Reconstitution of the RIG-I pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity.
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.
Zeng
Wenwen
W
Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148, USA.
Sun
Lijun
L
Jiang
Xiaomo
X
Chen
Xiang
X
Hou
Fajian
F
Adhikari
Anirban
A
Xu
Ming
M
Chen
Zhijian J
ZJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D002460:Cell Line; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D007113:Immunity, Innate; D050838:Interferon Regulatory Factor-3 / Q000276:immunology / Q000378:metabolism; D011122:Polyphosphates / Q000378:metabolism; D025821:Polyubiquitin / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000276:immunology; D012367:RNA, Viral / Q000276:immunology*; D011971:Receptors, Immunologic; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism; D000070584:Tripartite Motif Proteins; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
C494232:IRF3 protein, human; D050838:Interferon Regulatory Factor-3; D011122:Polyphosphates; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D011971:Receptors, Immunologic; D014157:Transcription Factors; D000070584:Tripartite Motif Proteins; D025821:Polyubiquitin; D000255:Adenosine Triphosphate; C097310:UBE2D3 protein, human; C471607:UBE2N protein, human; D044763:Ubiquitin-Conjugating Enzymes; C084523:TRIM25 protein, human; D044767:Ubiquitin-Protein Ligases; D051550:I-kappa B Kinase; C452820:RIGI protein, human; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases; C005692:triphosphoric acid
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