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. Author manuscript; available in PMC: 2017 Sep 23.
Published in final edited form as: Mol Cell. 2017 Jun 15;67(2):228–238.e5. doi: 10.1016/j.molcel.2017.05.022

Figure 4. CircRNA Stimulation of Innate Immune Genes Is Not Explained by Known RIG-I Ligands.

Figure 4

(A) Exogenous circRNA does not contain 5′ triphosphate. HeLa cells were transfected with 500 ng of circRNA or circRNA treated with phosphatase. Relative expression of the indicated mRNA and transfected RNA are measured by qRT-PCR. Means ± SEM are shown (n = 3).

(B) Exogenous circRNA does not contain long dsRNA duplex. In vitro selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) was conducted on linear and circRNA. SHAPE reactivity scores were calculated for each nucleotide. Black line denotes the longest consecutive dsRNA region.

(C) Transfection of circRNA made from ligating the ends of linear RNA stimulates innate immune genes. HeLa cells were mock transfected or transfected with 500 ng of linear or splint ligated circRNA encoding GFP-IRES. Relative expression of the indicated mRNA and transfected RNA are measured by qRT-PCR. Means ± SEM are shown (n = 3); *p < 0.05 throughout.

(D) CircRNA samples do not contain aberrant products that stimulate immune response. CircRNA was treated with RNase H in the presence of hybridizing oligonucleotide. HeLa cells were mock transfected or transfected with linear RNA, circRNA, or circRNA treated with RNase H and hybridizing oligonucleotide. Relative expression of the indicated mRNA and transfected RNA are measured by qRT-PCR. Means ± SEM are shown (n = 3).