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

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.

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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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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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