Gene expression not only depends on transcription in the nucleus, but also on post-transcriptional events in the cytoplasm, including mRNA degradation, translation, and storage. These processes are governed by the proteins which are bound to mRNAs and by their organization in complexes. They can be associated with particular localizations in ribonucleoprotein granules discovered recently, such as P-bodies. These granules, which are devoid of membrane, contain thousands of mRNAs associated with protein complexes (mRNPs). Their known components indicate their involvement in mRNA degradation, translational repression and RNAi pathway. Yet, so far, there is no evidence of their requirement for these processes, nor any understanding of how they could impact them. Nevertheless, they are present in all eukaryotes, animals and vegetals, from trypanosoma to mammals. The present project proposes to address the question from the granule point of view, by elucidating P-body composition, architecture and mechanism of assembly. Both P-bodies and their components will be studied, using multiscale approaches, made possible by the participation of four partners with distinct and complementary expertises. The coordinator, Dominique Weil, will study P-bodies in their cellular context, and use biochemical, proteomic and transcriptomic approaches to determine their composition. Eric Deprez, will focus on a protein that is essential for P-body assembly, and study the conformation of RNA-protein complexes made in vitro and in vivo by this protein, using biophotonics. Philippe Andrey, will generate a mathematical model of P-bodies, based on electron microscopy experimental data obtained by Gérard Pierron. One part of the project aims at characterizing the mRNP complexes which are present in P-bodies. On one side, as the DEAD-box protein Rck/p54 is essential for P-body assembly in mammals, its protein partners will be characterized, so as to know the distribution of Rck/p54 among mRNA degradation, translational repression, RNAi complexes, or others. The importance of these partners for P-body assembly will be investigated. On the other side, P-bodies will be purified to identify their protein and RNA components. They could contain all types of Rck/p54 complexes or only some, as well as, potentially, unrelated ones. Importantly, it will also elucidate which RNAs are targeted to these granules. A second part of the project aims at characterizing [Rck/p54 – RNA] complexes both in vitro and in vivo. Rck/p54 is an RNA-binding protein which unfolds RNA in vitro. This relaxed conformation of the RNA could play a central role in P-body architecture. Another feature of Rck/p54 is its propensity to homo-oligomerize, which could also participate to granule assembly. Here, we will determine the oligomeric status of RNA-bound Rck/p54, and we will analyze its RNA unfolding activity, in particular in the presence of its main partners. The last part of the project is to better understand P-body architecture using computational models of P-bodies with unfolded RNA and proteins as elementary bricks, and taking into account the new knowledge obtained on P-body content and mRNP conformation. The in silico model will be fitted using EM images of P-bodies in their cellular context, and spatial statistics tools adapted or created for this purpose. This study should provide new insight into P-body architecture and function, including their RNA content. This is of interest not only for these particular granules, but also for the related mRNP granules which are present in particular conditions (stress granules) or particular cell types (germ cells, neurons).