GENOME-WIDE SEARCH FOR FACTORS THAT CONTROL THE ASSEMBLY OF RNA GRANULES – RNAGRIMP

In vivo, mRNAs are packaged together with regulatory proteins into ribonucleoprotein particles (RNP) that control their fate and undergo extensive remodeling in response to developmental cues or environmental stresses. Cytoplasmic RNPs of different sizes, composition and regulatory properties have been described, including large macromolecular complexes such as P-bodies, stress granules, or germ cell granules. In neurons, so-called neuronal granules have been implicated in the long-distance transport of mRNAs to axons or dendrites, and in their local translation in response to external cues. To date, surprisingly little is known about the factors controlling the assembly and regulation of RNP granules, specifically neuronal granules. This has so far prevented a detailed understanding of the molecular mechanisms underlying the formation and turnover of these granules, and of how they are physiologically regulated.
Here, we propose to study the molecular bases underlying the assembly and regulation of RNA granules, using the highly conserved IMP-containing granules as a paradigm. Cytoplasmic RNP granules characterized by the presence of IMP family members (called IMP, IGF2BP, ZBP1, Vg1RBP or VICKZ) have been described in a wide-range of organisms and cell types, where they are implicated in subcellular mRNA targeting, and/or in the spatio-temporal regulation of mRNA translation. Surprisingly, although IMP neuronal granules are considered as a major class of neuronal granules, in vivo study of their function and regulation has lagged behind, and the physiological importance of IMP granules during brain maturation has for a long time remained unclear. Recently, IMP granules dynamically transported to axons have been observed in vivo in zebrafish embryonic neurons (F. Giudicelli, unpublished). Furthermore, it was discovered that IMP granules are actively and specifically transported to the axons of remodeling neurons during Drosophila brain maturation. Both the function and the transport of these granules are required for proper axonal remodeling (F. Besse; Medioni et al., 2014). The main objectives of this proposal are i) to systematically identify the molecular factors that regulate the assembly and the turnover of IMP-containing RNP complexes in Drosophila cultured cells, ii) to test their physiological importance in vivo, in the developing fly nervous system, and iii) to investigate their functional conservation in the zebrafish embryo.
Specifically, we propose to perform an unbiased genome-wide RNAi screen on Drosophila cultured cells to identify mutant conditions in which the organization and/or distribution of IMP-containing granules is altered. To quantitatively and statistically analyze mutant conditions, and to define precise and coherent classes of mutants, we will combine high throughput microscopy with the development of a computational pipeline optimized for automatic analysis and classification of images. The function of positive hits isolated in the screen will then be validated in vivo in Drosophila neurons using fly genetics and imaging techniques, and characterized at the molecular and cellular levels using biochemical assays, in vitro phase transition experiments and live-imaging. Finally, the functional conservation of identified regulators will be tested in zebrafish embryos combining gene inactivation and live-imaging techniques.
This integrative study will provide the first comprehensive analysis of the functional network that regulates the properties of the conserved IMP RNA granules. Our characterization of the identified regulators in vivo in neuronal cells will be of particular significance in the light of recent evidence linking the progression of several degenerative human diseases to the accumulation of non-functional RNA/protein aggregates. This work will thus shed new insight into the mechanisms controlling RNP particle assembly and disassembly in both wild-type and pathological contexts.