Extended Data Fig. 6: RIG-I monomer, but not the LLPS-forming oligomer, is targeted for poly-ubiquitination and degradation by E3 ligase MIB2.
From: Targeting a key disulfide linkage to regulate RIG-I condensation and cytosolic RNA-sensing
a, Normalized IFNB1 mRNA expression (determined by qPCR analysis) in RIG-I knockout HEK293T cells transfected with the indicated plasmids, followed by SeV infection for 12 h; n = 3. b, Microscale thermophoresis (MST) binding affinity between dsRNA and prokaryotic expressed GFP-RIG-I-WT, C810S or C813S mutant as indicated; n = 3. c, ATPase activities of RIG-I-WT, C810S, or C813S mutants were assessed in the presence of dsRNA; n = 3; P = 0.0015 for C810S vs. WT, P = 0.0012 for C813S vs. WT. d, Immunoblot (IB) analysis of HEK293T cells stably expressed with RIG-I-Strep WT, C810S, C813S, C864S or C869S and treated with Cycloheximide (CHX) (5 μM) for the indicated time periods (top). Quantified RIG-I-Strep band intensity was shown (bottom). n = 3. e, The representative RIG-I peptide carrying ubiquitin-conjugated Lys169, Lys256 and Lys652 were identified by mass spectrometry. f, Coomassie blue staining of purified Strep-tagged RIG-I WT and C864S. g, Fold change of IFN-β-luciferase (luc) activity in RIG-I knockout HEK293T cells transfected with control vector and the indicated expression plasmids, followed by SeV stimulation for 12 h (left). Immunoblot (IB) analysis of the RIG-I expression was shown (right); n = 3. h, Normalized IFNB1 and ISG56 mRNA expression (determined by qPCR analysis) in RIG-I knockout HEK293T cells transfected with the indicated plasmids, followed by SeV infection for 12 h; n = 3. i, IB analysis of HEK293T cells stably expressed with Flag-RIG-I-C864S and transfected with increased dosage of Myc-MIB2 WT or Myc-MIB2 C977S as indicated. Quantified Flag-RIG-I-C864S band intensity was shown (bottom). n = 3. j, In vitro binding between Flag-MIB2 and the purified RIG-I monomer or oligomer from Fig. 6n. k, Coomassie blue staining of Strep-tagged mCherry–MIB2. Data are representative of at least three independent experiments. Mean ± s.d., statistical analysis was performed using a two-tailed Student’s t-test (a,c,g,h), **P < 0.01, ****P < 0.0001. Exact P values, source numerical data and unprocessed blots are provided.
