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RNA Interference Induced by Transient or Stable Expression of Hairpin Structures of Double-Stranded RNA in Drosophila and Mammalian Cells

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Abstract

RNA interference (RNAi) may be induced by a plasmid with an inverted repeat (IR) sequence directing transcription of hairpin-type double-stranded RNA (dsRNA). This study examines the effects of changing various parameters of IR constructs on Drosophila and mammalian RNAi, using the dual luciferase system. RNAi activity was found to vary depending on IR length as well as the length and sequence of the internal loop separating sense and antisense sequences. Both transient and stable RNAi occurred in Drosophila cultured cells. Although transient DNA-mediated RNAi was noted in most mammalian cells, no mammalian cells stably possessing IR sequences and hence RNAi activity could be obtained. In Drosophila, DNA-mediated RNAi was considerably weaker than long-dsRNA-mediated RNAi. The cytological data indicated that this was most probably caused by abortive processing of hairpin RNA produced within cells. DNA-mediated RNAi was examined at the level of Drosophila individuals using extramacrochaetae as a model gene, and the presence of an intron sequence in the single-stranded loop region was shown to be essential for effective RNAi.

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REFERENCES

  1. Fire A., Xu S., Montgomery M.K., Koatas S.A., Driver S.E., Mello C.C. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 391, 806–811.

    Google Scholar 

  2. Montgomery M.K., Xu S., Fire A. 1998. RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA. 95, 15502–15507.

    Google Scholar 

  3. Bernstein E., Caudy A.A., Hammond S.M., Hannon G.J. 2001. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature. 409, 363–366.

    Google Scholar 

  4. Elbashir S.M., Leandeckel W., Tuschl T. 2001. RNA interference is mediated by 21-and 22-nucleotide RNAs. Genes Dev. 15, 188–200.

    Google Scholar 

  5. Wianny F., Zernicka-Goetz M. 2000. Specific interference with gene function by double-stranded RNA in early mouse development. Nature Cell. Biol. 21, 70–75.

    Google Scholar 

  6. Ui-Tei K., Zenno S., Miyata Y., Saigo, K. 2000. Sensitive assay of RNA interference in Drosophila and chinese hamster cultured cells using firefly luciferase gene as target. FEBS Lett. 479, 79–82.

    Google Scholar 

  7. McManus M.T., Sharp P.A. 2002. Gene silencing in mammals by small interfering RNAs. Nature Rev. Genetics. 3, 737–747.

    Google Scholar 

  8. Hamilton A.J. 1999. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science. 286, 950–952.

    Google Scholar 

  9. Zamore P.D. 2000. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell. 101, 25–33.

    Google Scholar 

  10. Doi N., Zenno S., Ueda R., Ohki-Hamazaki H., Ui-Tei K., Saigo K. 2003. Short-interfering-RNA-mediated gene silencing in mammalian cells requires Dicer and eIF2C translation initiation factors. Current Biol. 13, 41–46.

    Google Scholar 

  11. Martines J., Patkaniowska A., Urlaub H., Luhrmann R., Tuschl T. 2002. Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell. 110, 563–574.

    Google Scholar 

  12. Kennerdell J.R., Carthew R.W. 1998. Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled2 act in the wingless pathway. Cell. 95, 1017–1026.

    Google Scholar 

  13. Tavernarakis N., Wang S.L., Dorovkov M., Ryazanow A., Driscoll M. 2000. Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes. Nature Genet. 24, 180–183.

    Google Scholar 

  14. Kennerdell J.R., Carthew R.W. 2000. Heritable gene silencing in Drosophila using double-stranded RNA. Nature Biotechnol. 18, 896–898.

    Google Scholar 

  15. Kamiyama S., Suda T., Ueda R., Suzuki M., Okubo R., Kikuchi N., Chiba Y., Goto S., Toyoda H., Saigo K., Watanabe M., Narimatsu H., Jigami Y., Nishihara S. 2003. Molecular cloning and identification of 3′-phosphoadenosine 5′-phosphosulfate transporter. J. Biol. Chem. 278, 25958–25963.

    Google Scholar 

  16. Chuang C.F., Meyerowitz E.M. 2000. Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA. 97, 4985–4990.

    Google Scholar 

  17. Paddison P.J., Caudy A.A., Hannon G.J. 2002. Stable suppression of gene expression by RNAi in mammalian cells. Proc. Natl. Acad. Sci. USA. 99, 1443–1448.

    Google Scholar 

  18. Shi Y. 2003. Mammalian RNAi for the masses. Trends Genet. 19, 9–12.

    Google Scholar 

  19. Kuwabara T., Warashina M., Nakayama A., Ohkawa J., Taira K. 1999. tRNAVal-heterodimeric maxizymes with high potential as gene-inactivating agents: Simultaneous cleavage at two sites in HIV-1 tat mRNA in cultured cells. Proc. Natl. Acad. Sci. USA. 96, 1886–1891.

    Google Scholar 

  20. Bhanot P., Brink M., Samos C.H., Hsieh J.C., Wang Y., Machke J.P., Andrew D., Nathans J., Nusse R. 1996. A new member of the frizzled family form Drosophila functions as a Wingless receptor. Nature. 385, 225–230.

