Research Article

Real-time quantification of single RNA translation dynamics in living cells

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Science  17 Jun 2016:
Vol. 352, Issue 6292, pp. 1425-1429
DOI: 10.1126/science.aaf0899

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  • RE: Real-time quantification of single RNA translation dynamics in living cells

    The objective of this unique experiment by Morisaki et al. was to visualise and measure the real-time translation rate of single-molecules of mRNA in vivo. They combined dual-tag labelling of nascent peptide and mRNA, allowing calculation of ribosome elongation dwell time, and hence, translation rate. Furthermore, by cleverly labelling mRNAs encoding proteins with different (HA- and FLAG-) tags, the authors could use tri-colour imaging to investigate how different mRNAs and polysomes interacted.

    However, one potential oversight of the authors is in the method to construct the protein plasmids. Initially, the experiments were performed with the KDM5B protein construct. Following positive results, they were repeated with the shorter B-actin and H2B constructs, showing that the method worked for different sizes and classes of proteins. These other constructs gave similar results and showed that dwell time decreased with mRNA length. However, this is unsurprising given that PCR products were fused to the N-terminal of Beta-actin, followed by the Beta-actin zipcode and 24x-MS2 stem loops in the 3’-UTR, forming the tagged protein (SM-Beta-actin). Importantly, only the beta-actin coding region was then digested out to construct the SM-KDM5B and SM-H2B; all three proteins contained the same Beta-actin 3’-UTR. The 3’-UTR is critical for controlling translation, being a source of regulatory elements for mRNA stability, intracellular localisation and translation through RNA bi...

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    Competing Interests: None declared.

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