Fig 2: Left-sided asymmetric expression of RUVBL1 at the mouse embryonic node: (A) whole-mount in situ hybridization (WISH) of Ruvbl1 probes for whole-mount head-fold stage wild-type C3H/HeH mouse embryos showing widespread, ubiquitous Ruvbl1 expression and left-sided asymmetric distribution at the embryonic node. Grey frame indicates magnified inset (lower left) with a second example at lower right, with Ruvbl1 expression indicated by arrowheads. Scale bar = 100 μm. (B) Light-sheet microscopy images of whole-mount immunofluorescence for RUVBL1 (red) and DAPI (blue) in the wild-type C57BL/6 mouse E7.25 and E7.5 (late head fold and 2–4 somite stage) embryonic node, and E9.0 (6–8 somite stage) vasculature and heart loop. Left (L) and right (R) sides are indicated. RUVBL1 is asymmetrically distributed at the murine embryonic node (upper two panels). RUVBL1 is present in a widespread distribution throughout the atrial chambers, primitive ventricle and developing vasculature (lower two panels). Scale bars = 100 or 200 μm, as indicated. Abbreviations: rAC and IAC, right and left atrial chambers; EN, embryonic node; L, left; N, notochord; PV, primitive ventricle; R, right; ST, septum transversum.

Fig 3: Interactions of DNAAF1 with IFT88 and RUVBL1: (A) Schematic diagram to show DNAAF1 and IFT88 domains. Full-length DNAAF1 contains 6 leucine-rich repeats (LRR), a coiled-coil domain (CC) and a proline-rich domain (PRO). IFT88 contains 3 N-terminal and 7 C-terminal tetratricopeptide repeats (TPR). A dedicated yeast two-hybrid for DNAAF1-BD demonstrates interaction with full-length IFT88 and N-terminally truncated IFT88 (442–833). The interaction is not observed in C-terminal truncated IFT88 (1–449). (B) Confirmation of the interaction between DNAAF1 and IFT88, demonstrated by co-immunoprecipitation assays comprising over-expression of wild-type (wt; p.Leu191) or mutant (p.Phe191) FLAG-DNAAF1, pull-down of endogenous IFT88 by anti-IFT88 antibody and western blot (WB) with anti-FLAG. Input whole cell extracts indicate significantly reduced levels of DNAAF1 mutant (p.Phe191) compared to wild-type protein, with β-actin used a loading control. Negative control IgG lanes and IgG heavy chain (HC) bands are indicated. (C) Co-immunoprecipitation assays of over-expressed wild-type (wt; p.Leu191) or mutant (p.Phe191) FLAG-DNAAF1 and IFT88-eYFP, pull-down with anti-FLAG antibody and western blot (WB) with anti-GFP “Living Colors” antibody. Input whole cell extract and irrelevant control IgG lanes are indicated, with empty vector as the negative control. The largest IFT88 band that is immunoprecipitated by the wild-type DNAAF1 protein, is indicated by the arrowheads, and this interaction is lost with the mutant DNAAF1 protein. Non-specific protein bands, indicated by the asterisks, are likely to be due to cross-reaction of the secondary antibody with the anti-FLAG IgG used in these coIPs. (D) Interaction between DNAAF1 and RUVBL1 (Pontin) shown after over-expression of wildtype (p.Leu191) or mutant (p.Phe191) FLAG-DNAAF1, pull-down with anti-FLAG antibody and western blot (WB) blot with anti-RUVBL1 antibody.

Fig 4: DNAAF1-dependent left-sided asymmetric expression of RUVBL1 at the zebrafish Kupffer’s vesicle: (A) in situ hybridization (ISH) of Ruvbl1 expression at Kupffer’s vesicle (thin dashed black line) in wild-type heterozygous dnaaf1+/− and mutant homozygous dnaaf1−/− zebrafish embryos, at the 5 to 9 somite stages, visualized from dorsal views as indicated in the top left inset. Localized expression is indicated by arrowheads, and the mid-line of the notochord is shown by the thick dashed black line. Left (L) and right (R) sides are indicated. (B) Bar graph shows a significantly higher proportion of wild-type zebrafish (+/−) have left-sided localisation of ruvbl1 when compared to diffused central localization in mutant (−/−) embryos (P = 0.0023, unpaired Student’s t-test). (C) Additional examples of ruvbl1 expression at Kupffer’s vesicle in unaffected dnaaf1+/− heterozygous and dnaaf1−/− affected mutant zebrafish embryos.

Fig 5: Proposed model of DNAAF1 function in an RT2P-like complex and the IFT-B ciliary transport complex: Unfolded dynein arm components (brown) are folded via the action of DNAAF1 within an ODA pre-assembly module consisting of the AAA+ ATPases RUVBL1 and RUVBL2 (tan), RT2P-like complex proteins (grey) such as DNAAF1, DNAAF2/KTU, and other chaperone proteins (blue) such as SUGT1 and HSP90. In addition to dynein arm assembly through an RT2P-like complex, DNAAF1 also associates with the IFT-B complex (pink; represented by IFT88) allowing dynein arm assembly to be coupled to ciliary transport. RUVBL1 may also have a separate, direct gene regulatory role on asymmetrically expressed genes such as Nodal.