E.T. wrote the manuscript with input from S.B. and D.K. The authors declare no competing interests.
Funding Information:
We would like to thank the members of the Tanenbaum lab for helpful discussions. We would also like to thank Merlijn Staps for initial work on the computational pipeline, Ive Logister for help with the northern blot, and Tim Hoek for critical reading of the manuscript. This work was financially supported by an ERC starting grant ( ERC-STG 677936-RNAREG ), grants from the Netherlands Organization for Scientific Research (NWO) ( ALWOP.290 and NWO/016.VIDI.189.005 ), and by the Howard Hughes Medical Institute through an international research scholar grant to M.E.T. ( HHMI/IRS 55008747 ) and the Janelia Research Campus (to J.B.G. and L.D.L.). M.E.T. was also financially supported by the Oncode Institute .
Publisher Copyright:
© 2019 The Author(s)
PY - 2019/7/11
Y1 - 2019/7/11
N2 - 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. The MoonTag system is a fluorescence labeling system for visualizing translation of single mRNA molecules in live cells. Combining the MoonTag system with the orthogonal SunTag system enables simultaneous measurements of translation of two open reading frames in an mRNA and reveals that ribosomes differentially decode individual mRNA molecules.
AB - 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. The MoonTag system is a fluorescence labeling system for visualizing translation of single mRNA molecules in live cells. Combining the MoonTag system with the orthogonal SunTag system enables simultaneous measurements of translation of two open reading frames in an mRNA and reveals that ribosomes differentially decode individual mRNA molecules.
UR - http://www.scopus.com/inward/record.url?scp=85068011103&partnerID=8YFLogxK
U2 - 10.1016/j.cell.2019.05.001
DO - 10.1016/j.cell.2019.05.001
M3 - Article
C2 - 31178119
AN - SCOPUS:85068011103
SN - 0092-8674
VL - 178
SP - 458-472.e19
JO - Cell
JF - Cell
IS - 2
ER -