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Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells

Nature volume 411, pages 494–498 (2001)Cite this article

Abstract

RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene1,2,3,4. The mediators of sequence-specific messenger RNA degradation are 21- and 22-nucleotide small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs5,6,7,8,9. Here we show that 21-nucleotide siRNA duplexes specifically suppress expression of endogenous and heterologous genes in different mammalian cell lines, including human embryonic kidney (293) and HeLa cells. Therefore, 21-nucleotide siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.

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Figure 1: Reporter constructs and siRNA duplexes.
Figure 2: RNA interference by siRNA duplexes.
Figure 3: Effects of 21-nucleotide siRNAs, 50-bp, and 500-bp dsRNAs on luciferase expression in HeLa cells.
Figure 4: Silencing of nuclear envelope proteins lamin A/C in HeLa cells.

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References

  1. Fire, A. RNA-triggered gene silencing. Trends Genet. 15, 358–363 (1999).

    Article  CAS  Google Scholar 

  2. Sharp, P. A. RNA interference 2001. Genes Dev. 15, 485–490 (2001).

    Article  CAS  Google Scholar 

  3. Hammond, S. M., Caudy, A. A. & Hannon, G. J. Post-transcriptional gene silencing by double-stranded RNA. Nature Rev. Genet. 2, 110–1119 (2001).

    Article  CAS  Google Scholar 

  4. Tuschl, T. RNA interference and small interfering RNAs. Chem. Biochem. 2, 239–245 (2001).

    CAS  Google Scholar 

  5. Hamilton, A. J. & Baulcombe, D. C. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286, 950–952 (1999).

    Article  CAS  Google Scholar 

  6. Hammond, S. M., Bernstein, E., Beach, D. & Hannon, G. J. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404, 293–296 (2000).

    Article  ADS  CAS  Google Scholar 

  7. 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 

  8. Bernstein, E., Caudy, A. A., Hammond, S. M. & Hannon, G. J. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363–366 (2001).

    Article  ADS  CAS  Google Scholar 

  9. Elbashir, S. M., Lendeckel, W. & Tuschl, T. RNA interference is mediated by 21 and 22 nt RNAs. Genes Dev. 15, 188–200 (2001).

    Article  CAS  Google Scholar 

  10. Caplen, N. J., Fleenor, J., Fire, A. & Morgan, R. A. dsRNA-mediated gene silencing in cultured Drosophila cells: a tissue culture model for the analysis of RNA interference. Gene 252, 95–105 (2000).

    Article  CAS  Google Scholar 

  11. Clemens, J. C. et al. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc. Natl Acad. Sci. USA 97, 6499–6503 (2000).

    Article  ADS  CAS  Google Scholar 

  12. Ui-Tei, K., Zenno, S., Miyata, Y. & Saigo, K. Sensitive assay of RNA interference in Drosophila and Chinese hamster cultured cells using firefly luciferase gene as target. FEBS Lett. 479, 79–82 (2000).

    Article  ADS  CAS  Google Scholar 

  13. Wianny, F. & Zernicka-Goetz, M. Specific interference with gene function by double-stranded RNA in early mouse development. Nature Cell Biol. 2, 70–75 (2000).

    Article  CAS  Google Scholar 

  14. Svoboda, P., Stein, P., Hayashi, H. & Schultz, R. M. Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference. Development 127, 4147–4156 (2000).

    CAS  Google Scholar 

  15. Bahramian, M. B. & Zarbl, H. Transcriptional and posttranscriptional silencing of rodent alpha(I) collagen by a homologous transcriptionally self-silenced transgene. Mol. Cell. Biol. 19, 274–283 (1999).

    Article  CAS  Google Scholar 

  16. 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 

  17. Manche, L., Green, S. R., Schmedt, C. & Mathews, M. B. Interactions between double-stranded RNA regulators and the protein kinase DAI. Mol. Cell. Biol. 12, 5238–5248 (1992).

    Article  CAS  Google Scholar 

  18. Minks, M. A., West, D. K., Benvin, S. & Baglioni, C. Structural requirements of double-stranded RNA for the activation of 2′,5′-oligo(A) polymerase and protein kinase of interferon-treated HeLa cells. J. Biol. Chem. 254, 10180–10183 (1979).

