Live-cell single RNA imaging reveals bursts of translational frameshifting

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

As a first application of the MF tag, we focused on -1 programmed ribosomal frameshifting caused by the HIV-1 frameshift sequence (FSS). We inserted the FSS upstream of our MF tag and transiently transfected the resulting construct into U-2 OS cells. The FSS contains a slippery poly-U stretch nine nucleotides upstream of a stem loop. Two to ten hours post transfection, we observed cells with tens or hundreds of individual RNA diffusing throughout the nucleus and cytoplasm (Fig. 1B) . Nascent Chain To confirm these RNA were active translation sites, or polysomes, we performed two experiments. First, we re-imaged cells 12-24 hours after transfection. At these later time points, Fab and scFv began to accumulate in the cell membrane ( Fig. S1 and Movie this frame encoding a functional protein, as demonstrated by shifting the sequence by one nucleotide into the 0 frame ( Fig. S1 and Movie S2, right panels). Second, we treated cells with the translational inhibitor puromycin. Just minutes after treatment, we observed a dramatic decrease in the number of Fab-and/or scFv-labeled RNA, consistent with the premature release of nascent chains ( Fig. 1D and Movie S3). Together, these data provide strong evidence that we are able to detect single RNA frameshifting dynamics with the MF tag.