Fluid flow-induced left-right asymmetric decay of Dand5 mRNA in the mouse embryo requires a Bicc1-Ccr4 RNA degradation complex

Molecular left-right (L-R) asymmetry is established at the node of the mouse embryo as a result of the sensing of a leftward fluid flow by immotile cilia of perinodal crown cells and the consequent degradation of Dand5 mRNA on the left side. We here examined how the fluid flow induces Dand5 mRNA decay. We found that the first 200 nucleotides in the 3′ untranslated region (3′-UTR) of Dand5 mRNA are necessary and sufficient for the left-sided decay and to mediate the response of a 3′-UTR reporter transgene to Ca2+, the cation channel Pkd2, the RNA-binding protein Bicc1 and their regulation by the flow direction. We show that Bicc1 preferentially recognizes GACR and YGAC sequences, which can explain the specific binding to a conserved GACGUGAC motif located in the proximal Dand5 3′-UTR. The Cnot3 component of the Ccr4-Not deadenylase complex interacts with Bicc1 and is also required for Dand5 mRNA decay at the node. These results suggest that Ca2+ currents induced by leftward fluid flow stimulate Bicc1 and Ccr4-Not to mediate Dand5 mRNA degradation specifically on the left side of the node

Bicc1 ribonucleoprotein complexes specifying organ laterality are licensed by ANKS6-induced structural remodeling of associated ANKS3.

Organ laterality of vertebrates is specified by accelerated asymmetric decay of Dand5 mRNA mediated by Bicaudal-C1 (Bicc1) on the left side, but whether binding of this or any other mRNA to Bicc1 can be regulated is unknown. Here, we found that a CRISPR-engineered truncation in ankyrin and sterile alpha motif (SAM)-containing 3 (ANKS3) leads to symmetric mRNA decay mediated by the Bicc1-interacting Dand5 3' UTR. AlphaFold structure predictions of protein complexes and their biochemical validation by in vitro reconstitution reveal a novel interaction of the C-terminal coiled coil domain of ANKS3 with Bicc1 that inhibits binding of target mRNAs, depending on the conformation of ANKS3 and its regulation by ANKS6. The dual regulation of RNA binding by mutually opposing structured protein domains in this multivalent protein network emerges as a novel mechanism linking associated laterality defects and possibly other ciliopathies to perturbed dynamics in Bicc1 ribonucleoparticle (RNP) formation

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 p p 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.</p

Phenotypes in <i>Anks3</i>^Δ/Δ mutant mice.

Organ laterality of vertebrates is specified by accelerated asymmetric decay of Dand5 mRNA mediated by Bicaudal-C1 (Bicc1) on the left side, but whether binding of this or any other mRNA to Bicc1 can be regulated is unknown. Here, we found that a CRISPR-engineered truncation in ankyrin and sterile alpha motif (SAM)-containing 3 (ANKS3) leads to symmetric mRNA decay mediated by the Bicc1-interacting Dand5 3′ UTR. AlphaFold structure predictions of protein complexes and their biochemical validation by in vitro reconstitution reveal a novel interaction of the C-terminal coiled coil domain of ANKS3 with Bicc1 that inhibits binding of target mRNAs, depending on the conformation of ANKS3 and its regulation by ANKS6. The dual regulation of RNA binding by mutually opposing structured protein domains in this multivalent protein network emerges as a novel mechanism linking associated laterality defects and possibly other ciliopathies to perturbed dynamics in Bicc1 ribonucleoparticle (RNP) formation.</div

ANKS3 and ANKS6 modulate the binding of target mRNAs to HA-Bicc1 in HEK293T cells.

(A) Additional analysis of the RT-qPCR data shown in Fig 6B. Ratios of co-immunoprecipitated mRNAs over the input, normalized to the amount of HA-Bicc1 in the IP fraction and then expressed relative to the corresponding control condition without HA-Bicc1. β-actin mRNA served as a negative control to visualize the level of unspecific RNA binding by Bicc1. (B) Expression level of the dsVenus-Dand5 3′ UTR and HA-Bicc1 mRNAs relative to β-actin measured by RT-qPCR analysis in transfected HEK293T cells. These data are related to the co-immunoprecipitation results shown in Fig 6B. Data are means + SD from between 3 to 10 independent experiments. ns: nonsignificant, *p p p t test). Underlying data can be found in the S1 Raw Values file. (TIF)</p

Reporting of individual data points and statistical analysis shown in main and supplementary figures.

Reporting of individual data points and statistical analysis shown in main and supplementary figures.</p

Oligonucleotide primer sequences used in this study.

Oligonucleotide primer sequences used in this study.</p

Left/right patterning defects in Anks3^Δ/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 p S1 Raw Values file. LPM, lateral plate mesoderm. WISH, whole mount in situ hybridization.</p