DNAAF1 links heart laterality with the AAA+ ATPase RUVBL1 and ciliary intraflagellar transport

Abstract

DNAAF1 (LRRC50) is a cytoplasmic protein required for dynein heavy chain assembly and cilia motility, and DNAAF1 mutations cause primary ciliary dyskinesia (PCD; MIM 613193). We describe four families with DNAAF1 mutations and complex congenital heart disease (CHD). In three families, all affected individuals have typical PCD phenotypes. However, an additional family demonstrates isolated CHD (heterotaxy) in two affected siblings, but no clinical evidence of PCD. We identified a homozygous DNAAF1 missense mutation, p.Leu191Phe, as causative for heterotaxy in this family. Genetic complementation in dnaaf1-null zebrafish embryos demonstrated the rescue of normal heart looping with wild-type human DNAAF1, but not the p.Leu191Phe variant, supporting the conserved pathogenicity of this DNAAF1 missense mutation. This observation points to a phenotypic continuum between CHD and PCD, providing new insights into the pathogenesis of isolated CHD. In further investigations of the function of DNAAF1 in dynein arm assembly, we identified interactions with members of a putative dynein arm assembly complex. These include the ciliary intraflagellar transport protein IFT88 and the AAA+ (ATPases Associated with various cellular Activities) family proteins RUVBL1 (Pontin) and RUVBL2 (Reptin). Co-localization studies support these findings, with the loss of RUVBL1 perturbing the co-localization of DNAAF1 with IFT88. We show that RUVBL1 orthologues have an asymmetric left-sided distribution at both the mouse embryonic node and the Kupffer’s vesicle in zebrafish embryos, with the latter asymmetry dependent on DNAAF1. These results suggest that DNAAF1-RUVBL1 biochemical and genetic interactions have a novel functional role in symmetry breaking and cardiac development.

Role of the Novel Hsp90 Co-Chaperones in Dynein Arms’ Preassembly

“…The biochemical and immunofluorescence analyses indicate that DNAAF1/ODA7 and its orthologue in Trypanosoma brucei similar to other a DNAAFs, is localized in the cytoplasm [17,29]. DNAAF1 over-expressed in ciliated hTERT-RPE1 cells co-localized with IFT88 at the base of the primary cilium and RuvBL1 knockdown caused disruption of this co-localization [56]. In humans, cilia of the respiratory cells obtained from the patients carrying a DNAAF1 mutation (CILD13), lacked ODAs and IDAs subunits (DNAH5, DNAH9, DNAI2 and DNALI1) ( Table 2) [29,57].…”

“…Both RuvBL1 and RuvBL2 can also interact with DNAAF1/LRRC50/ODA7 (Leucine-Rich Repeat-Containing 50) ( Figure 2, Table 1) [56]. Mutations of DNAAF1/ODA7 in Chlamydomonas reduce flagella beat frequency and a block outer dynein arm assembly [13,68].…”

“…Interestingly, in humans the mutation in DNAAF1 was also associated with neural tube defects (DNAAF1 seems to be essential for the final step of neural tube closure) [69]. Similar to humans, mutations of DNAAF1 in mice and zebrafish cause changes typical of PCD in these species [56]. The biochemical analysis revealed that several heat shock proteins, including Hsp90, were more strongly associated with the mutanted DNAAF1 protein than the wild-type protein, which may be evidence of a possible instability of the mutated protein.…”

Rare Human Diseases: Model Organisms in Deciphering the Molecular Basis of Primary Ciliary Dyskinesia

“…Several hours later, cilia can be also observed in other locations, including pronephric ducts and brain ventricles [53,54]. Mutations in genes affecting cilia assembly or function frequently result in the characteristic curly body shape of the developing embryo, laterality defects caused by the dysfunction of cilia in the Kupffer's vesicle, heart looping, perturbation of otolith formation in the inner ear, the formation of cyst in kidneys, and hydrocephalus [42,53,55,56].…”

Rsph4a is essential for the triplet radial spoke head assembly of the mouse motile cilia

“…To date, numerous causal genes have been identified in PCD patients [1]. Typical PCD causal genes are involved in the assembly of the axonemal dynein complex of human motile cilia [2][3][4][5][6][7][8][9][10]. In mice and humans, multiple motile cilia exist in the trachea, brain/ependymal, oviduct, inner ear, nasal, and testis.…”