Figure 3

Site-specific C>U RNA editing in mRNAs co-purified with rA3G.

(a) Immunoblot showing WT A3G expressed in whole cell lysate (20 μg) of 293T cells and recombinant (r) WT A3G (500 ng) obtained from 293T cells (purchased from Origene, Rockville, MD). Full-length blot is presented in Supplementary Fig. S4 (b) Cytidine deamination activity of recombinant A3G was examined in an in vitro reaction with a 5′ fluorescent dye-labeled ssDNA substrate of 40 nucleotides (left panel). Full-length gel is presented in Supplementary Fig. S4. Sanger chromatograms of cDNAs of MED1 RNA from in vitro RNA editing assay containing rA3G in the presence of 100 nM (+) or 1 μM (++) and ART MED1 RNA (right panel). (c) Sanger chromatograms of cDNAs of A3G substrates (MED1, KIAA1715, SCD, ITFG1, RFX7, GOLGA5, CHMP4B, CLASP1) (left panel) and A3A substrates (VIM, ASCC2, TMEM179B, SDHB) (right panel) from in vitro RNA editing assay containing only rA3G or from in vitro RNA editing assay containing in vitro transcribed SDHB RNA and purified A3A protein (SDHB lower panel). Edited cytidines are highlighted in black.