Live-cell single RNA imaging reveals bursts of translational frameshifting

Abstract

Ribosomal frameshifting during the translation of RNA is implicated in both human disease and viral infection. While previous work has uncovered many mechanistic details about single RNA frameshifting kinetics in vitro, very 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 in living cells. Frameshifting RNA are preferentially in multi-RNA "translation factories," are translated at about the same All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/478040 doi: bioRxiv preprint 2 rate as non-frameshifting RNA (~2 aa/sec), 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 novel insight into retroviral frameshifting and could lead to new strategies to perturb the process in living cells.All rights reserved. No reuse allowed without permission.

Multi-Color Single-Molecule Imaging Uncovers Extensive Heterogeneity in mRNA Decoding

“…In this study, we show that multi-color translation imaging can be used to assess translation of the 3′ UTR, translation start site selection, and the dynamics of uORFs translation. A parallel study independently developed a multi-color translation reading frame imaging approach to visualize the kinetics of ribosome frameshifting on a viral RNA sequence (Lyon et al., 2018). Although the two studies investigate different biological processes, both studies uncover a high degree of translation heterogeneity among different mRNA molecules, and it is possible that widespread translational heterogeneity may be the norm rather than an exception for most aspects of mRNA translation.…”

High-throughput interrogation of programmed ribosomal frameshifting in human cells

“…Our approach aims at a systematic and comparative assessment of the commonalities and peculiarities in the regulation of frameshifting efficiency across PRF events. It is complementary to in-depth analyses of individual frameshifting sites yielding detailed structural or kinetic insights [69][70][71][72][73][74] . The high-throughput nature of our approach entails particular limitations.…”

Detection and quantification of single mRNA dynamics with the Riboglow fluorescent RNA tag

“…Translation of single mRNAs can be monitored by following the nascent peptide chain via a loaded affinity probe 22 or the SunTag [23][24][25] ; these studies from different labs yielded comparable protein translation kinetics (see ref 26 for a comparison). Other aspects of the mRNA life cycle, such as interactions with stress granules and P bodies 27 , association with the endoplasmic reticulum 28 , mRNA frame shifting 29 and nonsense-mediated mRNA decay 30 , were also characterized using a combination of MS2-based mRNA labeling and complementary fluorescent probes on the single molecule level. Flexibility of mRNA tagging with different fluorophores and labeling systems will be critical for combining different fluorescent probes and labeling systems in the future.…”