Figure 1. Real-time detection of translation on single mRNAs. Pichon et al. (2016) developed the Ki67 reporter (A), which contains 56 SunTag repeats at the N terminus of the ORF and several MS2 motifs in the 3′ UTR. MCP-RFP fusion protein (red) will bind the 3′ UTR, allowing visualization of the mRNA (red foci). If the mRNA is translating (B), scFv-sfGFP fusion (green) will bind the SunTag epitopes as they emerge from the ribosome. These yellow foci mark translating polysomes. Free Ki67 protein (C, small green foci) still bound to scFv-sfGFP is fainter than the translation sites, as they represent only one peptide and do not colocalize with the mRNA signal. In separate experiments, incorporating the SunTag into the endogenous DYNC1H1 transcript (D) revealed the presence of single polysomes (not depicted) and blobs containing three to seven DYNC1H1 polysomes. Pichon et al. (2016) show proof of concept that translating and nontranslating mRNAs can be tracked simultaneously in vivo. They also show that the SunTag can be used to track endogenous mRNAs, such as DYNC1H1.
Figure 1.

Real-time detection of translation on single mRNAs. Pichon et al. (2016) developed the Ki67 reporter (A), which contains 56 SunTag repeats at the N terminus of the ORF and several MS2 motifs in the 3′ UTR. MCP-RFP fusion protein (red) will bind the 3′ UTR, allowing visualization of the mRNA (red foci). If the mRNA is translating (B), scFv-sfGFP fusion (green) will bind the SunTag epitopes as they emerge from the ribosome. These yellow foci mark translating polysomes. Free Ki67 protein (C, small green foci) still bound to scFv-sfGFP is fainter than the translation sites, as they represent only one peptide and do not colocalize with the mRNA signal. In separate experiments, incorporating the SunTag into the endogenous DYNC1H1 transcript (D) revealed the presence of single polysomes (not depicted) and blobs containing three to seven DYNC1H1 polysomes. Pichon et al. (2016) show proof of concept that translating and nontranslating mRNAs can be tracked simultaneously in vivo. They also show that the SunTag can be used to track endogenous mRNAs, such as DYNC1H1.

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