Kinetics of CrPV and HCV IRES-mediated eukaryotic translation using single molecule fluorescence microscopy

Olivier Bugaud (1, 2) , Nathalie Barbier (3) , Hélène Chommy (1, 3) , Nicolas Fiszman (3) , Antoine Le Gall (3) , David Dulin (3) , Matthieu Saguy (1, 2) , Nathalie Westbrook (3) , Karen Perronet (3) , Olivier Namy (1, 2)

1 I2BC - Institut de Biologie Intégrative de la Cellule
2 GST - Génomique, Structure et Traduction
3 Laboratoire Charles Fabry / Biophotonique

Protein synthesis is a complex multi-step process involving many factors that need to interact in a coordinated manner to properly translate the messenger RNA. As translating ribosomes cannot be synchronized over many elongation cycles, single molecule studies have been introduced to bring a deeper understanding of prokaryotic translation dynamics. Extending this approach to eukaryotic translation is very appealing, but initiation and specific labelling of the ribosomes are much more complicated. Here we use a non-canonical translation initiation based on internal ribosome entry sites (IRES) and we monitor the passage of individual, unmodified mammalian ribosomes at specific fluorescent milestones along mRNA. We explore initiation by two types of IRES, the intergenic IRES of Cricket Paralysis virus (CrPV) and the hepatitis C (HCV) IRES, and show that they both strongly limit the rate of the first elongation steps compared to the following ones suggesting that those first elongation cycles do not correspond to a canonical elongation. This new system opens the possibility to study both IRES-mediated initiation and elongation kinetics of eukaryotic translation and will undoubtedly be a valuable tool to investigate the role of translation machinery modifications in human diseases.

Domaines: Biophysique

Olivier Bugaud, Nathalie Barbier, Hélène Chommy, Nicolas Fiszman, Antoine Le Gall, et al.. Kinetics of CrPV and HCV IRES-mediated eukaryotic translation using single molecule fluorescence microscopy. RNA, 2017, 23 (11), pp.1626-1635. ⟨10.1261/rna.061523.117⟩. ⟨hal-01575997⟩