Live-cell single RNA imaging reveals bursts of translational frameshifting

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

To directly address these sorts of questions, we have developed technology to visualize and quantify single RNA frameshifting dynamics in living cells. Using multi-frame repeat epitopes, complementary high-affinity fluorescent probes that selectively bind the epitopes, and multicolor single-molecule microscopy, we are able to simultaneously monitor the translation of single RNA into two unique nascent polypeptide chains encoded in shifted open reading frames. Application to the HIV-1 FSS uncovers unexpected heterogeneity in the production of frameshifted product and implicates a novel bursty frameshifting mechanism. Besides frameshifting, our technology can now be used to examine other translational regulatory dynamics, including upstream open reading frame selection, non-canonical initiation, and ribosomal shunting. We anticipate multi-frame nascent chain tracking will be a powerful new tool to dissect complex translational regulatory dynamics in living cells and organisms.