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An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells

Nature volume 404, pages 293–296 (2000)Cite this article

Abstract

In a diverse group of organisms that includes Caenorhabditis elegans , Drosophila, planaria, hydra, trypanosomes, fungi and plants, the introduction of double-stranded RNAs inhibits gene expression in a sequence-specific manner1,2,3,4,5,6,7. These responses, called RNA interference or post-transcriptional gene silencing, may provide anti-viral defence, modulate transposition or regulate gene expression1,6,8,9,10. We have taken a biochemical approach towards elucidating the mechanisms underlying this genetic phenomenon. Here we show that ‘loss-of-function’ phenotypes can be created in cultured Drosophila cells by transfection with specific double-stranded RNAs. This coincides with a marked reduction in the level of cognate cellular messenger RNAs. Extracts of transfected cells contain a nuclease activity that specifically degrades exogenous transcripts homologous to transfected double-stranded RNA. This enzyme contains an essential RNA component. After partial purification, the sequence-specific nuclease co-fractionates with a discrete, ∼25-nucleotide RNA species which may confer specificity to the enzyme through homology to the substrate mRNAs.

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Figure 1: RNAi in S2 cells.
Figure 2: RNAi in vitro.
Figure 3: Substrate requirements of the RISC.
Figure 4: The RISC contains a potential guide RNA.

References

  1. Sharp, P. A. RNAi and double-strand RNA. Genes Dev. 13, 139–141 (1999).

    CAS  Article  Google Scholar 

  2. Sanchez-Alvarado, A. & Newmark, P. A. Double-stranded RNA specifically disrupts gene expression during planarian regeneration. Proc. Natl Acad. Sci. USA 96, 5049– 5054 (1999).

    ADS  CAS  Article  Google Scholar 

  3. Lohmann, J. U., Endl, I. & Bosch, T. C. Silencing of developmental genes in Hydra. Dev. Biol. 214, 211–214 (1999).

    CAS  Article  Google Scholar 

  4. Cogoni, C. & Macino, G. Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase. Nature 399, 166–169 ( 1999).

    ADS  CAS  Article  Google Scholar 

  5. Waterhouse, P. M., Graham, M. W. & Wang, M. B. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc. Natl Acad. Sci. USA 95, 13959– 13964 (1998).

    ADS  CAS  Article  Google Scholar 

  6. Montgomery, M. K. & Fire, A. Double-stranded RNA as a mediator in sequence-specific genetic silencing and co-suppression. Trends Genet. 14, 225–228 (1998).

    Article  Google Scholar 

  7. Ngo, H., Tschudi, C., Gull, K. & Ullu, E. Double-stranded RNA induces mRNA degradation in Trypanosoma brucei. Proc. Natl Acad. Sci. USA 95, 14687–14692 (1998).

    ADS  CAS  Article  Google Scholar 

  8. Tabara, H. et al. The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99, 123– 132 (1999).

    CAS  Article  Google Scholar 

  9. Ketting, R. F., Haverkamp, T. H. A., van Luenen, H. G. A. M. & Plasterk, R. H. A. mut-7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner Syndrome helicase and RnaseD. Cell 99, 133–141 (1999).

    CAS  Article  Google Scholar 

  10. Ratcliff, F., Harrison, B. D. & Baulcombe, D. C. A similarity between viral defense and gene silencing in plants. Science 276, 1558– 1560 (1997).

    CAS  Article  Google Scholar 

  11. Kennerdell, J. R. & Carthew, R. W. Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. Cell 95, 1017–1026 (1998).

    CAS  Article  Google Scholar 

  12. Misquitta, L. & Paterson, B. M. Targeted disruption of gene function in Drosophila by RNA interference: a role for nautilus in embryonic somatic muscle formation. Proc. Natl Acad. Sci. USA 96, 1451–1456 (1999).

    ADS  CAS  Article  Google Scholar 

  13. Kalejta, R. F., Brideau, A. D., Banfield, B. W. & Beavis, A. J. An integral membrane green fluorescent protein marker, Us9-GFP, is quantitatively retained in cells during propidium iodine-based cell cycle analysis by flow cytometry. Exp. Cell. Res. 248, 322– 328 (1999).

    CAS  Article  Google Scholar 

  14. Wolf, D. A. & Jackson, P. K. Cell cycle: oiling the gears of anaphase. Curr. Biol. 8, R637– R639 (1998).

    Article  Google Scholar 

  15. Kramer, E. R., Gieffers, C., Holz, G., Hengstschlager, M. & Peters, J. M. Activation of the human anaphase-promoting complex by proteins of the CDC20/fizzy family. Curr. Biol. 8, 1207–1210 (1998).

    CAS  Article  Google Scholar 

  16. Shuttleworth, J. & Colman, A. Antisense oligonucleotide-directed cleavage of mRNA in Xenopus oocytes and eggs. EMBO J. 7, 427–434 (1988).

    CAS  Article  Google Scholar 

  17. Tabara, H., Grishok, A. & Mello, C. C. RNAi in C. elegans: soaking in the genome sequence. Science 282, 430– 432 (1998).

    CAS  Article  Google Scholar 

  18. Bosher, J. M., Dufourcq, P., Sookhareea, S. & Labouesse, M. RNA interference can target pre-mRNA. Consequences for gene expression in a Caenorhabditis elegans operon. Genetics 153 , 1245–1256 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  Article  Google Scholar 

  20. Jones, L. A., Thomas, C. L. & Maule, A. J. De novo methylation and co-suppression induced by a cytoplasmically replicating plant RNA virus. EMBO J. 17, 6385–6393 (1998).

    CAS  Article  Google Scholar 

  21. Jones, L. A. et al. RNA–DNA interactions and DNA methylation in post-transcriptional gene silencing. Plant Cell 11, 2291– 2301 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Schneider, I. Cell lines derived from late embryonic stages of Drosophila melanogaster . J. Embryol. Exp. Morpho. 27, 353– 365 (1972).

    CAS  Google Scholar 

  23. Di Nocera, P. P. & Dawid, I. B. Transient expression of genes introduced into cultured cells of Drosophila. Proc. Natl Acad. Sci. USA 80, 7095– 7098 (1983).

    ADS  CAS  Article  Google Scholar 

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Acknowledgements

We thank C.Velinzon and L. Rodgers for assistance with flow cytometry. Materials and advice were provided by A. Krainer, J. Yin and A. Nicholson. D.B. is supported by the Hugh and Catherine Stevenson Fund. G.J.H. is a Pew Scholar in the Biomedical Sciences. This work was supported in part by grants from the NIH (G.J.H.) and the US Army Breast Cancer Research Program (G.J.H.).

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Authors and Affiliations

  1. Genetica, Inc., P.O. Box 99, Cold Spring Harbor, 11724 , New York, USA

    Scott M. Hammond & David Beach

  2. Graduate Program in Genetics, State University of New York at Stony Brook, Stony Brook,, 11794, New York, USA

    Emily Bernstein

  3. Wolfson Institute for Biological Sciences, University College London, Gower Street, London, WC1E 6BT, UK

    David Beach

  4. Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, 11724, New York, USA

    Emily Bernstein & Gregory J. Hannon

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  1. Scott M. Hammond
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Correspondence to Gregory J. Hannon.

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Hammond, S., Bernstein, E., Beach, D. et al. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404, 293–296 (2000). https://doi.org/10.1038/35005107

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