Figure 3. ADAR1-induced A-to-I RNA editing of GM2A regulates GM2A expression.

See also Supplemental Figure 4. (A) Volcano plot of relative gene expression in GSCs transduced with shCONT or shADAR1. Purple and blue points designate upregulated and downregulated target genes, respectively, upon genetic targeting of ADAR1. Red points denote genes with editing sites. (B) Venn diagram of the overlapping genes.Blue circle: downregulated genes with ADAR1 depletion, purple circle: genes with negative prognostic significance in GBM in TCGA, orange circles: genes with alterations in editing levels. The rectangle shows 10 overlapping genes. (C) Top: Correlation between expression levels of ADAR1 and overall editing level of GM2A in GSCs. Bottom: Correlation between expression levels and overall editing levels of GM2A in GSCs. (D) Immunoblotting of ADAR1 and GM2A in GSCs transduced with shCONT or shADAR1. β-Actin served as loading control. (E) Sequence chromatograms of GM2A transcripts in 3691 GSCs transduced with shCONT, shADAR1, or shADARB1. Arrowheads indicate edited positions. Percentages indicate the calculated frequency of editing at selected positions. (F) Immunoblotting of ADAR1 IP in indicated GSCs. β-Actin served as a nonspecific control. (G) ADAR1 RNA immunoprecipitation (RIP) to pull down GM2A mRNA in indicated GSCs. ACTB served as nonspecific control. +RT and –RT: with and without RTase in reverse transcription. (H) ADAR1 and GM2A mRNA expression in indicated GSCs transduced with shCONT or shADAR1 with simultaneous transduction with vector control, wild-type ADAR1, or editing-dead E912A ADAR1 mutant. n = 4. Quantitative data from 4 independent experiments are shown as mean ± SD. Statistical significance was determined by ANOVA. *P < 0.05, ***P < 0.001, ****P < 0.0001. (I) Immunoblotting of ADAR1 and GM2A in indicated GSCs performed in H. β-Actin served as loading control. (J) Sequence chromatograms of the GM2A transcript performed in I. Arrowheads indicate edited positions.