Fig 2: Left/right patterning defects in Anks3Δ/Δ mutant mice.(A) Lungs, hearts, thoracic, and abdominal blood vessels of representative Anks3 Δ/Δ pups and heterozygous control litter mates on postnatal day P0. Schematic depictions of organ patterning are shown below. R, L: right and left pulmonary lobes, respectively. (B) Schematic ventral view of a mouse embryo at E8.0 (left) and WISH analysis of Nodal mRNA expression in Anks3Δ/Δ embryos and wild-type control litter mates at 4- and 7-somite stages (right). Scale bars, 500 μm or 100 μm for the magnified node regions (insets). The Nodal pattern (left, bilateral, or absent) in LPM and the number of embryos corresponding to each phenotype are indicated. (C) dsVenus fluorescence at the node of Anks3Δ/Δ and wild-type control embryos harboring the NDE-Hsp-dsVenus-Dand5 3′ UTR transgene. The dsVenus coding sequence fused to the DNA sequence for the 3′ UTR of mouse Dand5 mRNA is under the control of the mouse Hsp68 promoter and 4 copies of the crown cell-specific enhancer (NDE) of mouse Nodal. Images are representative of at least 6 embryos per genotype at the 3- to 5-somite stage. The graphs represent the global dsVenus intensity and fluorescence ratios on the right versus left side of the node for each genotype. Blue circles indicate individual values. Data are means ± SD. Statistical significance was determined using the Mann–Whitney U test, with ns: nonsignificant, ***p < 0.001, ****p < 0.0001. Scale bar, 50 μm. Underlying data can be found in the S1 Raw Values file. LPM, lateral plate mesoderm. WISH, whole mount in situ hybridization.

Fig 3: ANKS3 binds to the Bicc1 KH domains and competes with RNA binding.(A) Yeast two-hybrid mapping of the interaction between a fusion of ANKS3 with the DNA-binding domain of Gal4 (Gal4-BD) and human BICC1 KH domains fused to Gal4 activation domain (Gal4-AD). Controls in nonselective medium without leucine (L) and tryptophan (T) are shown in the first column (LT−). Interactions were revealed at the indicated concentrations of 3AT in triple selective medium (LTH−) lacking histidine. (B) Pull-down of ANKS3-Flag from HEK293T cell extracts by glutathione sepharose beads coated with recombinant domains of Bicc1 fused to GST. (C, D) 3D model of structured Bicc1 interfaces with ANKS3 predicted by AlphaFold viewed from above the RNA-binding KH domain surfaces (C) or sideways (D). The structured domains are annotated. For the sake of clarity, intrinsically disordered regions are not shown. Note the interaction between the Bicc1 KH domains and a C-terminal coiled coil of ANKS3 (Cter). (E) Cartoon depicting the domain organization of full-length ANKS3 and its truncated variants. (F) Left: Pull-down of full-length and truncated ANKS3-Flag in HEK293T cell extracts by GST-Bicc1 or GST alone (control). Right: Quantification of bound ANKS3-Flag (100%) and of its indicated truncation mutants. (G) Left: pull-down of ANKS3-Flag in HEK293T cell extracts by GST-Bicc1-KH in presence or absence of v5-ANKS6. The amounts of v5-ANKS6 that were pulled down from the HEK293T cell extracts are quantified below. Right: Quantification of bound ANKS3-Flag normalized to the pull-down without ANKS6 (100%). (H) Cartoon depicting the protein–protein interactions observed in panel G. Data are means + SD from 3 independent experiments. ns: nonsignificant, *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test). Underlying data can be found in the S1 Raw Images and S1 Raw Values files. 3AT, 3-aminotriazole; Bicc1, Bicaudal-C1; GST, glutathione S-transferase; KH, K-homology; SAM, sterile alpha motif.

Fig 4: ANKS3 stimulates the RNA-binding activity of endogenous Bicc1 in IMCD3 cells.(A) RT-qPCR analysis of endogenous Anks3 mRNA in IMCD3 cells treated with or without 0.25 μg/ml doxycycline (Dox) to induce the expression of Anks3 shRNA. The levels of Anks3 relative to β-actin mRNAs is expressed as a percentage of the baseline in untreated cells. (B) Western blot of endogenous Bicc1 in cytoplasmic extracts (inputs) and immunoprecipitates of IMCD3 cells treated with or without doxycycline to induce Anks3 shRNA. γ-tubulin was a loading control. The levels of Bicc1 protein are expressed as a fold change of the baseline in untreated cells. For the inputs, the fold change was calculated after normalization to the γ-tubulin signal that served as internal control. (C) RT-qPCR analysis of the indicated mRNAs in IMCD3 before and after Anks3 depletion in IMCD3 cells. The values are expressed as the fold change normalized to the untreated condition. The y-axis represents the amount of mRNA in Bicc1 immunoprecipitates normalized to Bicc1 protein in the IP fraction. Data are means + SD from 4 independent experiments. ns: nonsignificant, *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test). Underlying data can be found in the S1 Raw Images and S1 Raw Values files. Bicc1, Bicaudal-C1; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

Fig 5: Model of the licensing of Bicc1 RNP formation by ANKS3 and ANKS6.(A) In absence of ANKS6, ANKS3 interacts with both ML and EH surfaces of the Bicc1 SAM domain. In parallel, the coiled coil of the ANKS3 Cter associates with the KH domains and inhibits RNA binding, while the Nter tends to attenuate this effect. (B) The incorporation of ANKS6 induces a topological remodeling of the complex. Due to a 10-fold higher affinity, ANKS6 hijacks the EH surface of the ANKS3 SAM domain [17,23]. In parallel, ANKS6 cooperates with the ANKS3 Nter to clamp down the coiled coil and to license RNA binding. Bicc1, Bicaudal-C1; EH, end-helix; KH, K-homology; ML, mid-loop; SAM, sterile alpha motif.