Real-Time Imaging of Translation on Single mRNA Transcripts in Live Cells

Abstract

SUMMARY Translation is under tight spatial and temporal controls to ensure protein production in the right time and place in cells. Methods that allow real-time, high-resolution visualization of translation in live cells are essential for understanding the spatiotemporal dynamics of translation regulation. Based on multivalent fluorescence amplification of the nascent polypeptide signal, we develop a method to image translation on individual mRNA molecules in real time in live cells, allowing direct visualization of translation events at the translation sites. Using this approach, we monitor transient changes of translation dynamics in responses to environmental stresses, capture distinct mobilities of individual polysomes in different subcellular compartments, and detect 3’UTR-dependent local translation and active transport of polysomes in dendrites of primary neurons.

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).…”

Limitless translation limits translation

“…Single-molecule imaging of translation on individual mRNA molecules, in real time and in live cells, might eventually allow simultaneous observation of mRNAs and their protein products 11 . ■…”

Technologies of High-Throughput Tissue-Specific Gene Expression Profiling

“…Recombinant technology allows a fluorescent protein to ligate covalently to the product of target gene [3,4] . Generally, gene profiling technologies based on fluorescence microscopy are not only intuitive but also precise enough to reach single molecular level accuracy [5] . However, these technologies are expensive and labor intensive in large scale gene expression profiling projects, let alone that image analysis is not automated enough to REVIEW improve the throughput.…”