Although messenger RNA (mRNA) translation is a fundamental biological process, it has never been imaged in real time in vivo with single-molecule precision. To achieve this, we developed nascent chain tracking (NCT), a technique that uses multi-epitope tags and antibody-based fluorescent probes to quantify protein synthesis dynamics at the single-mRNA level. NCT reveals an elongation rate of ~10 amino acids per second, with initiation occurring stochastically every ~30 seconds. Polysomes contain ~1 ribosome every 200 to 900 nucleotides and are globular rather than elongated in shape. By developing multicolor probes, we showed that most polysomes act independently; however, a small fraction (~5%) form complexes in which two distinct mRNAs can be translated simultaneously. The sensitivity and versatility of NCT make it a powerful new tool for quantifying mRNA translation kinetics.
Antibody derivatives, such as antibody fragments (Fab) and single-chain variable fragments (scFv), are now being used to image traditionally hard-to-see protein subpopulations, including nascent polypeptides being translated and post-translationally modified proteins. This has allowed researchers to directly image and quantify for the first time (1) translation initiation and elongation kinetics with single-transcript resolution and (2) the temporal ordering and kinetics of post-translational histone and RNA polymerase II modifications. Here we review these developments and discuss the strengths and weaknesses of live-cell imaging with antibody-based probes. Further development of these probes will increase their versatility and open up new avenues of research for dissecting complex gene regulatory dynamics.
Highlights d A multi-frame tag can monitor single-RNA translation in two open reading frames d A bursty model captures frameshift kinetics for the HIV-1 frameshift sequence (FSS) d Frameshifting persists on a subset of RNA and can be stimulated by an RNA oligo d Frameshifted ribosomes take longer to clear the FSS and cause ribosome traffic jams
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.
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