Live-Cell Single RNA Imaging Reveals Bursts of Translational Frameshifting

• Published in: Molecular cell Vol. 75; no. 1; pp. 172 - 183.e9
• Main Authors: Lyon, Kenneth, Aguilera, Luis U., Morisaki, Tatsuya, Munsky, Brian, Stasevich, Timothy J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 11.07.2019
• Subjects: Base Pairing; Cell Line, Tumor; computational modeling; fluorescence microscopy; frameshifting; Frameshifting, Ribosomal; HIV-1; HIV-1 - genetics; HIV-1 - metabolism; Humans; live-cell imaging; Models, Genetic; Nucleic Acid Conformation; Oligonucleotide Probes - chemical synthesis; Oligonucleotide Probes - genetics; Oligonucleotide Probes - metabolism; Oligopeptides - genetics; Oligopeptides - metabolism; Open Reading Frames; Osteoblasts - metabolism; Osteoblasts - virology; ribosomal traffic jams; RNA translation; RNA, Viral - chemistry; RNA, Viral - genetics; RNA, Viral - metabolism; Single Molecule Imaging - methods; single-molecule imaging; Staining and Labeling - methods; stochastic gene expression; translational heterogeneity; HIV-1; frameshifting; stochastic gene expression; single-molecule imaging; live-cell imaging; RNA translation; computational modeling; translational heterogeneity; fluorescence microscopy; ribosomal traffic jams
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2019.05.002

Ribosomal frameshifting during the translation of RNA is implicated in human disease and viral infection. While previous work has uncovered many details about single RNA frameshifting kinetics in vitro, little is known about how single RNA frameshift in living systems. To confront this problem, we have developed technology to quantify live-cell single RNA translation dynamics in frameshifted open reading frames. Applying this technology to RNA encoding the HIV-1 frameshift sequence reveals a small subset (∼8%) of the translating pool robustly frameshift. Frameshifting RNA are translated at similar rates as non-frameshifting RNA (∼3 aa/s) and can continuously frameshift for more than four rounds of translation. Fits to a bursty model of frameshifting constrain frameshifting kinetic rates and demonstrate how ribosomal traffic jams contribute to the persistence of the frameshifting state. These data provide insight into retroviral frameshifting and could lead to alternative strategies to perturb the process in living cells. [Display omitted] •A multi-frame tag can monitor single-RNA translation in two open reading frames•A bursty model captures frameshift kinetics for the HIV-1 frameshift sequence (FSS)•Frameshifting persists on a subset of RNA and can be stimulated by an RNA oligo•Frameshifted ribosomes take longer to clear the FSS and cause ribosome traffic jams A multi-frame tag and stochastic model visualize and quantify bursty frameshifting of single RNA encoding the HIV-1 frameshift sequence (FSS). Frameshifting involves elongation pauses that induce ribosomal traffic jams. Frameshifting occurs in slowly diffusing RNA clusters and can be stimulated by an RNA oligo encoding the FSS.