Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Activation of the interferon system by short-interfering RNAs

Abstract

RNA interference (RNAi) is a powerful tool used to manipulate gene expression or determine gene function1,2. One technique of expressing the short double-stranded (ds) RNA intermediates required for interference in mammalian systems is the introduction of short-interfering (si) RNAs3,4,5,6. Although RNAi strategies are reliant on a high degree of specificity, little attention has been given to the potential non-specific effects that might be induced. Here, we found that transfection of siRNAs results in interferon (IFN)-mediated activation of the Jak–Stat pathway and global upregulation of IFN-stimulated genes. This effect is mediated by the dsRNA-dependent protein kinase, PKR, which is activated by 21-base-pair (bp) siRNAs and required for upregulation of IFN-β in response to siRNAs. In addition, we show by using cell lines deficient in specific components mediating IFN action that the RNAi mechanism itself is independent of the interferon system. Thus, siRNAs have broad and complicating effects beyond the selective silencing of target genes when introduced into cells. This is of critical importance, as siRNAs are currently being explored for their potential therapeutic use7,8.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Intracellular effects of siRNA.
Figure 2: Global upregulation of ISGs in response to GAPDH siRNA in RCC1 cells.
Figure 3: PKR is necessary for the non-specific siRNA-induced signalling events.
Figure 4: RNA interference of GL3 luciferase expression by 21-bp GL3 siRNA.

References

  1. Zamore, P.D., Tuschl, T., Sharp, P.A. & Bartel, D.P. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 25–33 (2000).

    Article  CAS  Google Scholar 

  2. Hannon, G.J. RNA interference. Nature 418, 244–251 (2002).

    Article  CAS  Google Scholar 

  3. Elbashir, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).

    Article  CAS  Google Scholar 

  4. Donze, O. & Picard, D. RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase. Nucleic Acids Res. 30, e46 (2002).

    Article  Google Scholar 

  5. Xia, H., Mao, Q., Paulson, H.L. & Davidson, B.L. siRNA-mediated gene silencing in vitro and in vivo. Nature Biotechnol. 20, 1006–1010 (2002).

    Article  CAS  Google Scholar 

  6. Hutvagner, G. & Zamore, P.D. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 297, 2056–2060 (2001).

    Article  Google Scholar 

  7. Jacque, J., Triques, K. & Stevenson, M. Modulation of HIV-1 replication by RNA interference. Nature 418, 435–438 (2002).

    Article  CAS  Google Scholar 

  8. Gitlin, L., Karelsky, S. & Andino, R. Short interfering RNA confers intracellular antiviral immunity in human cells. Nature 418, 430–434 (2002).

    Article  CAS  Google Scholar 

  9. Haque, S.J. & Williams, B.R.G. Signal transduction in the interferon system. Semin. Oncol. 25, 14–22 (1998).

    CAS  PubMed  Google Scholar 

  10. Stark, G.R., Kerr, I.M., Williams, B.R., Silverman, R.H. & Schreiber, R.D. How cells respond to interferons. Annu. Rev. Biochem. 67, 227–264 (1998).

    Article  CAS  Google Scholar 

  11. Srivastava, S.P., Kumar, K.U. & Kaufman, R.J. Phosphorylation of eukaryotic translation factor 2 mediates apoptosis in response to activation of the double-stranded RNA-dependent protein kinase. J. Biol. Chem. 273, 2416–2423 (1998).

    Article  CAS  Google Scholar 

  12. Kumar, A., Haque, J., Lacoste, J., Hiscott, J. & Williams, B.R.G. Double-stranded RNA-dependent protein kinase activates transcription factor NF-κB by phosphorylating IκB. Proc. Natl Acad. Sci. USA 91, 6288–6292 (1994).

    Article  CAS  Google Scholar 

  13. Williams, B.R., Gilbert, C.S. & Kerr, I.M. The respective roles of the protein kinase and pppA2′p5′A2′p5 A-activated endonucleases in the inhibition of protein synthesis by double-stranded RNA in rabbit reticulocyte lysates. Nucleic Acids Res. 6, 1335–1350 (1979).

    Article  CAS  Google Scholar 

  14. Williams, B.R.G. PKR; a sentinel kinase for cellular stress. Oncogene 18, 6112–6120 (1999).

    Article  CAS  Google Scholar 

  15. Silverman, R.H. in Ribonucleases: structure and function (eds G. D'Alessio and J. F. Riordan) Ch. 16, 515–551 (Academic Press, St Louis, 1997).

    Book  Google Scholar 

  16. Pellegrini, S., John, J., Shearer, M., Kerr, I.M. & Stark, G.R. Use of a selectable marker regulated by α interferon to obtain mutations in the signaling pathway. Mol. Cell Biol. 9, 4605–4612 (1989).

    Article  CAS  Google Scholar 

  17. Tavernarakis, N., Wang, S.L., Dorovkov, M., Ryazanov, A. & Driscoll, M. Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes. Nature Genet. 24, 180–183 (2000).

    Article  CAS  Google Scholar 

  18. Alexopoulou, L., Holt, A.C., Medzhitov, R. & Flavell, R.A. Recognition of double-stranded RNA and activation of NF-κB by Toll-like receptor 3. Nature 413, 732–738 (2001).

    Article  CAS  Google Scholar 

  19. Matsumoto, M., Kikkawa, S., Kohase, M., Miyake, K. & Seya, T. Establishment of a monoclonal antibody against human Toll-like receptor 3 that blocks double-stranded RNA-mediated gene silencing. Biochem. Biophys. Res. Commun. 293, 1364–1369 (2002).

    Article  CAS  Google Scholar 

  20. Yang, et al. Deficient signaling in mice devoid of the double-stranded RNA dependent protein kinase, PKR. EMBO J. 14, 6095–6106 (1995).

    Article  CAS  Google Scholar 

  21. Zhou, A. et al. Interferon action and apoptosis are defective in mice devoid of 2′-5′-oligoadenylate-dependent RNase L. EMBO J. 16, 3297–3304 (1997).

    Google Scholar 

  22. Zhou, A., Paranjape, J.M., Der, S.D., Williams, B.R.G. & Silverman, R.H. Interferon action in triply deficient mice reveals the existence of alternative antiviral pathways. Virology 258, 435–440 (1999).

    Article  CAS  Google Scholar 

  23. Goh, K.C., Haque, S.J. & Williams, B.R.G. p38 MAP kinase is required for Stat1 serine phosphorylation and transcriptional activation induced by interferons. EMBO J. 18, 5601–5608 (1999).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We would like to thank Z. Wang for synthesizing luciferase siRNAs, P. Stanhope-Baker for providing the Supplementary Information on the non-specific effects of the WT-1 siRNAs and A. Sadler for comments on the manuscript. This work was supported by National Institutes of Health (NIH) grants RO1 AI34039 and PO1 CA62220.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Bryan R.G. Williams.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information, Fig. S1

Supplementary Information, Fig. S2 (PPT 1722 kb)

Supplementary Information, Fig. S3

Supplementary Information, Fig. S4

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sledz, C., Holko, M., de Veer, M. et al. Activation of the interferon system by short-interfering RNAs. Nat Cell Biol 5, 834–839 (2003). https://doi.org/10.1038/ncb1038

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb1038

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing