Figure 9. MDA-5 deletion diminishes interferon-stimulated gene (ISG) expression in endothelial cells (ECs) with reduced short interspersed nuclear element (SINE) RNA editing caused by ADAR1 deficiency.

(A) ECs isolated from wild type (WT), inducible ADAR1 KO (i-KO), and inducible ADAR1/MDA-5 double knockout (i-d-KO) mice were cultured in vitro with tamoxifen (TM) induction for ADAR1 gene deletion. RNA editing in SINE RNA transcripts was assessed through RT-PCR and Sanger sequencing, and compared between wild-type, i-KO, and i-d-KO ECs. Panel (A) shows the chromatographs of Sanger sequencing result of EC RNAs. The multiple editing sites in the specific B1 SINE element located in the 3′ untranslated region of the Slfn5 gene were indicated by arrows. Obvious editing, as shown by the G peaks (black) together with A peak (green), presents in wild type EC RNAs. The editing levels (G peak areas) in i-d-KO ECs were same as the i-KO ECs, which were dramatically lower than the WT ECs. (B) ECs isolated from WT, EC-KO, and EC d-KO mice were analyzed for ISG expression. Compared to the EC-KO mice, ISG expression was significantly decreased in d-KO mice as shown by the seven representative ISGs. P < 0.05, n = 3 (WT) and 3–6 (i-KO and d-KO). (C) ISG expression was also tested in i-d-KO ECs after the ADAR1 gene was deleted; ISG levels were significantly lower than the controls. P < 0.05, n = 3 (WT) and 3–4 (i-KO and d-KO). (D). ADAR1 and MDA-5 gene deletion was monitored in the tested ECs with TM induction. ADAR1 gene was efficiently deleted in both i-KO and i-d-KO ECs after TM induction, whereas the MDA-5 gene was deleted in i-d-KO ECs with and without TM induction.