Reconstitution of the RIG-I Pathway Reveals a Signaling Role of Unanchored Polyubiquitin Chains in Innate Immunity

SummaryRIG-I detects invading viral RNA and activates the transcription factors NF-κB and IRF3 through the mitochondrial protein MAVS. Here we show that RNA bearing 5′-triphosphate strongly activates the RIG-I–IRF3 signaling cascade in a reconstituted system composed of RIG-I, mitochondria, and cytosol. Activation of RIG-I requires not only RNA but also polyubiquitin chains linked through lysine 63 (K63) of ubiquitin. RIG-I binds specifically to K63-polyubiquitin chains through its tandem CARD domains in a manner that depends on RNA and ATP. Mutations in the CARD domains that abrogate ubiquitin binding also impair RIG-I activation. Remarkably, unanchored K63-ubiquitin chains, which are not conjugated to any target protein, potently activate RIG-I. These ubiquitin chains function as an endogenous ligand of RIG-I in human cells. Our results delineate the mechanism of RIG-I activation, identify CARD domains as a ubiquitin sensor, and demonstrate that unanchored K63-polyubiquitin chains are signaling molecules in antiviral innate immunity.

Graphical AbstractFigure optionsDownload high-quality image (169 K)Download as PowerPoint slideHighlights
► In vitro reconstitution of the RIG-I pathway from viral RNA binding to IRF3 activation
► The tandem CARD domains of RIG-I bind to K63-polyubiquitin chains
► Unanchored K63-polyubiquitin chains potently activate RIG-I
► RIG-I activation requires sequential binding of RNA and K63-polyubiquitin chains