Fig. 6

circCDYL2 recruits EIF3D protein to promote RAD51 translation initiation. A. RNA pulldown showed the interaction between circCDYL2 and EIF3D protein in HNE and CNE2 cells. B. Immunofluorescence demonstrated co-localization of circCDYL2 and EIF3D proteins in HNE and CNE2 cells. Blue, DAPI; Red, Dig-labeled circCDYL2; Green, anti-EIF3D antibody; Yellow, the co-localization of circCDYL2 and EIF3D protein. Scale bar = 10 μm. C. RNA immunoprecipitation (RIP) assay showed EIF3D proteins interacted with circCDYL2 in HNE2 and CNE2 cells. D. RIP experiments showed that EIF3D protein interacted with RAD51 mRNA in HNE2 and CNE2 cells. E. CircRIP experiments showed that circCDYL2 bound to RAD51 mRNA in HNE2 and CNE2 cells. F. RIP assays showed circCDYL2 enhanced the binding of EIF3D protein with RAD51 mRNA in NPC cells after overexpression or knockdown of circCDYL2. G. Polysome profiling was used to analyze the expression level of RAD51 mRNA in NPC cells after simultaneous knockdown of circCDYL2 with overexpression of EIF3D or overexpression of circCDYL2 with knockdown of EIF3D, respectively, using GAPDH mRNA as a control. H. Western blotting showed that EIF3D partially reverses the regulation of circCDYL2 on the expression of RAD51 protein in NPC cells after simultaneous knockdown of circCDYL2 with overexpression of EIF3D or overexpression of circCDYL2 with knockdown of EIF3D, respectively. Data were represented as mean ± SD. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001