Fig. 3: circCDK13 interacts with IGF2BP3 in an m6A-dependent manner.
a Schematic of RNA pull-down assay. b Silver staining of proteins pulled down by biotin-labeled probe specific for circCDK13 and control probe. c Venn diagram of RNA pull-down protein products after LC-MS/MS analysis. d Venn diagram of the overlapping RBPs between circCDK13-interacting proteins identified by LC-MS/MS and RBPs predicted to bind to circCDK13 by catRAPID omics v2.0. e Mountain plot representing the minimum free energy (MFE, red), the thermodynamic ensemble (green) and the centroid structures (blue) of circCDK13. f Graphical representation of the molecular docking between circCDK13 and IGF2BP3 protein using NPDock. g The interaction between circCDK13 and IGF2BP3 was verified by RNA pull-down and western blot assays. GAPDH was used as a negative control. h RIP assay validated the interaction between circCDK13 and IGF2BP3. i RT-qPCR analysis of relative enrichment of circCDK13 in IGF2BP3 RIP products. j FISH and IF co-staining showing co-localization of circCDK13 (red) with IGFB2P3 (green) in HDFs and HEKs. Scale bar, 50 μm. k RNA pull-down and western blot assays confirmed that IGF2BP3 bound to circCDK13 through the KH domains. l Prediction of circCDK13-IGF2BP3 interaction using the catPAPID algorithm and schematic of IGF2BP3 with functional protein domains. m Analysis for the circCDK13 enrichment, relative to input. RIP assay was performed using HA antibody in HDFs and HEKs. n Gene-specific m6A RT-qPCR assay detected m6A modification of circCDK13 in HDFs and HEKs with or without METTL3/14 knockdown. o RNA pull-down and western blot assays were used to detect the interaction between circCDK13 and IGF2BP3 in the indicated group. p−s The half-life of circCDK13 after treatment with 5 μg/L actinomycin D for the indicated times in HDFs and HEKs with knockdown or overexpression of IGF2BP3. t The protein level of IGF2BP3 in HDFs and HEKs with knockdown or overexpression of circCDK13 treated with cycloheximide (25 μg/ml) for 12 h. In (b, g, h, k, o, t) three independent experiments were performed and similar results were obtained. Comparisons were performed by one-way ANOVA followed by Dunnett’s multiple comparisons test in (q, s) and two-tailed Student’s t test in (i, m, n, p, r). Data are presented as mean values ± SD (n = 3 biologically independent samples). Source data are provided as a Source Data file.
