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A structural basis for discriminating between self and nonself double-stranded RNAs in mammalian cells

Nature Biotechnology volume 24, pages 559–565 (2006)Cite this article

Abstract

Nonspecific effects triggered by small interfering RNAs (siRNAs) complicate the use of RNA interference (RNAi) to specifically downregulate gene expression1,2,3,4,5. To uncover the basis of these nonspecific activities, we analyzed the effect of chemically synthesized siRNAs on mammalian double-stranded RNA (dsRNA)-activated signaling pathways. siRNAs ranging from 21 to 27 nucleotides (nt) in length activated the interferon system when they lacked 2-nt 3′ overhangs, a characteristic of Dicer products. We show that the recognition of siRNAs is mediated by the RNA helicase RIG-I and that the presence of 3′ overhangs impairs its ability to unwind the dsRNA substrate and activate downstream signaling to the transcription factor IRF-3. These results suggest a structural basis for discrimination between microRNAs that are endogenous Dicer products, and nonself dsRNAs such as by-products of viral replication. These findings will enable the rational design of siRNAs that avoid nonspecific effects or, alternatively, that induce bystander effects to potentially increase the efficacy of siRNA-based treatments of viral infections or cancer.

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Figure 1: Analysis of size and structure dependence for activation of dsRNA signaling and RNAi by siRNAs.
Figure 2: Cell type differences in responses to chemically synthesized siRNAs and the role of the RNA helicase RIG-I.
Figure 3: The RNA helicase RIG-I interacts with chemically synthesized siRNAs in vitro despite the presence of 3′ overhangs but is activated preferentially by the blunt siRNAs.
Figure 4: Modulating the activation of dsRNA signaling and RNAi through modifications of the siRNA ends.

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Acknowledgements

We would like to thank Patricia Stanhope-Baker, Michelle Holko, Anthony Sadler and Mark Whitmore for helpful comments and Patricia Kessler and Scott D. Rose for valuable assistance. We are also grateful to Michael Gale Jr. for providing the sequences for DDX58 primers, James Finke and Patricia Rayman, Joe DiDonato and the DiDonato laboratory, Ganes Sen and the Sen laboratory and Yan Xu for providing reagents. This work was supported by National Institutes of Health grants RO1 AI34039 and PO1 CA 62220.

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Authors and Affiliations

  1. Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, 44195, Ohio, USA

    Joao Trindade Marques, Thalie Devosse, Die Wang, Maryam Zamanian-Daryoush, Paul Serbinowski & Bryan RG Williams

  2. Department of Molecular Biology, University of Aarhus, Aarhus, DK-8000, Denmark

    Rune Hartmann

  3. Laboratory of Molecular Genetics, Institute for Virus Research, Kyoto University, Kyoto, 606-8507, Japan

    Takashi Fujita

  4. Integrated DNA Technologies, Inc., Coralville, 52241, Iowa, USA

    Mark A Behlke

  5. Monash Institute of Medical Research, Monash University, 246 Clayton Road, Clayton, VIC. 3168, Melbourne, Australia

    Bryan RG Williams

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  1. Joao Trindade Marques
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Corresponding author

Correspondence to Bryan RG Williams.

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Competing interests

M.A.B. is employed by Integrated DNA Technologies Inc. (IDT), which offers oligonucleotides for sale similar to the ones described in the manuscript. IDT is not, however, a publicly traded company and the author does not own any equity in IDT.

Supplementary information

Supplementary Fig. 1

The recognition of siRNAs containing blunt ends is TLR independent. (PDF 435 kb)

Supplementary Fig. 2

Chemically synthesized siRNAs with poor quality control can induce the activation of dsRNA signaling independently of size and structure. (PDF 706 kb)

Supplementary Fig. 3

Silencing RIG-I expression using siRNAs. (PDF 583 kb)

Supplementary Fig. 4

In vitro binding and activation of purified PKR by chemically synthesized siRNAs is independent of size or structure. (PDF 391 kb)

Supplementary Table 1

siRNAs targeting GFP (PDF 53 kb)

Supplementary Table 2

siRNAs targeting STAT1 (PDF 37 kb)

Supplementary Table 3

Mass spectrometry and HPLC analysis of siRNAs (PDF 51 kb)

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Marques, J., Devosse, T., Wang, D. et al. A structural basis for discriminating between self and nonself double-stranded RNAs in mammalian cells. Nat Biotechnol 24, 559–565 (2006). https://doi.org/10.1038/nbt1205

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