Visualization of single endogenous polysomes reveals the dynamics of translation in live human 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.
Institut de Génétique Moléculaire de Montpellier, UMR5535 CNRS, 34293 Montpellier Cedex 5, France; Université de Montpellier, 34090 Montpellier, France xavier.pichon@igmm.cnrs.fr edouard.bertrand@igmm.cnrs.fr.
Xavier
X
Pichon
Institut de Génétique Moléculaire de Montpellier, UMR5535 CNRS, 34293 Montpellier Cedex 5, France; Université de Montpellier, 34090 Montpellier, France.
Amandine
http://orcid.org/0000-0001-7896-8490
A
Bastide
Institut de Génétique Moléculaire de Montpellier, UMR5535 CNRS, 34293 Montpellier Cedex 5, France; Université de Montpellier, 34090 Montpellier, France.
Adham
http://orcid.org/0000-0002-9324-9037
A
Safieddine
Institut de Génétique Moléculaire de Montpellier, UMR5535 CNRS, 34293 Montpellier Cedex 5, France; Université de Montpellier, 34090 Montpellier, France.
Racha
R
Chouaib
Unité Imagerie et Modélisation, Institut Pasteur, UMR3691, Centre National de la Recherche Scientifique, 75015 Paris, France.
Aubin
http://orcid.org/0000-0001-8060-6616
A
Samacoits
Institut de Génétique Moléculaire de Montpellier, UMR5535 CNRS, 34293 Montpellier Cedex 5, France; Université de Montpellier, 34090 Montpellier, France.
Eugenia
E
Basyuk
Institut de Génétique Moléculaire de Montpellier, UMR5535 CNRS, 34293 Montpellier Cedex 5, France; Université de Montpellier, 34090 Montpellier, France.
Marion
M
Peter
Unité Imagerie et Modélisation, Institut Pasteur, UMR3691, Centre National de la Recherche Scientifique, 75015 Paris, France.
Florian
http://orcid.org/0000-0002-9622-4396
F
Mueller
Institut de Génétique Moléculaire de Montpellier, UMR5535 CNRS, 34293 Montpellier Cedex 5, France; Université de Montpellier, 34090 Montpellier, France xavier.pichon@igmm.cnrs.fr edouard.bertrand@igmm.cnrs.fr.
Edouard
http://orcid.org/0000-0003-0772-0601
E
Bertrand
D003593:Cytoplasm / Q000378:metabolism; D056445:Cytoplasmic Dyneins / Q000096:biosynthesis* / Q000235:genetics; D004058:Diffusion; D036681:Fluorescence Recovery After Photobleaching; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D018613:Microscopy, Confocal; D010441:Peptide Chain Elongation, Translational; D011132:Polyribosomes / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D012319:RNA Polymerase II / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
C511240:DYNC1H1 protein, human; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; C026452:messenger ribonucleoprotein; D012319:RNA Polymerase II; D056445:Cytoplasmic Dyneins