Journal: PLOS Biology

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

doi: 10.1371/journal.pbio.3002302

Figure Lengend Snippet: (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 and files. 3AT, 3-aminotriazole; Bicc1, Bicaudal-C1; GST, glutathione S-transferase; KH, K-homology; SAM, sterile alpha motif.

Article Snippet: Extracts were centrifuged twice at 10,000 × g for 5 min at 4°C and 5% of each set aside as controls for the “input.” For immunoprecipitation, 20 μL of Protein G-sepharose beads (GE Healthcare) coated with 12 μL rabbit anti-Bicc1, or 20 μL of mouse anti-HA beads (Sigma) and preabsorbed with RNAse-free BSA (800 μg/mL) were incubated with the remainder of each extract for 2 h at 4°C on a rotating wheel, then rinsed 4 times 10 min in wash buffer (20 mM Tris-HCl (pH 7.4), 2 mM MgCl 2 , 200 mM NaCl, 1 mM DTT, and 0.1% NP-40).

Techniques: Binding Assay, Activation Assay, Recombinant, RNA Binding Assay, Sterility

Journal: PLOS Biology

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

doi: 10.1371/journal.pbio.3002302

Figure Lengend Snippet: (A) Structure of Bicc1-ANKS3-ANKS6 complexes predicted by AlphaFold, viewed from above the RNA-binding KH domain surfaces (left) or from the side (right). The structured domains are annotated. For the sake of clarity, intrinsically disordered regions are not shown. (B) Left: Pull-down of ANKS3-Flag full-length or ∆Nter alone, or together with v5-ANKS6 by GST-KH. Note that GST-KH was so abundant in bead eluates that it is faintly visible also in the anti-Flag western blot as a shady band closely above ANKS3 ΔNter marked by a blue asterisk. The amounts of v5-ANKS6 relative to ANKS3-Flag that were pulled down from the HEK293T cell extracts are quantified below. Right: Quantification of GST-KH binding to the indicated ANKS3 truncation mutants versus full-length ANKS3-Flag (100%). 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 and files. Bicc1, Bicaudal-C1; EH, end-helix; GST, glutathione S-transferase; KH, K-homology; ML, mid-loop; SAM, sterile alpha motif.

Article Snippet: Extracts were centrifuged twice at 10,000 × g for 5 min at 4°C and 5% of each set aside as controls for the “input.” For immunoprecipitation, 20 μL of Protein G-sepharose beads (GE Healthcare) coated with 12 μL rabbit anti-Bicc1, or 20 μL of mouse anti-HA beads (Sigma) and preabsorbed with RNAse-free BSA (800 μg/mL) were incubated with the remainder of each extract for 2 h at 4°C on a rotating wheel, then rinsed 4 times 10 min in wash buffer (20 mM Tris-HCl (pH 7.4), 2 mM MgCl 2 , 200 mM NaCl, 1 mM DTT, and 0.1% NP-40).

Techniques: RNA Binding Assay, Western Blot, Binding Assay, Sterility

Journal: PLOS Biology

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

doi: 10.1371/journal.pbio.3002302

Figure Lengend Snippet: (A) Western blot analysis of ANKS3-Flag from HEK293T cell extracts before (input) and after pull-down by in vitro reconstituted RNPs of recombinant GST-Bicc1 that were pre-assembled with saturating amounts of the fluorescently labeled Dand5-3′ UTR RNA fragment 66–110 or, as a control for nonspecific binding, fragment 226–270 (*). Coomassie blue staining of GST-Bicc1 protein retained by the beads (bottom panel) and the fluorescence of bound RNA (middle panels) are shown below. The quantification of the amounts of target RNA (*) retained by the GST-Bicc1 beads before and after incubation with ANKS3-Flag is shown below the gels. The values for the amounts of pulled down ANKS3-Flag shown in the histogram to the right were normalized to controls without target RNA (100%). (B) Cartoon summarizing the result shown in panel A. (C) Western blot analysis of ANKS3-Flag before (input) and after pull-down by the SAM polymerization mutant GST-Bicc1 mutD versus WT GST-Bicc1 that were saturated with fluorescently labeled Dand5-3′ UTR RNA fragment 66–110 as in (A). Pull-downs of RNA and ANKS3-Flag were quantified as in (A). Values for the amount of bound RNA are relative to the pull-down by WT GST-Bicc1 (100%). (D) Cartoon summarizing the result shown in panel C. 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 and files. GST, glutathione S-transferase; KH, K-homology; RNP, ribonucleoparticle; SAM, sterile alpha motif; WT, wild-type.

Article Snippet: Extracts were centrifuged twice at 10,000 × g for 5 min at 4°C and 5% of each set aside as controls for the “input.” For immunoprecipitation, 20 μL of Protein G-sepharose beads (GE Healthcare) coated with 12 μL rabbit anti-Bicc1, or 20 μL of mouse anti-HA beads (Sigma) and preabsorbed with RNAse-free BSA (800 μg/mL) were incubated with the remainder of each extract for 2 h at 4°C on a rotating wheel, then rinsed 4 times 10 min in wash buffer (20 mM Tris-HCl (pH 7.4), 2 mM MgCl 2 , 200 mM NaCl, 1 mM DTT, and 0.1% NP-40).

