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

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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. 1

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

  2. 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).

  3. 3

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

  4. 4

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

  5. 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).

  6. 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).

  7. 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).

  8. 8

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

  9. 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).

  10. 10

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

  11. 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).

  12. 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).

  13. 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).

  14. 14

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

  15. 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).

  16. 16

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

  17. 17

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

  18. 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).

  19. 19

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

  20. 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).

  21. 21

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

  22. 22

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

  23. 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).

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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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Correspondence to Gregory J. Hannon.

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