Fig. 5: CircKIF18A prevents UBE3A-mediated degradation of MCM7 in NPCs.

a A schematic showing six constructs, including full-length circKIF18A and truncations with different regions of circKIF18A. b RIP showed that the full-length and truncation 2 (circ-T2) mutant of circKIF18A interacted with MCM7 in HEK-293T cells. HEK-293T cells were separately transfected with plasmids containing full-length circKIF18A, its truncations and empty vector (n = 3; NS no significance; *P < 0.05; **P < 0.01). c Schematic illustration of full-length MCM7 and its three constructs containing different domains. d The results of RIP showed that only the CD1 mutant construct did not interact with circKIF18A. HEK-293T cells were separately transfected with plasmids containing full-length Myc-MCM7; plasmids containing the Myc-ND1, Myc-CD1, and Myc-MD1 mutant constructs; or empty vector (n = 3; NS no significance; *P < 0.05; **P < 0.01). e The effect of UBE3A on the MCM7 protein level in circKIF18A-silenced NPCs was detected using western blot analysis (n = 3, three different donors). f IP analysis of the interaction between UBE3A and MCM7 in NPCs transfected with increasing concentrations of the circKIF18A lentivirus (n = 3, three different donors). g RIP confirmed that circKIF18A directly interacted with MCM7 but not UBE3A in NPCs (n = 3, three different donors for three individual experiments; NS no significance; *P < 0.05; **P < 0.01). h and i IP analysis of the interaction between UBE3A and MCM7 in HEK-293T cells transfected with plasmids encoding circKIF18A, Myc-MCM7 or HA-UBE3A in NPCs (n = 3). j Sequence alignment between the UBE3A-binding motif in MCM7 and those in other known UBE3A substrates. k HEK-293T cells were transfected with plasmids encoding Myc-MCM7, Myc-MCM7△L2G, HA-UBE3A, or circKIF18A. Subsequent western blot analyses were performed to determine Myc-tagged MCM7 and HA-tagged UBE3A levels (n = 3). All data are presented as the mean ± SD values.