Reconstitution of the RIG-I pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity

Cell. 2010 Apr 16;141(2):315-30. doi: 10.1016/j.cell.2010.03.029.

Abstract

RIG-I detects invading viral RNA and activates the transcription factors NF-kappaB 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Cell Line
  • DEAD Box Protein 58
  • DEAD-box RNA Helicases / immunology
  • DEAD-box RNA Helicases / metabolism*
  • Humans
  • I-kappa B Kinase / metabolism
  • Immunity, Innate*
  • Interferon Regulatory Factor-3 / immunology
  • Interferon Regulatory Factor-3 / metabolism
  • Polyphosphates / metabolism
  • Polyubiquitin / metabolism
  • RNA, Double-Stranded / immunology
  • RNA, Viral / immunology*
  • Signal Transduction*
  • Transcription Factors / metabolism
  • Tripartite Motif Proteins
  • Ubiquitin-Conjugating Enzymes / metabolism*
  • Ubiquitin-Protein Ligases / metabolism

Substances

  • IRF3 protein, human
  • Interferon Regulatory Factor-3
  • Polyphosphates
  • RNA, Double-Stranded
  • RNA, Viral
  • Transcription Factors
  • Tripartite Motif Proteins
  • Polyubiquitin
  • Adenosine Triphosphate
  • UBE2D3 protein, human
  • UBE2N protein, human
  • Ubiquitin-Conjugating Enzymes
  • TRIM25 protein, human
  • Ubiquitin-Protein Ligases
  • I-kappa B Kinase
  • DDX58 protein, human
  • DEAD Box Protein 58
  • DEAD-box RNA Helicases
  • triphosphoric acid