Visualization of single endogenous polysomes reveals the dynamics of translation in live human cells

• DOI: 10.1083/jcb.201605024
• Publication Date: 2016-09-05T14:07:28Z
• Authors: Xavier Pichon; Amandine Bastide; Adham Safieddine; Racha Chouaib; Aubin Samacoits; Eugenia Basyuk; Marion Peter; Florian Mueller; Edouard Bertrand
• Keywords: Cytoplasmic Dyneins; Technology; MESH: Protein Transport; Cytoplasm; Time Factors; Recombinant Fusion Proteins; Biophysics; Peptide Chain Elongation, Translational; [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC]; Transfection; Diffusion; MESH: Recombinant Fusion Proteins; Image Processing, Computer-Assisted; MESH: Microscopy, Confocal; Humans; MESH: Peptide Chain Elongation, Translational; Research Articles; MESH: Humans; Microscopy, Confocal; MESH: Cytoplasm; MESH: Transfection; MESH: Time Factors; MESH: Cytoplasmic Dyneins; MESH: Diffusion; RNA Biology; MESH: Image Processing, Computer-Assisted; MESH: RNA Polymerase II; MESH: Ribonucleoproteins; Protein Transport; Ribonucleoproteins; Polyribosomes; MESH: HeLa Cells; RNA Polymerase II; MESH: Polyribosomes; MESH: Fluorescence Recovery After Photobleaching; Fluorescence Recovery After Photobleaching; HeLa Cells

Translation is an essential step in gene expression. In this study, we used an improved SunTag system to label nascent proteins and image translation of single messenger ribonucleoproteins (mRNPs) in human cells. Using a dedicated reporter RNA, we observe that translation of single mRNPs stochastically turns on and off while they diffuse through the cytoplasm. We further measure a ribosome density of 1.3 per kilobase and an elongation rate of 13–18 amino acids per second. Tagging the endogenous POLR2A gene revealed similar elongation rates and ribosomal densities and that nearly all messenger RNAs (mRNAs) are engaged in translation. Remarkably, tagging of the heavy chain of dynein 1 (DYNC1H1) shows this mRNA accumulates in foci containing three to seven RNA molecules. These foci are translation sites and thus represent specialized translation factories. We also observe that DYNC1H1 polysomes are actively transported by motors, which may deliver the mature protein at appropriate cellular locations. The SunTag should be broadly applicable to study translational regulation in live single cells.