Techniques: Western Blot, In Vitro, Recombinant, Labeling, Binding Assay, Staining, Fluorescence, Incubation, Mutagenesis, Sterility

Journal: PLOS Biology

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

doi: 10.1371/journal.pbio.3002302

Figure Lengend Snippet: (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 and files. Bicc1, Bicaudal-C1; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

Article Snippet: Extracts were centrifuged twice at 10,000 × g for 5 min at 4°C and 5% of each set aside as controls for the “input.” For immunoprecipitation, 20 μL of Protein G-sepharose beads (GE Healthcare) coated with 12 μL rabbit anti-Bicc1, or 20 μL of mouse anti-HA beads (Sigma) and preabsorbed with RNAse-free BSA (800 μg/mL) were incubated with the remainder of each extract for 2 h at 4°C on a rotating wheel, then rinsed 4 times 10 min in wash buffer (20 mM Tris-HCl (pH 7.4), 2 mM MgCl 2 , 200 mM NaCl, 1 mM DTT, and 0.1% NP-40).

Techniques: Quantitative RT-PCR, Expressing, shRNA, Western Blot, Real-time Polymerase Chain Reaction

Journal: PLOS Biology

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

doi: 10.1371/journal.pbio.3002302

Figure Lengend Snippet: (A) Top: Representative western blots of the protein fractions in RNA co-immunoprecipitates from cytoplasmic extracts of HEK293T cells expressing the dsVenus- Dand5 -3′ UTR reporter and HA-Bicc1 alone or in combination with full-length or truncated ANKS3-Flag. Three transfection doses (×1, ×2, and ×4) were used for ANKS3 ΔCter. The amounts of soluble ANKS3-Flag relative to HA-Bicc1 in the IP fractions are quantified below the blots. Bottom: Below the immunoblots, RT-qPCR analysis shows the ratios of co-immunoprecipitated Dand5 -3′ UTR reporter mRNA and HA-Bicc1 transcript normalized to their amounts in inputs and to HA-Bicc1 protein in the IP, relative to the control HA-Bicc1 IP without ANKS3 (100%). (B) Top: Representative western blots of the protein fractions in RNA co-immunoprecipitates from cytoplasmic extracts of HEK293T cells expressing the dsVenus- Dand5 3′ UTR reporter and HA-Bicc1 alone or in combination with ANKS3-Flag, or with its truncated form (∆Nter), and with or without v5-ANKS6. The amounts of soluble ANKS3-Flag relative to HA-Bicc1 in the IP fractions are quantified below the blots. Bottom: The RT-qPCR analysis shown below indicates the ratios of co-immunoprecipitated Dand5 -3′ UTR reporter mRNA and HA-Bicc1 transcript normalized to their amounts in inputs and to HA-Bicc1 protein in the IP, relative to the control HA-Bicc1 IP without ANKS3 (100%). Data are means + SD from at least 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 and files. Bicc1, Bicaudal-C1; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

Article Snippet: Extracts were centrifuged twice at 10,000 × g for 5 min at 4°C and 5% of each set aside as controls for the “input.” For immunoprecipitation, 20 μL of Protein G-sepharose beads (GE Healthcare) coated with 12 μL rabbit anti-Bicc1, or 20 μL of mouse anti-HA beads (Sigma) and preabsorbed with RNAse-free BSA (800 μg/mL) were incubated with the remainder of each extract for 2 h at 4°C on a rotating wheel, then rinsed 4 times 10 min in wash buffer (20 mM Tris-HCl (pH 7.4), 2 mM MgCl 2 , 200 mM NaCl, 1 mM DTT, and 0.1% NP-40).

Techniques: Western Blot, Expressing, Transfection, Quantitative RT-PCR, Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: PLOS Biology

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

doi: 10.1371/journal.pbio.3002302

Figure Lengend Snippet: (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 [ , ]. 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.

Article Snippet: Extracts were centrifuged twice at 10,000 × g for 5 min at 4°C and 5% of each set aside as controls for the “input.” For immunoprecipitation, 20 μL of Protein G-sepharose beads (GE Healthcare) coated with 12 μL rabbit anti-Bicc1, or 20 μL of mouse anti-HA beads (Sigma) and preabsorbed with RNAse-free BSA (800 μg/mL) were incubated with the remainder of each extract for 2 h at 4°C on a rotating wheel, then rinsed 4 times 10 min in wash buffer (20 mM Tris-HCl (pH 7.4), 2 mM MgCl 2 , 200 mM NaCl, 1 mM DTT, and 0.1% NP-40).

Techniques: RNA Binding Assay, Sterility