    CAS  Google Scholar 

  19. Clemens, M. & Williams, B. Inhibition of cell-free protein synthesis by pppA2′p5′A2′p5′A: a novel oligonucleotide synthesized by interferon-treated L cell extracts. Cell 13, 565–572 (1978).

    Article  CAS  Google Scholar 

  20. Macejak, D. G. et al. Inhibition of hepatitis C virus (HCV)-RNA-dependent translation and replication of a chimeric HCV poliovirus using synthetic stabilized ribozymes. Hepatology 31, 769–776 (2000).

    Article  CAS  Google Scholar 

  21. Kehlenbach, R. H., Dickmanns, A. & Gerace, L. Nucleocytoplasmic shuttling factors including Ran and CRM1 mediate nuclear export of NFAT In vitro. J. Cell. Biol. 141, 863–874 (1998).

    Article  CAS  Google Scholar 

  22. Kreis, T. & Vale, R. Guidebook to the Cytoskeletal and Motor Proteins, Parts 2b and 3a (Oxford Univ. Press, Oxford, 1999).

    Google Scholar 

  23. Sullivan, T. et al. Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy. J. Cell Biol. 147, 913–920 (1999).

    Article  CAS  Google Scholar 

  24. Wassenegger, M. RNA-directed DNA methylation. Plant Mol. Biol. 43, 203–220 (2000).

    Article  CAS  Google Scholar 

  25. Mette, M. F., Aufsatz, W., van der Winden, J., Matzke, M. A. & Matzke, A. J. M. Transcriptional silencing and promoter methylation triggered by double-stranded RNA. EMBO J. 19, 5194–5201 (2000).

    Article  CAS  Google Scholar 

  26. Wang, M.-B., Wesley, S. V., Finnegan, E. J., Smith, N. A. & Waterhouse, P. M. Replicating satellite RNA induces sequence-specific DNA methylation and truncated transcripts in plants. RNA 7, 16–28 (2001).

    Article  CAS  Google Scholar 

  27. Razin, A. CpG methylation, chromatin structure and gene silencing—a three-way connection. EMBO J. 17, 4905–4908 (1998).

    Article  CAS  Google Scholar 

  28. Röber, R. A., Gieseler, R. K., Peters, J. H., Weber, K. & Osborn, M. Induction of nuclear lamins A/C in macrophages in in vitro cultures of rat bone marrow precursor cells and human blood monocytes, and in macrophages elicited in vivo by thioglycollate stimulation. Exp. Cell Res. 190, 185–194 (1990).

    Article  Google Scholar 

  29. Harborth, J., Wang, J., Gueth-Hallonet, C., Weber, K. & Osborn, M. Self assembly of NuMA: multiarm oligomers as structural units of a nuclear lattice. EMBO J. 18, 1689–1700 (1999).

    Article  CAS  Google Scholar 

  30. Parrish, S., Fleenor, J., Xu, S., Mello, C. & Fire, A. Functional anatomy of a dsRNA trigger: Differential requirement for the two trigger strands in RNA Interference. Mol. Cell 6, 1077–1087 (2000).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank J. Martinez, J. Ludwig and D. Bartel for comments on the manuscript; L. Fredel for help with image processing; H.-J. Dehne for technical assistance; F. Döring, R. Nehring, D. Ingelfinger and C. Schneider for supplying cell lines; A. Dickmanns for the gift of the plasmid pAD3; and R. Lührmann for support. This work was funded by a Biofuture grant of the Bundesministerium für Bildung und Forschung.

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Authors and Affiliations

  1. Department of Cellular Biochemistry, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen, D-37077, Germany

    Sayda M. Elbashir, Winfried Lendeckel, Abdullah Yalcin & Thomas Tuschl

  2. Department of Biochemistry and Cell Biology, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen, D-37077, Germany

    Jens Harborth & Klaus Weber

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  1. Sayda M. Elbashir
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Correspondence to Thomas Tuschl.

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Elbashir, S., Harborth, J., Lendeckel, W. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001). https://doi.org/10.1038/35078107

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