Compartmentation and intracellular traffic of mRNPs

In eukaryotic cells, cytoplasmic mRNAs can be translated, degraded or stored, depending on the proteins which are bound to them. These proteins mediate a variety of post-transcriptional regulation pathways, which affect mRNA metabolism at various levels and are essential for many facets of cell physiology. Remarkably, many implicated factors accumulate in cytoplasmic membrane-less ribonucleoprotein (RNP) granules, which form following liquid-liquid phase separation: P-bodies, stress granules, germ granules, neuronal granules, etc. In spite of different names, morphology and exact composition, depending on the model organism, the cell type and the environmental conditions, most of these granules contain repressed mRNAs. The main issues concerning these granules are: How do they assemble? What is their protein and RNA content? What is their function in RNA metabolism? What is their function in cell physiology? In other words, what is the added function of these mRNP macro-aggregates over diffuse mRNPs?

We tackle these questions using a combination of experimental approaches including biochemical and cell imaging techniques, as well as proteomic and transcriptomic analyses. Most our studies focus on P-bodies in human cells.

- How do they assemble? We have shown that three proteins are required for P-body assembly in human cells: the RNA helicase DDX6 and its partners LSM14A and 4E-T [1]. We are focusing on these key proteins and their interacting proteins [2–8] to understand the molecular mechanisms involved and their regulation.

- What is their protein and RNA content? What is their function in RNA metabolism? We have pioneered a novel method to purify P-bodies from cells, called Fluorescence Activated Particle Sorting (FAPS). It enabled us to identify their protein and RNA content by mass spectrometry and RNAseq, respectively. Their analysis, combined with polysome profiling experiments, led us to conclude that P-bodies are primarily involved in mRNA storage [9–11]. This approach now opens the possibility to investigate the dynamics of the P-body content in various conditions, cell environments and cell types.

- What is their function if cell physiology? Established cell lines grow well in the absence of DDX6 expression, and therefore in the absence of P-bodies. However, in human, several mutations in the DDX6 gene are responsible for mental retardation and some developmental defects. We have shown that the DDX6 mutations lead to a strong P-body defect in the patient cells, as well as to some mRNA post-transcriptional deregulation [12]. These findings constitute a unique entry point to understand the function of P-bodies and/or DDX6 during development.

1 Ayache, J. et al. (2015) Mol. Biol. Cell 26, 2579–2595

2 Souquere, S. et al. (2015) Nucl. Austin Tex 6, 326–338

3 Kamenska, A. et al. (2016) Nucleic Acids Res. 44, 6318–6334

4 Vindry, C. et al. (2017) Cell Rep. 20, 1187–1200

5 Chauderlier, A. et al. (2018) Biochim. Biophys. Acta 1861, 762–772

6 Vindry, C. et al. (2019) Wiley Interdiscip. Rev. RNA DOI: 10.1002/wrna.1557

7 Courel, M. et al. (2018) bioRxiv DOI: 10.1101/373498

8 Garcia-Jove Navarro, M. et al. (2019) Nat. Commun. 10, 3230

9 Hubstenberger, A. et al. (2017) Mol. Cell 68, 144-157.e5

10 Standart, N. and Weil, D. (2018) Trends Genet. 34, 612–626

11 Courel, M. et al. (2018) Med. Sci. MS 34, 306–308

12 Balak, C. et al. (2019) Am. J. Hum. Genet. DOI: 10.1016/j.ajhg.2019.07.010

Collaborations

  • Chris Balak: Tgen, Phoenix, Arizona, US
  • Edouard Bertrand: IGMM, Montpellier, France
  • Patrick Brest: ICAN, Nice, France
  • Zoher Gueroui: ENS Paris, France
  • Arnaud Hubstenberger: Valrose, Nice, France
  • Antonin Morillon: Institut Curie, France
  • Julien Mozziconacci: LPTMC, UPMC, Paris, France
  • Gérard Pierron: Institut Gustave Roussy, Villejuif, France
  • Amélie Piton: IGBMC, Strasbourg, France
  • Hugues Roest Crollius: ENS Paris, France
  • Nancy Standart: University of Cambridge, UK