Live-cell single RNA imaging reveals bursts of translational frameshifting

Kenneth Lyon; Luis U. Aguilera; Tatsuya Morisaki; Brian Munsky; Timothy J. Stasevich

A multi-frame tag to monitor single RNA translation in two reading frames simultaneously We created a multi-frame (MF) tag to monitor, in living cells, the translation of single RNAs with overlapping open reading frames (ORFs). The tag builds off earlier technology to visualize translation using repeat FLAG or SunTag epitopes labeled by fluorescent Fab or scFv, respectively (Lyon and Stasevich, 2017; Morisaki and Stasevich, 2018) . In the MF tag, FLAG epitopes in the 0 frame are separated from one another by SunTag epitopes in the -1 frame. With this arrangement, single RNAs with ribosomes translating the 0 frame will produce FLAG epitopes labeled by Fab, while those with ribosomes translating the -1 frame will produce SunTag epitopes labeled by scFv. Thus, depending on the chosen frame(s), polysomes will appear all green (all ribosomes translating the 0 frame), all blue (all ribosomes translating the -1 frame), or some combination of the two (Fig. 1A) .