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 . We are focusing on these key proteins and their
interacting proteins [2–8] to understand the molecular mechanisms involved and
- 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 . These findings constitute
a unique entry point to understand the function of P-bodies and/or DDX6 during
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