Dynamics of Translation of Single mRNA Molecules In Vivo

Abstract

SUMMARY Regulation of mRNA translation, the process by which ribosomes decode mRNAs into polypeptides, is used to tune cellular protein levels. Currently, methods for observing the complete process of translation from single mRNAs in vivo are unavailable. Here, we report the long-term (>1 hr) imaging of single mRNAs undergoing hundreds of rounds of translation in live cells, enabling quantitative measurements of ribosome initiation, elongation, and stalling. This approach reveals a surprising heterogeneity in the translation of individual mRNAs within the same cell, including rapid and reversible transitions between a translating and non-translating state. Applying this method to the cell-cycle gene Emi1, we find strong overall repression of translation initiation by specific 5′ UTR sequences, but individual mRNA molecules in the same cell can exhibit dramatically different translational efficiencies. The ability to observe translation of single mRNA molecules in live cells provides a powerful tool to study translation regulation.

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.…”

Tracking translation of single mRNA molecule in live cells

“…(3) Even for the transcript, whose overall translational level is highly repressed by its 5 0 UTR, has a small fraction (*2 %) that is highly translated. The amount of ribosomes associated with this subset of the transcript (*2 %) is higher than the amount of ribosomes associated with the rest of the transcript [6]. (4) Tracking real-time movements of mRNAribosome complexes expressing cytosolic proteins reveals three different behaviors: stationary, sub-diffusive, and diffusive.…”

“…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]. (3) Even for the transcript, whose overall translational level is highly repressed by its 5 0 UTR, has a small fraction (*2 %) that is highly translated.…”

“…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.…”