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Cinétique de la traduction eucaryote

Cinétique de la traduction eucaryote / Eukaryotic translation kinetics

Expérience de fluorescence en molécule unique

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permanent staff: Karen Perronet

PhD student : Charlène Valadon

Former members: Nathalie Barbier (thesis in pdf), Nicolas Fiszman(thesis in pdf), Antoine Le Gall (thesis in pdf), David Dulin (thesis in pdf), Hélène Chommy (postdoc), Hélène Walbott (postdoc), Philippe Bouyer (now in LP2N in Bordeaux), Nathalie Westbrook

Introduction

Protein synthesis is a key process for all living organisms. It is orchestrated by the ribosome, a macromolecule capable of reading the genetic code carried by the messenger RNA (mRNA), to translate it into a chain of amino acids. Since this mechanism involves multiple asynchronous steps, observing the translation at the scale of a single ribosome provides a complete view of the process without blurring it by ensemble measurements.

We work closely with Olivier Namy's "Genomics, Structure and Translation" team at I2BC. We have developed a reporter assay that functions as a ribosome "chronometer", based on a simple design where no biological species need to be modified to be fluorescently labelled. This experiment uses mammalian ribosomes, which are more complex than the prokaryotic ribosomes used in the majority of single molecule studies. The principle of the experiment is shown in Figure 1: an mRNA is complexed with a ribosome and labelled with two small fluorescent RNA probes (called R and G primers) hybridized along its coding sequence. Translation begins with the injection of the cell extract (rabbit reticulocyte lysate, RRL). During translation, the ribosome, thanks to its helicase activity, detaches the probes to continue reading the mRNA code. Translation kinetics is obtained by measuring the departure times for each probe.

Principe du suivi de la cinétique du ribosome

 

Figure 1. Principle of the observation of the translation of a eukaryotic ribosome.

Our results on the delay in the kinetics of initiation due to viral IRES structures have been published in RNA on August 2nd, 2017. The article is accessible on line (here)

Thanks to this technique, we have started a new collaboration with a team at CRCL in Lyon to extend our studies to specific processes taking place in cancerous cells. Our objective is to characterize the impact of different modifications on RNAs (especially ribosomal and transfer RNAs) on the initiation and elongation kinetics of translation. This project has just been funded by an ANR.

Set-up

To conduct single molecule studies on the ribosome, we have developed a total internal reflection fluorescence microscopy set-up. It includes 3 different lasers that allow the use of a wide range of fluorophores. The device is fully synchronized, thanks to electronics developed in the laboratory. It is possible to take image sequences by alternating excitation wavelengths, to co-locate fluorescent markers in real time. The sample is illuminated only during the image acquisition time to avoid premature photobleaching of fluorophores, and the appropriate filters are selected automatically. In addition, a focus holding system has been developed to ensure proper focus despite the wavelength change and residual chromatism of the lens, and despite thermal drifts or product injection into the microfluidic system constituting the sample to be observed.

Collaborations:

  • Institute for Integrative Biology of the Cell (I2BC) in Gif/Yvette and Orsay: Olivier Namy (eukaryotic ribosome)
  • Centre de Recherche en Cancérologie de Lyon (CRCL) : Jean-Jacques Diaz et Frédéric Catez
  • David Dulin, Erlangen University
  • François Marquier, Laboratoire Aimé Cotton
  • Géraud Bouwmans et Laurent Bigot, PhLAM et FiberTech Lille

Support:

- ANR project Actimeth

- LABEX NanoSaclay

- Université Paris Saclay, Initiative Doctorale Interdisciplinaire

Selected Publications:

  1. O. Bugaud, N. Barbier, H. Chommy, N. Fiszman, A. Le Gall, D. Dulin, M. Saguy, N. Westbrook, K. Perronet, O. Namy, "Kinetics of CrPV and HCV IRES-mediated eukaryotic translation using single molecule fluorescence microscopy", RNA 2017. 23: 1626-1635
  2. A. Le Gall, D. Dulin, G. Clavier, R. Méallet-Renault, P. Bouyer, K. Perronet, N. Westbrook, "Improved photon yield from a green dye with a reducing and oxidizing system", ChemPhysChem 12: 1657–1660 (2011).

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