Abstract
Double-stranded RNA (dsRNA) can suppress gene expression by inducing mRNA degradation of the homologous gene, known as RNA interference (RNAi). First, an RNase III-like dsRNA-specific endonuclease, Dicer, cleaves long dsRNA into 21- to 23-nucleotide (nt) small, interfering RNAs (siRNAs). Second, each resulting siRNA is incorporated into an RNA-induced silencing complex (RISC), which consists of eIF2C1, eIF2C2, Gemin3 (an RNA helicase), Gemin4 and other proteins. The siRNA guides the RISC to the homologous mRNA. Finally, the endoribonuclease in the RISC cleaves the targeted mRNA at the vicinity of the binding site, followed by further degradation of the mRNA by an exoribonuclease. As a result, gene silencing is established.
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References
Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498.
Sui, G., Soohoo, C., Affarel, B., et al. (2002) A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl. Acad. Sci. USA 99, 5515–5520.
Brummelkamp, T. R., Bernards, R., and Agami, R. (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550–553.
Paddison, P. J., Caudy, A. A., and Hannon, G. J. (2002) Stable suppression of gene expression by RNAi in mammalian cells. Proc. Natl. Acad. Sci. USA 99, 1443–1448.
Paul, C. P., Good, P. D., Winer, I., and Engelke, D. R. (2002) Effective expression of small interfering RNA in human cells. Nat. Biotechnol. 20, 505–508.
McManus, M. T., Petersen, C. P., Haines, B. B., Chen, J., and Sharp, P. A. (2002) Gene silencing using micro-RNA designed hairpins. RNA 8, 842–850.
Yu, J. Y., DeRuiter, S. L., and Turner, D. L. (2002) RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA 99, 6047–6052.
Miyagishi, M. and Taira, K. (2002) U6 promoter-driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells. Nat. Biotechnol. 20, 497–500.
Lee, N. S., Dohjima, T., Bauer, G., et al. (2002) Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat. Biotechnol. 20, 500–505.
Gupta, S., Schoer, R. A., Egan, J. E., Hannon, G. J., and Mittal, V. (2004) Inducible, reversible, and stable RNA interference in mammalian cells. Proc. Natl. Acad. Sci. USA 101, 1927–1932.
Kasim, V., Miyagishi, M., and Taira, K. (2004) Control of siRNA expression using the Cre-loxP recombination system. Nucleic Acids Res. 32, e66.
Higuchi, M., Tsutsumi, R., Higashi, H., and Hatakeyama, M. (2004) Conditional gene silencing utilizing the lac repressor reveals a role of SHP-2 in cagA-positive Helicobacter pylori pathogenicity. Cancer Sci. 95, 442–447.
Czauderna, F., Santel, A., Hinz, M., et al. (2003) Inducible shRNA expression for application in a prostate cancer mouse model. Nucleic Acids Res. 31, e127.
Matsukura, S., Jones, P. A., and Takai, D. (2003) Establishment of conditional vectors for hairpin siRNA knockdowns. Nucleic Acids Res. 31, e77.
Tuschl, T. (2002) Expanding small RNA interference. Nat. Biotechnol. 20, 446–448.
Bogenhagen, D. F., Sakonju, S., and Brown, D. D. (1980) A control region in the center of the 5S RNA gene directs specific initiation of transcription: II. The 3′ border of the region. Cell 19, 27–35
Schwarz, D. S., Hutvagner, G., Du, T., Xu, Z., Aronin, N., and Zamore, P. D. (2003) Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208.
Khvorova, A., Reynolds, A., and Jayasena, S. D. (2003) Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209–216.
Reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W. S., and Khvorova, A. (2004) Rational siRNA design for RNA interference. Nat. Biotechnol. 22, 326–330.
Mittal, V. (2004) Improving the efficiency of RNA interference in mammals. Nat. Rev. Genet. 5, 355–365.
Ui-Tei, K., Naito, Y., Takahashi, F., et al. (2004) Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res. 32, 936–948.
Wang, L. and Mu, F. Y. (2004) A Web-based design center for vector-based siRNA and siRNA cassette. Bioinformatics 4, 4.
McManus, M. T., Haines, B. B., Dillon, C. P., et al. (2002) Small interfering RNAmediated gene silencing in T lymphocytes. J. Immunol. 169, 5754–5760.
Martinez, J., Patkaniowska, A., Urlaub, H., Luhrmann, R., and Tuschl, T. (2002) Singlestranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 110, 563–574.
Jackson, A. L., Bartz, S. R., Schelter, J., et al. (2003) Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21, 635–637.
Scacheri, P. C., Rozenblatt-Rosen, O., Caplen, N. J., et al. (2004) Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Proc. Natl. Acad. Sci. USA 101, 1892–1897.
Sambrook, J., Fritsch, E. F., and Maniatis, T., eds. (1990) Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. p. 1.86.
Paddison, P. J., Silva, J. M., Conklin, D. S., et al. (2004) A resource for large-scale RNA-interference-based screens in mammals. Nature 428, 427–431.
Tiscornia, G., Singer, O., Ikawa, M., and Verma, I. M. (2003) A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA. Proc. Natl. Acad. Sci. USA 100, 1844–1848.
Rubinson, D. A., Dillon, C. P., Kwiatkowski, A. V., et al. (2003) A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat. Genet. 33, 401–406.
Matta, H., Hozayev, B., Tomar, R., Chugh, P., and Chaudhary, P. M. (2003) Use of lentiviral vectors for delivery of small interfering RNA. Cancer Biol. Ther. 2, 206–210.
Devroe, E. and Silver, P. A. (2002) Retrovirus-delivered siRNA. BMC Biotechnol. 2, 15.
Shen, C. and Reske, S. N. (2004) Adenovirus-delivered siRNA. Methods Mol. Biol. 252, 523–532.
Zhao, L. J., Jian, H., and Zhu, H. (2003) Specific gene inhibition by adenovirus-mediated expression of small interfering RNA. Gene 316, 137–141.
Tomar, R. S., Matta, H., and Chaudhary, P. M. (2003) Use of adeno-associated viral vector for delivery of small interfering RNA. Oncogene 22, 5712–5715.
Acknowledgments
The authors are grateful to Dr. Sidney Altman for providing the U6 promoter plasmid pmU6. We thank our colleagues in the Shi lab for helpful discussion. We also thank Dr. Nathan R. Wall for critically reading the manuscript and Dr. Peter Mulligan for working out the “three-fragment” ligation. G.C.S is supported by a Viral Oncology Training Grant from the NIH (T32CA09031). This work was supported by a grant from the NIH (GM53874) to YS.
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Sui, G., Shi, Y. (2005). Gene Silencing by a DNA Vector-Based RNAi Technology. In: Carmichael, G.G. (eds) RNA Silencing. Methods in Molecular Biology™, vol 309. Humana Press. https://doi.org/10.1385/1-59259-935-4:205
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DOI: https://doi.org/10.1385/1-59259-935-4:205
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