• Publié par : Christian DELAMARCHE
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P-bodies

Objective : To determine the structural organisation of P-bodies (“processing bodies”) and identify their links with mRNAs and ribosomes

 

P-bodies are dynamic cytoplasmic granules where non-translated eukaryotic mRNAs accumulate with a core of proteins to form machinery for general translation repression and mRNA decay. These mRNAs can either be decapped and degraded within the P-bodies or exit to return to translation. Therefore, a dynamic equilibrium of mRNAs between polysomes and the P-bodies allows gene expression to be fine-tuned. Since their discovery (Sheth and Parker, 2003), several studies using fluorescence-based strategies have uncovered their pivotal role in mRNA metabolism, particularly during translation repression and/or mRNA degradation. Yet, P-bodies still remain a “black box” in which numerous proteins accumulate next to RNAs to regulate their fate by unknown mechanisms. During this project, our main goal will be to determine the structural organisation of the P-bodies and to identify the molecular elements involved during the anchorage and degradation of mRNA. In order to visualize P-bodies within their native cellular environment, we recently introduced an alternative laboratory method for cryofixation, namely, high/-/pressure freezing. To my knowledge, such a method has never been used to analyse structures like P-bodies. After ultra-rapid freezing, the cells are immobilized within milliseconds, allowing the instantaneous fixation of all molecules in their native architecture. After vitreous sectioning of the cells, the P-bodies are immunostained using specific antibodies, which will hopefully allow rapid unravelling of the 3D ultrastructure of the P-bodies and the localization of mRNAs and ribosomes in their native environment (Figure). In collaboration with hospital clinicians from the Regional Hospital of Pontchaillou (Rennes) we will seek for P-bodies into breast cancer tissues.

 

 

Figure: Electron tomography reveals two levels of compaction in P-bodies. P-bodies were localized using immunogold labeling with anti-hDcp1a and then processed for electron tomography. (A, B, C) digital sections through the 3D map of one P-body. (D) Final 3D surface-rendered reconstruction of a P-body showing peripheral ramifications and connections to ribosomes. P-body is shown in blue, ribosomes in yellow, and mitochondria in green. Arrowhead: peripheral ramifications. Scale bar: 100 nm. (N Cougot, A Cavalier, D Thomas, R Gillet, submitted)