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. 2021 Jul 28;81(19):5074–5088. doi: 10.1158/0008-5472.CAN-20-4321

Figure 4.

Figure 4.

circBART2.2 binds to RIG-I protein through the helicase domain. A, Biotin-labeled or unbiotin-labeled circBART2.2 probes were transfected into HONE1 cells after the overexpression of circBART2.2. The cell lysates were incubated with biotin-affinity magnetic beads for 2 hours. The precipitated proteins were resolved by SDS-PAGE, followed by Coomassie blue staining. Next, the differential band in the biotin-labeled circBART2.2 probe lane was identified by LC-MS/MS. The unbiotin-labeled circBART2.2 probe served as a control. RIG-I was also identified. B, The expression of RIG-I in 52 NPC tissues and 36 noncancerous NPE tissues by IHC using an anti-RIG-I antibody. P < 0.0001. C, Binding of circBART2.2 to RIG-I protein was detected in HONE1 and HK1 cells after overexpression of circBART2.2 using RNA pulldown assays with a biotin-labeled circBART2.2 probe. The unbiotin-labeled circBART2.2 probe was used for control. D, Direct binding of RIG-I protein to circBART2.2 was evaluated in HONE1 and HK1 cells after overexpression of circBART2.2 or in EBV-positive HONE1-EBV and HK1-EBV cells by RNA immunoprecipitation using anti–RIG-I antibody, followed by qPCR analysis for circBART2.2. CircPVT1 was used as a negative control. ***, P < 0.001. E, The binding between circBART2.2 and the helicase domain of RIG-I protein was detected in HONE1 and HK1 cells after cotransfection of the circBART2.2 overexpression vector and the RIG-I full-length or truncated fragments using RNA pulldown assays with a biotin-labeled circBART2.2 probe. F, The binding between circBART2.2 and the helicase domain of RIG-I protein was examined in HONE1 and HK1 cells after the cotransfection of the circBART2.2 overexpression vector and the Flag-tagged RIG-I full-length or truncated fragments by RNA immunoprecipitation using anti-Flag-RIG-I antibody. IgG was used as a control. G, The 114–165 nt of circBART2.2 was crucial for the binding between circBART2.2 and RIG-I proteins. HONE1 and HK1 cells were transfected with the full-length circBART2.2 (WT) or deletion mutants (367–11 nt for DEL1, 62–89 nt for DEL2, 114–165 nt for DEL3). RNA pulldown assays were performed using a biotin-labeled circBART2.2 probe, followed by Western blotting using anti–RIG-I antibody. Unbiotin-labeled circBART2.2 or mutant probes were used as controls. H, RIG-I protein directly binds to the 114–165 nt of circBART2.2 in HONE1 and HK1 cells. Cells were cotransfected with the FLAG-tagged RIG-I vector and the full-length circBART2.2 (WT) or deletion mutants (DEL1, DEL2, and DEL3). RNA immunoprecipitation was performed using anti-Flag-RIG-I antibody, followed by qPCR analysis for circBART2.2. NS, not significant; ***, P < 0.001.