siRNA enhances DNA-mediated interferon lambda-1 response through crosstalk between RIG-I and IFI16 signalling pathway.
In addition to silencing specific genes, small interfering RNA (siRNA) transfection is also associated with the non-specific induction of inflammatory cytokines and type I interferon. Those so-called "off-target" effects have considerable implications for the interpretation of in vitro studies and clinical application of siRNA. The present study attempted to develop a better understanding of the mechanism involved in these off target effects. Synthesized siRNA significantly enhances DNA-mediated interferon lambda-1 response (IFN-λ1/IL-29), a newly characterized antiviral interferon in non-immune or primary immune cells. This enhancement was most pronounced by double-stranded siRNA with at least a 2-nucleotide overhang at one 3' terminus in a dose-dependent manner, while the presence of DNA was indispensable. A pull-down assay using biotinylated siRNA- or DNA-conjugated beads indicated that retinoic acid-inducible gene I (RIG-I) and interferon gamma-inducible protein 16 (IFI16) were involved in the sensing of siRNA and DNA, respectively. Co-immunoprecipitation analysis further revealed that RIG-I and IFI16 formed a complex via siRNA, and the dissociation of IFI16 from this complex in the presence of DNA activated the downstream STING-TBK1-IRF3 (stimulator of interferon genes - tank-binding kinase 1 - interferon regulatory factor 3) pathway, shedding light on a new physiological signalling pathway to activate innate immunity. Collectively, these findings may provide rational information for siRNA-induced innate immunity, with important implications for developing siRNA-based reagents to control human diseases.
Laboratory of Human Retrovirology, Clinical Services Program, Applied and Developmental Directorate, SAIC-Frederick, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA, Laboratory of Proteomics and Analytical Technologies, Advanced Technology Program, SAIC-Frederick, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA and Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Hongyan
H
Sui
Ming
M
Zhou
Qian
Q
Chen
H Clifford
HC
Lane
Tomozumi
T
Imamichi
D002460:Cell Line; D002478:Cells, Cultured; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D006367:HeLa Cells; D018259:Herpesvirus 1, Human / Q000502:physiology; D006801:Humans; D050838:Interferon Regulatory Factor-3 / Q000378:metabolism; D007372:Interferons; D007378:Interleukins / Q000096:biosynthesis*; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D034741:RNA, Small Interfering / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D011971:Receptors, Immunologic; D015398:Signal Transduction; D000095022:Interferon Lambda
D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases; D004247:DNA; D050838:Interferon Regulatory Factor-3; D007372:Interferons; D007378:Interleukins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D034741:RNA, Small Interfering; D011971:Receptors, Immunologic; C000624353:interferon-lambda, human; D000095022:Interferon Lambda; C494232:IRF3 protein, human; C076700:IFI16 protein, human; C403191:TBK1 protein, human; C452820:RIGI protein, human