Fig. 6: The direct interaction between circMAN1A2(2,3,4,5) and CENPB mRNA enhances the IGF2BP2-mediated CENPB mRNA stability.

a Schematic of in-cell AMT-crossed RNA pull-down assay. b CircMAN1A2(2,3,4,5) interacted with CENPB mRNA in vivo, as revealed by in-cell AMT-crossed RNA pull-down assay. GAPDH mRNA is a negative control. c Schematic of in vitro RNA pull-down assay. d IvcMAN1A2(2,3,4,5) interacted with CENPB-3′UTR in vitro, as revealed by in vitro RNA pull-down assay. e Prediction of RNA interaction sequences between CENPB mRNA and circMAN1A2(2,3,4,5) by IntaRNA. The yellow highlight indicates seed region of interacted sequence, which exactly covers the BSJ site of circMAN1A2(2,3,4,5). The pink and red lines indicate LNA pairing to exon2 and BSJ sequence of circMAN1A2(2,3,4,5), respectively. The green line indicates LNA pairing to 3′UTR of CENPB mRNA. f LNA-blocked RNA pull-down assay revealed the specific binding sites of CENPB-3′UTR on circMAN1A2(2,3,4,5). g LNA-blocked RNA pull-down assay revealed the specific binding sites of circMAN1A2(2,3,4,5) on CENPB-3′UTR. h LNA-BSJ reduced CENPB mRNA stability in DLD-1 cells (n = 3 independent experiments). LNA-NC and LNA-BSJ were transfected into DLD-1 for 48 h and then were treated with DRB (100 μM) for 0, 0.5, 1, 2, 3, and 4 h followed by RNA isolation and RT-qPCR. i LNA-BSJ reduced CENPB mRNA level in DLD-1 cells. j KD of circMAN1A2(2,3,4,5) reduced CENPB mRNA stability in DLD-1 cells (n = 3 independent experiments). k KD of circMAN1A2(2,3,4,5) reduced the association of IGF2BP2 on CENPB mRNA in DLD-1 cells, as revealed by native RIP assay. l A proposed model of circMAN1A2(2,3,4,5)-IGF2BP2-CENPB-3′UTR interaction. Through directly RNA-RNA interaction, circMAN1A2(2,3,4,5) enhanced the association of IGF2BP2 on CENPB mRNA, which impacting its mRNA stability and expression. RNA levels were detected by RT-qPCR (b, d, f–k), normalized to ACTB (i) and presented as mean ± s.d. (b, d, f–k), with biologically individual data points shown (b, d, f, g, i, k). P values were determined by ordinary two-way ANOVA with Dunnett’s post-hoc comparisons versus scramble control at each row factor level (adjusted P values) (b, d, f–h, j, k) and unpaired two-tailed Student’s t-test (i). Source data are provided as a Source Data file.