ADAR1 edits cellular transcripts forming duplex RNA structures.

(A) Western blot analysis of untreated and IFN A/D–treated ADAR1-modified cells. Cells were treated with 1,000 U/ml IFN A/D for 24 h. (B) Ratios of absolute numbers of identified variants in HeLa and ADAR1KO cells. A>G and U>C variants show higher enrichment in HeLa cells. Average and standard deviation of numbers of variants are indicated. P values of each variant were determined by one-sample, two-tailed Student’s t test against an expected value of 1 (***, P ≤ 0.0001). Underlying values can be found in S1 Data. (C) Relative contribution of ADAR1 isoforms (p150 and p110) to editing in the top 156 genes and correlation with editing in exons/UTRs or introns. Values are calculated based on the number of editing sites detected by GIREMI in HeLa, p150KO, and ADAR1KO cells. Non-ADAR1, percentage of editing sites remaining in ADAR1KO cells. (D) Coverage plot of the 3′ UTR region of VOPP1. The region in the red box shows strong enrichment of U>C transitions. Nucleotide positions with variant frequencies ≥10% are color-coded: A, green; C, blue; G, orange; U, red. (E) Editing score analysis of the boxed region of (D) in HeLa, p150KO, ADAR1KO, and HeLa cells treated with 1,000 U/ml IFN A/D (top to bottom). Gray line indicates total coverage (“Cov.”) in the region. Repetitive sequences are indicated at the bottom: positive sense in blue and negative sense in red. (F) Correlation of editing scores of the VOPP1-region in HeLa + IFN A/D against untreated HeLa cells (gray dots, black line), HeLa versus p150KO (red dots and line), and HeLa versus ADAR1KO (blue dots and line). (G) Relative editing rates in cell lines and primary RNAseq data. Editing rates are normalized to coverage and length of the analyzed regions. Editing rate of each gene in HeLa cells is set to 100%. ADAR1, adenosine deaminase acting on RNA 1; ADAR1KO, fully ADAR1-deficient; GIREMI, Genome-independent Identification of RNA Editing by Mutual Information; IFN A/D, recombinant type-I interferon-alpha; lin. regr., linear regression; N/A, no RNAseq data available because of low or no coverage; p150KO, selectively ADAR1p150-deficient; p150mutLV, catalytically inactive ADAR1p150; p150wtLV, wild-type ADAR1p150; PKR, protein kinase R; pPKR, phospho-PKR; RNAseq, RNA sequencing; UTR, untranslated region; VOPP1, vesicular, overexpressed in cancer, prosurvival protein 1.