    Google Scholar 

  21. Brand A., Perrimon N. 1993. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 118, 401–415.

    Google Scholar 

  22. Gingeras T.R., Sciaky D., Gelinas R.E., Bing-Dong J., Yen C.E., Kelly M.M., Bullock P.A., Parsons B.L., O'Neill K.E., Roberts R.J. 1982. Nucleotide sequences from the adenovirus-2 genome. J. Biol. Chem. 25, 13475–13491.

    Google Scholar 

  23. Sugaya R., Ishimaru S., Hosoya T., Saigo K., Emori Y. 1994. A Drosophila homolog of human proto-oncogene ret transiently expressed in embryonic neuronal precursor cells including neuroblasts and CNS cells. Mech. Dev. 45, 139–145.

    Google Scholar 

  24. Takahashi F., Endo S., Kojima T., Saigo K. 1996. Regulation of cell-cell contacts in developing Drosophila eyes by Dsrc41, a new, close relative of vertebrate c-src. Genes Dev. 10, 1645–1656.

    Google Scholar 

  25. Southern E.M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503.

    Google Scholar 

  26. Staehling-Hampton K., Jackson P.D., Clark M.J., Brand A.H., Hoffmann F.M. 1994. Specificity of bone morphogenetic protein-related factors: cell fate and gene expression changes in Drosophila embryos induced by decapentaplegic but not 60A. Cell Growth Differ. 5, 585–593.

    Google Scholar 

  27. Zhang H., Kolb F.A., Brondani V., Billy E., Filipowicx W. 2002. Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. EMBO J. 21, 5875–5885.

    Google Scholar 

  28. Smith N.A., Singh S.P., Wang M.B., Stoutjesdijk P.A., Green A.G., Waterhouse P.M. 2000. Total silencing by intron-spliced hairpin RNAs. Nature. 407, 319–320.

    Google Scholar 

  29. Hammond S.M., Boettcher S., Caudy A.A., Kobayashi R., Hannon G.J. 2001. Argonaute2, a link between genetic and biochemical analyses of RNAi. Science. 293, 1146–1150.

    Google Scholar 

  30. Cabrera C.V., Alonso M.C., Huikeshoven H. 1994. Regulation of scute function by extramacrochaete in vitro and in vivo. Development. 120, 3595–3603.

    Google Scholar 

  31. Sato M., Kojima T., Michiue T., Saigo K. 1999. Bar homeobox genes are latitudinal prepattern genes in the developing Drosophila notum whose expression is regulated by the concerted functions of decapentaplegic and singles. Development. 126, 1456–1466.

    Google Scholar 

  32. Leulier F., Vidal S., Saigo K., Ueda R., Lemaire B. 2002. Inducible expression of double-stranded RNA reveals a role for dFADD in the regulation of the antibacterial response in Drosophila adults. Current Biol. 12, 996–1000.

    Google Scholar 

  33. Inaki M., Kojima T., Ueda R., Saigo K. 2002. Requirements of high levels of Hedgehog signaling activity for medial-region cell fate determination in Drosophila legs: identification of pxb, a putative Hedgehog signaling attenuator gene repressed along the anterior-posterior compartment boundary. Mech. Dev. 116, 3–18.

    Google Scholar 

  34. Takaemae H., Ueda R., Okubo R., Nakato H., Izumi S., Saigo K., Nishihara S., Saigo K. 2003. Proteoglycan UDP-galactose:beta-xylose beta 1.4-galactosyltransferase I is essential for viability in Drosophila melanogaster. J. Biol. Chem. 278, 15571–15578.

    Google Scholar 

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Authors and Affiliations

  1. Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

    K. Ui-Tei, S. Zenno, F. Takahashi, N. Doi, Y. Naito & K. Saigo

  2. Mitsubishi-Kagaku Institute of Life Sciences, 11 Minami-ooya, Machida-shi, Tokyo, 194-8511, Japan

    K. Ui-Tei, R. Ueda, F. Takahashi, M. Yamamoto & N. Hashimoto

  3. UPBSB, School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

    K. Ui-Tei & F. Takahashi

  4. Genetic Strain Research Center, National Institute of Genetics, Mishima, Shizuoka, 411-8540, Japan

    R. Ueda & K. Takahashi

  5. Laboratory for Stem Cell Translational Research, Center for Developmental Biology, RIKEN, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan

    T. Hamada

  6. Development and Differentiation Laboratory, Developmental Biology Department, Insect and Animal Sciences Division, National Institute of Agrobiological Sciences, Ikenodai 2, Kukisaki, Inashiki, 305-0901, Ibaraki, Japan

    T. Tokunaga

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  1. K. Ui-Tei
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  2. R. Ueda
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  3. S. Zenno
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  4. F. Takahashi
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  5. N. Doi
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  6. Y. Naito
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  7. M. Yamamoto
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  8. N. Hashimoto
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  9. K. Takahashi
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  10. T. Hamada
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  11. T. Tokunaga
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  12. K. Saigo
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Ui-Tei, K., Ueda, R., Zenno, S. et al. RNA Interference Induced by Transient or Stable Expression of Hairpin Structures of Double-Stranded RNA in Drosophila and Mammalian Cells. Molecular Biology 38, 228–238 (2004). https://doi.org/10.1023/B:MBIL.0000023739.63178.70

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  • DOI: https://doi.org/10.1023/B:MBIL.0000023739.63178.70

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