RNA helicase DDX6 governs ASC speck formation in P-bodies and the transition to stress granules via phase separation during inflammasome activation

Abstract The recruitment and condensation of apoptosis-associated speck-like protein containing a CARD (ASC) are critical for ASC speck formation and inflammasome activation. However, how this process occurs efficiently in vivo remains unclear. Here, we identified the RNA helicase DDX6 as an ASC-interacting protein through immunoprecipitation‒mass spectrometry (IP‒MS) analysis. DDX6 promotes the activation of both NLRP3 and AIM2 inflammasomes by facilitating the recruitment of ASC to these receptors through its RNA helicase activity. Mechanistically, DDX6 functions as a scaffold protein for processing body (P-body) assembly and drives ASC speck formation in P-bodies via liquid‒liquid phase separation (LLPS). We report that membrane integrity is associated with stress granule (SG) formation and that in Caspase-1 –/– , Gsdmd –/– , or NINJ1-inhibited cells, DDX6 mediates initial ASC speck assembly in P-bodies, followed by their transition to SGs during inflammasome activation. DDX6 deficiency in macrophages increases host susceptibility to Listeria infection. Our results establish that DDX6 orchestrates ASC recruitment, speck formation, and subsequent transition through LLPS-mediated mechanisms, offering new insights into inflammasome assembly and potential therapeutic approaches for inflammasome-related diseases.