m⁶A methylation inhibits recruitment of the Dand5 3′UTR to the left–right determinant Bicc1

m⁶A methylation of the AGACGUGAC motif inhibits the binding of Bicc1 to the proximal Dand5 3′UTR. (A) In vitro–transcribed Dand5 3′UTR_66–110 RNA and variant versions used as competitors in EMSA assays to assess the binding of Bicc1. The secondary structure and minimum free energy (ΔG) of the Dand5 3′UTR_66–110 RNA were predicted by the RNAfold server (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi). (B) Principle of the competitive EMSA experiment: increasing amounts of unlabeled competitors were added to preassembled complexes between recombinant GST-KH fusion protein and Dand5 3′UTR_66–110 RNA marked with a fluorescent probe (green bold line) complementary to an extrinsic sequence used as a 3′ tag (black bold line). Destruction of the fluorescent ribonucleoparticle (*RNP) by the competitor RNA indicates specific interaction. (C) EMSA analysis of competitive binding of Bicc1 KH domains to synthetic transcripts of unmodified (unmod.) or m⁶A-methylated (m⁶A) Dand5 3′UTR_66-110 against the fluorescent unmodified Dand5 3′UTR_66–110 RNA probe. A Bicc1 binding null mutant (Mut1) (Minegishi et al. 2021) was used as a negative control to show absence of competition. (D) Graph showing the % of residual RNP relative to the indicated competitor RNA concentration, and the half maximal inhibitory concentrations (IC50) of each competitor. P-values (asterisks) above the curves refer to differences between each variant versus the unmodified RNA, whereas differences between variants ([n.s.] not significant) are marked by brackets. (E) Cartoon depicting the GST pull-down experiment presented in F and G. (F) Western blot analysis of ANKS3-FLAG from HEK293T cell extracts before (input) and after pull-down by recombinant GST-Bicc1 preincubated or not with saturating amounts of unmodified or m⁶A Dand5 3′UTR_66–110 RNA. Coomassie blue staining of GST-Bicc1 protein retained by the beads (bottom panel) is shown below. (G) Histogram showing the amounts of pulled down ANKS3-FLAG shown in panel F experiment. Values were normalized to the control without target RNA (100%). Data are means ± SD from three independent experiments. (ns) Nonsignificant, (*) P < 0.05, (**) P < 0.01, (***) P < 0.001 (Student's t-test).