Figure 4. m⁶A-modified circFOREIGN evades non-self detection through the binding of YTHDF2.

A. YTHDF2 is necessary for differential response to unmodified vs. m⁶A-modified circFOREIGN. Transfection of unmodified or m⁶A-modified circFOREIGN into YTHDF2−/− HeLa cells stimulate immune response. Left: Model showing the responses to unmodified or m⁶A-modified circFOREIGN. Right: Graph showing gene expression of innate immune genes 24 hours following RNA transfection. Relative expression of the indicated mRNA and transfected RNA are measured by qRT-PCR, results normalized to expression following mock transfection. Means ± SEM are shown (n = 3). Student’s t-test, comparing circFOREIGN with 0% m⁶A to indicated RNA transfection.

B. Ectopic expression of YTHDF2 rescues response to unmodified vs. m⁶A-modified circFOREIGN in YTHDF2 KO HeLa cells. Left: Model showing response to m⁶A-modified circFOREIGN following rescue. Right: Graph showing gene expression of innate immune genes 24 hours following RNA transfection. Relative expression of the indicated mRNA and transfected RNA are measured by qRT-PCR, results normalized to expression following mock transfection. Means ± SEM are shown (n = 3). *p<0.05, Student’s t-test, comparing 0% m⁶A circFOREIGN to 1% m⁶A circFOREIGN.

C. Tethering of YTHDF2 to unmodified circFOREIGN mask circRNA immunity. Left: Model showing in vivo tethering of protein to RNA via lambdaN and BoxB leading to attenuation of immunogencity. Right top: Protein domain architecture of full-length wild-type YTHDF2 with and without lambdaN tethering tag, and YTHDF2 N-terminal domain with and without lambdaN tethering tag. Right bottom: RIP-qPCR enrichment of indicated YTH protein followed by qRT-PCR of circRNA-BoxB or control actin RNA. Means ± SEM are shown (n = 3). *p<0.05, Student’s t-test, comparing YTHDF2 N-terminus with lambdaN tethering to YTHDF2 N-terminus without tethering.

D. Transfection of unmodified circBoxB tethered to full length wild-type YTHDF2 into wild-type HeLa cells attenuates immune response. Graph showing gene expression of innate immune genes 24 hours following RNA transfection. Relative expression of the indicated mRNA and transfected RNA are measured by qRT-PCR, results normalized mock transfection. Wild-type YTHDF2-lambdaN (grey) was ectopically expressed as immunogenicity negative control. Transfection with solely circBoxB (purple) serves as immunogenicity positive control. Means ± SEM are shown (n = 3). *p<0.05, Student’s t-test, comparing circBoxB with wild-type YTHDF2 with lambdaN tethering to wild-type YTHDF2 without tethering.

E. Transfection of unmodified circBoxB tethered to N-terminal domain of YTHDF2 into YTHDF2 KO cells is insufficient to attenuate immune response. Graph showing gene expression of innate immune genes 24 hours following RNA transfection. Relative expression of the indicated mRNA and transfected RNA are measured by qRT-PCR, results normalized mock transfection. N-terminal domain of YTHDF2-lambdaN (black) was ectopically expressed as immunogenicity negative control. Means ± SEM are shown (n = 3). Student’s t-test, comparing circBoxB with YTHDF2 N-terminus with lambdaN tethering to YTHDF2 N-terminus without tethering.