P-body structure and assembly – PBODYSTRUC

DDX6 interactome will be identified by tandem affinity purification of DDX6 RNP complexes from a human epithelial cell line, coupled to bulk peptide analysis by mass spectrometry. Protein function in P-body assembly will be assessed by silencing experiments as previously performed for DDX6 (Serman et al. 2007). P-bodies will be purified by differential centrifugation followed by a cutting edge particle purification method, and their proteins and RNA will be identified by mass spectrometry and RNAseq analysis.
As to the conformation of RNP complexes, time resolved fluorescence anisotropy will be used to determine the oligomerization status of recombinant DDX6 bound to RNA in vitro, while homoFRET will provide this information in the cells, both in the cytosol and in P-bodies. Then, a fluorescence correlation spectroscopy assay will be set up to measure RNA unfolding upon protein binding in vitro. This will allow measuring DDX6 helicase activity, and the enhancing or inhibiting contribution of its partners. RNP organization within P-bodies will be analyzed by immunodetection of DDX6 and its partners in electron microscopy, followed by spatial statistical analysis of the images. The informations obtained in vitro and in electron microscopy will be used to compute a virtual model of P-body organization.

The DDX6 protein is required for P-body assembly in human cells. Its protein partners were identified by tandem affinity purification coupled to mass spectrometry. The resulting interactome confirms the place of this helicase at the interface between RNA degradation and translational repression in human epithelial cells. Three complexes were prominent: the decapping complex linked to RNA degradation, a CPEB-like complex linked to mRNA translation repression, and a poorly characterized ATXN2/2L complex. Only the two former complexes accumulate in P-bodies, showing the existence of distinct DDX6 complexes in and out of P-bodies. Two new key proteins for P-body assembly were identified in the CPEB-like complex (Ayache et al. 2015).
The project was an opportunity to set-up a procedure for double immunolabeling with same species antibodies in electron microscopy (Souquère et al. 2015).

The identification of DDX6 partners will be a resource for future studies. In the frame of this project it allowed identifying two other proteins involved in P-body assembly. Future studies will determine whether the three proteins act together or separately to recruit mRNAs into P-bodies.
The protocol set up for double immunolabeling in electron microscopy will have applications beyond the project presented here.

Publications dans des journaux internationaux
1. S. Souquere, D. Weil#, and G. Pierron#. 2015. Comparative ultrastructure of CRM1-Nucleolar bodies (CNoBs), Intranucleolar bodies (INBs) and hybrid PML/p62 bodies reveals a new facet of nuclear body plurality. Nucleus 6, 326-38.
2. J. Ayache*, M. Bénard*, M. Ernoult-Lange, N. Minshall, N. Standart, M. Kress, and D. Weil. 2015. P-body assembly requires DDX6 repression complexes rather than decay or Ataxin2/2L complexes. Mol Biol Cell.26, 2579-95.

Communications dans des congrès internationaux
1. 06/2015: EMBO Meeting: RNA localization and local translation (Crete) – poster
2. 07/2015: Translation UK (Aberdeen, UK) – communication orale et poster
3. 10/2015: Euronet RNP and disease (Marrakech, Maroc) – communication orale et poster
4. 10/2015: Hallmark of cancer: focus on RNA (Institut Curie, Paris) – poster

Communications dans des congrès nationaux
1. 10/2015: Colloque inaugural de l’IBPS (Paris) – poster
2. 03/2016: 10ème rencontre du SIFRARN (Toulouse) - communication orale et 2 posters

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).