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Caractérisation fonctionnelle de la protéine ANKS3 impliquée dans les ciliopathies rénales et étude de son rôle dans la régulation des ARNs

Abstract : Nephronophtisis (NPH) is an autosomal recessive tubulo-interstitial nephropathy characterized by the a massive interstitial fibrosis and cyst formation. NPH is the major reason for end-stage renal disease in childhood. It can be isolated or syndromic, associated with extrarenal manifestations. NPH belongs to the ciliopathies, a group of multisystemic diseases, caused by mutations in genes encoding proteins of the primary cilia. The primary cilia acts on the surface of most cells as a mechano and chemosensor controlling signaling pathways essential for tissue development and homeostasis (Shh, Wnt, PC2 / Ca2+ signaling). To date twenty-three causatives genes have been identified for NPH (including 14 in our laboratory). These codes for ciliary proteins implicated in the control of cilia protein composition and ciliary signaling, at the base of the cilium (transition zone (TZ)), at the level of the Inversin compartment (IC), or during intraflagellar transport. We identified a homozygous missense mutation in a new gene responsible for NPH associated with liver fibrosis. ANKS3 code a protein known to interact with several NPH proteins such as NPHP1 (TZ), ANKS6 and NEK8 (IC) as well as BICC1, a ribonucleoprotein mutated in renal cystic dysplasia. My thesis project has been focused on the characterization of ANKS3 function and the impact of human mutation in cellular processes relevant for renal ciliopathies. These studies were performed from different cellular models: patient fibroblasts, renal tubular cells IMCD3 depleted for Anks3 (ANKS3_KD) re-expressing the wild-type or mutated form. My work demonstrated that ANSK3 mutation decreases its interaction with the TZ component, NPHP1 and IC one, NEK8. In addition, the absence or expression of the mutated form of ANKS3 affects the cilia length and the IC biogenesis. Indeed, the IC components are abnormally relocated all along the primary cilia. Moreover, a disruption of the ciliary signaling Shh pathway and the localization of the PC2 protein at the primary cilia were observed in the ANKS3_KD or mutant cells. This last observation is confirmed by zebrafish mutated for anks3 (TALEN) model, which exhibits a decrease in ciliary motility associated with calcium signaling defetcs in the Kupffer vesicle, leading to laterality defects (situs inversus). In addition to these ciliary defects, the loss or mutation of ANKS3 causes apico-basal polarity defects in IMCD cells, with a decrease in cell height and delay in tight junction formation, a phenotype reminiscent of NPHP1 mutated models. Consistent with these results we observed a decrease in the stability of Nphp1 transcripts in ANKS3_KD and mutant cells. The re-expression of NPHP1 in these cells rescues the polarity and cilia length defects demonstrating the ANKS3 implication in the Nphp1 transcripts regulation in these phenotypes. To elucidate the mechanism by which ANKS3 regulates the Nphp1 and possibly other ciliary RNAs stability, we investigated the implication of its partner BICC1, which is known to bind and regulate RNAs. Transcriptomic analyzes notably RNAseq and RIPseq on ANKS3-KD and mutated cells in the presence or absence of BICC1, demonstrate that the absence of ANKS3 or a mutant form triggers BICC1 interaction with the targeted mRNA and the RISC-associated protein AGO2, thus addressing ciliary gene transcripts to degradation. This finding allows us to place ANKS3/BICC1 complex as a key regulator of ciliopathy-related transcripts that orchestrates the biogenesis and function of primary cilia.
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Gweltas Odye. Caractérisation fonctionnelle de la protéine ANKS3 impliquée dans les ciliopathies rénales et étude de son rôle dans la régulation des ARNs. Biologie cellulaire. Université Sorbonne Paris Cité, 2018. Français. ⟨NNT : 2018USPCB029⟩. ⟨tel-02490646⟩

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