Real-time quantification of single RNA translation dynamics in living cells

Abstract

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.

Kinetics of CrPV and HCV IRES-mediated eukaryotic translation using single-molecule fluorescence microscopy

“…Using the CrPV IRES, we measured the speed of eukaryotic ribosomes and were able to identify two characteristic times: a very long time (40 sec) that corresponds to the first (or the first plus the sec) translocation step, and a short time (1.4 sec) for all remaining translocations. This translocation time is in good agreement with recent in vivo experiments that indicated a ribosome translocation rate around 3 ± 1 amino acids per second (Morisaki et al 2016;Wang et al 2016;Wu et al 2016;Yan et al 2016).…”

“…This value is slightly slower than the one obtained by the ribosome profiling approach (0.2 sec per codon) (Ingolia et al 2012). However, with nonpurified ribosomes, the translation rate increases threefold (0.5 ± 0.2 sec per codon), which is in good agreement with the kinetics already obtained in vivo (Morisaki et al 2016;Wang et al 2016;Wu et al 2016;Yan et al 2016). Although the kinetics of initiation has recently been addressed in a heterogeneous cell-free system , the consequences of the IRES-dependent initiation for the elongation cycles have never been evaluated in an unmodified translation system.…”

“…Although several clever approaches have been developed to study eukaryotic translation by single-molecule FRET (Ferguson et al 2015), all of them rely on modifying either the ribosome itself or translational factors like tRNA to attach a fluorescent dye. An approach with unmodified translational components is of great interest to analyze eukaryotic translation dynamics, as shown by the recent in vivo quantification of translational speed (Morisaki et al 2016;Murray et al 2016;Wang et al 2016;Wu et al 2016).…”

Tracking translation of single mRNA molecule in live cells

“…(1) Only a proportion of mRNAs are under active translation, whereas the rest ones are not translated. The percentage of actively translated mRNA varies from 4 % to 86 %, which is positively correlated with the size of transcript [3]. (2) Each individual mRNA can cycle between translating and nontranslating states at a time scale of hours, which suggests that mRNAs go through one or several translational shutdown and re-initiation in their lifetime [6].…”

“…However, methods to track translation at single molecule level in vivo have been lacking. On May 5th 2016, four independent groups [3][4][5][6] reported a set of novel tools to capture translational dynamics of individual mRNAs in live cells.…”