Genetic and physical interaction between the NPHP5 and NPHP6 gene products

Abstract

Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease, caused by mutations of at least nine different genes. Several extrarenal manifestations characterize this disorder, including cerebellar defects, situs inversus and retinitis pigmentosa. While the clinical manifestations vary significantly in NPHP, mutations of NPHP5 and NPHP6 are always associated with progressive blindness. This clinical finding suggests that the gene products, nephrocystin-5 and nephrocystin-6, participate in overlapping signaling pathways to maintain photoreceptor homeostasis. To analyze the genetic interaction between these two proteins in more detail, we studied zebrafish embryos after depletion of NPHP5 and NPHP6. Knockdown of zebrafish zNPHP5 and zNPHP6 produced similar phenotypes, and synergistic effects were observed after the combined knockdown of zNPHP5 and zNPHP6. The N-terminal domain of nephrocystin-6-bound nephrocystin-5, and mapping studies delineated the interacting site from amino acid 696 to 896 of NPHP6. In Xenopus laevis, knockdown of NPHP5 caused substantial neural tube closure defects. This phenotype was copied by expression of the nephrocystin-5-binding fragment of nephrocystin-6, and rescued by co-expression of nephrocystin-5, supporting a physical interaction between both gene products in vivo. Since the N- and C-terminal fragments of nephrocystin-6 engage in the formation of homo- and heteromeric protein complexes, conformational changes seem to regulate the interaction of nephrocystin-6 with its binding partners.

Nephrocystin-3 is required for ciliary function in zebrafish embryos

“…Injection of this morpholino into fertilized eggs resulted in phenotypes resembling those observed in other knockdown models of zebrafish nephrocystin orthologs (23,26): body curvature was observed in 51% (n ϭ 125) of the morphant embryos injected with 8 ng of AUG-MO as early as 24 hpf (Fig. 2, A and D); after 36 hpf, morphants started to develop hydrocephalus and by 48 h, 98% (n ϭ 154) of embryos showed hydrocephalus (Fig.…”

“…ment, we characterized the zebrafish ortholog of NPHP3 by morpholino oligo-mediated knockdown. We found that disruption of zebrafish nphp3 led to many defects that have been shared by several zebrafish models of NPHP, BBS and MKS (2,3,23,24,26,33,36). These phenotypes, including body curvature, hydrocephalus, situs inversus, and pronephric cysts, have also been reported for zebrafish morphants of ciliary genes (14,15,19,27,30,31), suggesting an essential role of nphp3 in ciliary function.…”

“…Morpholino-mediated knockdown of many NPHP, Bardet-Biedl syndrome (BBS), and cystic kidney disease genes in zebrafish have produced phenotypes consistent with symptoms observed in human patients (9,14,19,(23)(24)(25)(26)(27)36). Here, we report the functional characterization of zebrafish nphp3 ortholog via morpholino oligo-mediated knockdown.…”

Open Sesame: How Transition Fibers and the Transition Zone Control Ciliary Composition

“…3). An important hub in this network is Cep290, which is part of the MKS complex but also binds Nphp5, a basal body and TZ protein that associates with two NPHP complex components (Schäfer et al 2008; Sang et al 2011; Barbelanne et al 2013; Barbelanne et al 2015; Gupta et al 2015). Inversin is another such hub, linking the MKS and NPHP complexes to the inversin/Nphp3/Nek8/Anks6/Anks3 complex (Sang et al 2011; Hoff et al 2013; Leettola et al 2014; Czarnecki et al 2015; Yakulov et al 2015).…”

Nephronophthisis

“…The primary cilium of renal epithelial cells corresponds to the connecting cilia of the photoreceptors of the retina [77]. In addition to nephrocystin-5, expression of nephrocystin-6 was also shown in the connecting cilium of the photoreceptors and nephrocystin-5 and 6 were shown to interact with each other [29,78]. …”