In many eukaryotes, expression of nuclear-encoded mRNA can be strongly inhibited by the presence of a double-stranded RNA (dsRNA) corresponding to exon sequences in the mRNA (refs 1,2). The use of this “RNA interference” (RNAi) in mammalian studies had lagged well behind its utility in lower animals because uninterrupted RNA duplexes longer than 30 base pairs trigger generalized cellular responses through activation of dsRNA-dependent protein kinases3. Recently it was demonstrated4 that RNAi can be made to work in cultured human cells by introducing shorter, synthetic duplex RNAs (∼20 base pairs) through liposome transfection. We have explored several strategies for expressing similar short interfering RNA (siRNA) duplexes within cells from recombinant DNA constructs, because this might allow long-term target-gene suppression in cells, and potentially in whole organisms. Effective suppression of target gene product levels is achieved by using a human U6 small nuclear RNA (snRNA) promoter to drive nuclear expression of a single RNA transcript. The siRNA-like parts of the transcript consists of a 19–base pair siRNA stem with the two strands joined by a tightly structured loop and a U1–4 3′ overhang at the end of the antisense strand. The simplicity of the U6 expression cassette and its widespread transcription in human cell types suggest that this mode of siRNA delivery could be useful for suppressing expression of a wide range of genes.
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Zamore, P.D. RNA interference: listening to the sound of silence. Nat. Struct. Biol. 8, 746–750 (2001).
Bernstein, E., Denli, A.M. & Hannon, G.J. The rest is silence. RNA 7, 1509–1521 (2001).
Gil, J. & Esteban, M. Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): mechanism of action. Apoptosis 5, 107–114 (2000).
Elbashir, S.M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).
Good, P.D. et al. Expression of small, therapeutic RNAs in human cell nuclei. Gene Ther. 4, 45–54 (1997).
Bertrand, E. et al. The expression cassette determines the functional activity of ribozymes in mammalian cells by controlling their intracellular localization. RNA 3, 75–88 (1997).
Cheong, C., Varani, G. & Tinoco, I. Jr. Solution structure of an unusually stable RNA hairpin, 5′-GGAC(UUCG)GUCC. Nature 346, 680–682 (1990).
Elbashir, S.M., Lendeckel, W. & Tuschl, T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188–200 (2001).
Nykänen, A., Haley, B. & Zamore, P.D. ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309–321 (2001).
Lipardi, C., Wei, Q. & Paterson, B.M. RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell 107, 297–307 (2001).
Chatterjee, S., Johnson, P.R. & Wong, K.K. Jr. Dual-target inhibition of HIV-1 in vitro by means of an adeno-associated virus antisense vector. Science 258, 1485–1488 (1992).
Kunkel, G.R., Maser, R.L., Calvet, J.P. & Pederson, T. U6 small nuclear RNA is transcribed by RNA polymerase III. Proc. Natl. Acad. Sci. USA 83, 8575–8579 (1986).
Kunkel, G.R. & Pederson, T. Upstream elements required for efficient transcription of a human U6 RNA gene resemble those of U1 and U2 genes even though a different polymerase is used. Genes Dev. 2, 196–204 (1988).
Danzeiser, D.A., Urso, O. & Kunkel, G.R. Functional characterization of elements in a human U6 small nuclear RNA gene distal control region. Mol. Cell. Biol. 13, 4670–4678 (1993).
This work was supported by NIH grant AI40936 to D.R.E. and the Medical Scientist Training Program at the University of Michigan. We thank Gary R. Kunkel for the cloned, human U6 snRNA gene, and Michael Imperiale and Eric Fearon for other materials.
D.R.E., C.P.P., and P.D.G have filed a patent application for expression of si-like RNAs in mammalian cells using U6 promoter cassettes, entitled “Intracellular Expression and Delivery of siRNAs in Mammalian Cells.”
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Paul, C., Good, P., Winer, I. et al. Effective expression of small interfering RNA in human cells. Nat Biotechnol 20, 505–508 (2002) doi:10.1038/nbt0502-505
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