Fig. 4. USP18-dependent ISGylation of ADAR leads to enzymatic activity inhibition and dsRNA accumulation.

ADAR knockdown using siRNA renders HAP1 WT sensitive to growth inhibition by type I IFN as shown by the growth curves in (a). b Immunoblots of a time-course experiment showing an increase in modified ADAR and PKR and activation of PKR (phosphorylated PKR in Thr 446) USP18-deficient cells treated with IFN for the indicated times. c A plot of the ADAR GlyGly-modified lysine residues and their ion intensities measured by MS. d dsRNA bound ADAR2 deaminase domain structure (PDB code 5ED2). Lysine residues corresponding to the ADAR1 GlyGly sites are represented with sticks. The Zn²⁺ cation is represented as a grey sphere (top panel). Table showing the ADAR1 potential GlyGly sites indicating their domain location and their contribution to RNA binding. A score of 1 indicates the highest contribution and 3 the lowest and away from the RNA binding pocket. DD = deaminase domain. dsRBD = dsRNA binding domain (lower panel). e, f Analysis of the dsRNA levels after IFN treatment in WT and KO cells by immunofluorescence using specific antibodies (n = 6 per condition).