Real-time quantification of single RNA translation dynamics in living cells.
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.
Department of Biochemistry and Molecular Biology and Institute for Genome Architecture and Function, Colorado State University, Fort Collins, CO, USA.
Tatsuya
T
Morisaki
Department of Biochemistry and Molecular Biology and Institute for Genome Architecture and Function, Colorado State University, Fort Collins, CO, USA.
Kenneth
K
Lyon
Department of Biochemistry and Molecular Biology and Institute for Genome Architecture and Function, Colorado State University, Fort Collins, CO, USA.
Keith F
KF
DeLuca
Department of Biochemistry and Molecular Biology and Institute for Genome Architecture and Function, Colorado State University, Fort Collins, CO, USA.
Jennifer G
JG
DeLuca
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.
Brian P
BP
English
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.
Zhengjian
Z
Zhang
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.
Luke D
LD
Lavis
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.
Jonathan B
JB
Grimm
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.
Sarada
S
Viswanathan
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.
Loren L
LL
Looger
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.
Timothee
T
Lionnet
Department of Biochemistry and Molecular Biology and Institute for Genome Architecture and Function, Colorado State University, Fort Collins, CO, USA.
Timothy J
TJ
Stasevich
D000906:Antibodies / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D056484:Jumonji Domain-Containing Histone Demethylases / Q000096:biosynthesis / Q000235:genetics; D007700:Kinetics; D057054:Molecular Imaging / Q000379:methods*; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis*; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics; D059010:Single-Cell Analysis; D013997:Time Factors
D000906:Antibodies; D005456:Fluorescent Dyes; D009687:Nuclear Proteins; D012333:RNA, Messenger; D012097:Repressor Proteins; D049452:Green Fluorescent Proteins; D056484:Jumonji Domain-Containing Histone Demethylases; C119611:KDM5B protein, human