TRIM21 promotes autophagic degradation of IRF3 dimers and attenuates type I IFN production. (A) Confocal microscopy of HeLa cells coexpressing mCherry-TRIM21, Flag-IRF3, and GFP-LC3B in the presence of bafilomycin A1. White outline, cell boundary. Arrows; colocalization. (B) Confocal microscopy of HEK293 cells coexpressing mCherry-TRIM21, Flag-IRF3, and GFP-ULK1. Arrows, colocalization. (C) Co-immunoprecipitation analysis of IRF3-ULK1 complexes in the presence and absence of TRIM21. Lysates from HEK293 cells transiently expressing Myc-ULK1, Flag-IRF3, and either GFP-TRIM20 or GFP were immunoprecipitated with anti-Myc, and immunoblots were probed as indicated. (D) Levels of dimerized IRF3 were assessed by native PAGE of extracts from THP-1 cells subjected to TRIM21 or control knockdowns and stimulated for 12h by herring testis DNA (HT-DNA) transfected into the cells in the presence of 200 U/ml IFN-γ. (E) Effects of autophagy inhibition (bafilomycin A1) on TRIM21-dependent IRF3 dimer degradation in THP-1 cells. (F) Effects of TRIM21 knockdown on IFN-β mRNA levels after stimulation of THP-1 cells with IFN-γ and HT-DNA. (G) Model of TRIMs’ roles in regulation of inflammation by precision autophagy. TRIM20 targets the inflammasome components for autophagic degradation, whereas TRIM21 targets IRF3 dimer, to suppress inflammasome activity and type I IFN response, respectively. TRIM20 and TRIM21, responding to IFN-γ, directly bind their respective cargo and recruit autophagic machinery to execute degradation. Dashed arrow, cooperation between TRIM20 and TRIM21 may play a role in autophagic responses to IFN-γ. Bars, 10 µm. Data, means ± SE; n ≥ 3; *, P < 0.05; ^†, P ≥ 0.05 (ANOVA). IP, immunoprecipitation; Scr, scrambled; WB, Western blot.