Role of the Prion-like domain of Imp in neuronal RNP granule regulation

Abstract : Eukaryotic mRNAs are bound by RNA Binding Proteins (RBP) and packaged into diverse range of macromolecular assemblies named RNP granules. In neurons, transport RNP granules are implicated in the transport of specific mRNAs to axons or dendrites, and in their local translation in response to external cues. Although little is known about the assembly and regulation of these granules in vivo, growing evidence indicates that the presence of Prion Like domains (PLD) within RBPs favours multivalent protein–protein and protein-RNA interactions, promoting the transition of soluble complexes into RNP granules. The conserved RBP Imp is as a core component of RNP granules that are actively transported to axons upon neuronal remodelling in Drosophila. Furthermore, Imp function was shown to be required for axonal remodelling during Drosophila nervous system maturation. Analyses of the domain architecture of the Imp protein revealed that, in addition to four RNA binding domains (RBD), Imp contains a Cterminal domain showing a striking enrichment in Glutamines and Serines, which is one of the characteristics of a PLD. During my PhD, I explored the function of the PLD in the context of granule assembly and transport. In cultured cells, I observed that Imp granules assembled in the absence of the PLD, however their number and size were increased. Proteins with scrambled PLD sequence accumulated in granules of normal size and number, implying that the degree of disorder of this domain, and not its sequence, is essential for granule homeostasis. Moreover, FRAP experiments, performed on cultured cells and in vivo, revealed that Imp PLD is important to maintain the turnover of these granules. In vivo, this domain is both necessary and sufficient for efficient transport of Imp granules to axons. These defects are associated with a reduction on the number of motile granules in axons. Furthermore, mutant forms lacking the PLD do not rescue the axon remodelling defects observed upon imp loss of function. Finally, a swapping experiment in which I moved Imp PLD from the C-terminus to the N-terminus of the protein revealed that the functions of Imp PLD in granule transport and homeostasis are uncoupled, and that PLD-dependent modulation of Imp granule properties is dispensable in vivo. Together, my results show that Imp PLD of is not required for the assembly of RNP granules, but rather regulates granule number and dynamics. Furthermore, my work uncovered an unexpected in vivo function for a PLD in axonal transport and remodelling during nervous system maturation.
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Jeshlee Cyril Vijayakumar. Role of the Prion-like domain of Imp in neuronal RNP granule regulation. Cellular Biology. Université Côte d'Azur, 2018. English. ⟨NNT : 2018AZUR4099⟩. ⟨tel-02273547⟩

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