RNA Therapeutics pp 237-253

Part of the Methods in Molecular Biology book series (MIMB, volume 629)

Inhibition of the microRNA Pathway in Zebrafish by siRNA

  • Anders Fjose
  • Xiao-Feng Zhao
Protocol

Abstract

The microRNA (miRNA) pathway and the phenomenon of RNA interference (RNAi), which have both been shown to involve targeting of mRNAs by small RNA molecules, are interconnected and depend partly on the same cellular machinery. RNAi in vertebrates was first reported in zebrafish (Danio rerio) 10 years ago. However, reliable RNAi-based gene silencing techniques, based on injection of small interfering RNAs (siRNAs) into zygotes, have not been established for this important vertebrate model because of unspecific developmental defects. We have recently shown that these side effects can be attributed to inhibition of the miRNA pathway by siRNAs at early embryonic stages. This review highlights these findings and the function of microRNAs in zebrafish development.

Key words

RNAi siRNA microRNA miR-430 zebrafish 

References

  1. 1.
    Fire, A., Xu, S., Montgomery, M.K. et al. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391, 806–811.PubMedCrossRefGoogle Scholar
  2. 2.
    Bernstein, E., Caudy, A.A., Hammond, S.M. et al. (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, 409, 363–366.PubMedCrossRefGoogle Scholar
  3. 3.
    Lau, N.C., Lim, L.P., Weinstein, E.G. et al. (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science, 294, 858–862.PubMedCrossRefGoogle Scholar
  4. 4.
    Reinhart, B.J., Slack, F.J., Basson, M. et al. (2000) The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature, 403, 901–906.PubMedCrossRefGoogle Scholar
  5. 5.
    Wightman, B., Ha, I., and Ruvkun, G. (1993) Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell, 75, 855–862.PubMedCrossRefGoogle Scholar
  6. 6.
    Lee, R.C., Feinbaum, R.L., and Ambros, V. (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell, 75, 843–854.PubMedCrossRefGoogle Scholar
  7. 7.
    Zeng, Y., Yi, R., and Cullen, B.R. (2003) MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. Proc Natl Acad Sci USA, 100, 9779–9784.PubMedCrossRefGoogle Scholar
  8. 8.
    Rana, T.M. (2007) Illuminating the silence: understanding the structure and function of small RNAs. Nat Rev Mol Cell Biol, 8, 23–36.PubMedCrossRefGoogle Scholar
  9. 9.
    Kim, D.H., Behlke, M.A., Rose, S.D. et al. (2005) Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat Biotechnol, 23, 222–226.PubMedCrossRefGoogle Scholar
  10. 10.
    Baulcombe, D. (2004) RNA silencing in plants. Nature, 431, 356–363.PubMedCrossRefGoogle Scholar
  11. 11.
    Stefani, G. and Slack, F.J. (2008) Small non-coding RNAs in animal development. Nat Rev Mol Cell Biol, 9, 219–230.PubMedCrossRefGoogle Scholar
  12. 12.
    Kloosterman, W.P. and Plasterk, R.H. (2006) The diverse functions of microRNAs in animal development and disease. Dev Cell, 11, 441–450.PubMedCrossRefGoogle Scholar
  13. 13.
    Dillon, C.P., Sandy, P., Nencioni, A. et al. (2005) RNAi as an experimental and therapeutic tool to study and regulate physiological and disease processes. Annu Rev Physiol, 67, 147–173.PubMedCrossRefGoogle Scholar
  14. 14.
    Beal, J. (2005) Silence is golden: can RNA interference therapeutics deliver? Drug Discov Today, 10, 169–172.PubMedCrossRefGoogle Scholar
  15. 15.
    Hammond, S.M. (2005) Dicing and slicing: the core machinery of the RNA interference pathway. FEBS Lett, 579, 5822–5829.PubMedCrossRefGoogle Scholar
  16. 16.
    Yi, R., Qin, Y., Macara, I.G. et al. (2003) Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev, 17, 3011–3016.PubMedCrossRefGoogle Scholar
  17. 17.
    Lewis, B.P., Burge, C.B., and Bartel, D.P. (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell, 120, 15–20.PubMedCrossRefGoogle Scholar
  18. 18.
    Doench, J.G. and Sharp, P.A. (2004) Specificity of microRNA target selection in translational repression. Genes Dev, 18, 504–511.PubMedCrossRefGoogle Scholar
  19. 19.
    Pillai, R.S., Bhattacharyya, S.N., and Filipowicz, W. (2007) Repression of protein synthesis by miRNAs: how many mechanisms? Trends Cell Biol, 17, 118–126.PubMedCrossRefGoogle Scholar
  20. 20.
    Yekta, S., Shih, I.H., and Bartel, D.P. (2004) MicroRNA-directed cleavage of HOXB8 mRNA. Science, 304, 594–596.PubMedCrossRefGoogle Scholar
  21. 21.
    Williams, A.E. (2008) Functional aspects of animal microRNAs. Cell Mol Life Sci, 65, 545–562.PubMedCrossRefGoogle Scholar
  22. 22.
    Thatcher, E.J., Bond, J., Paydar, I. et al. (2008) Genomic organization of zebrafish microRNAs. BMC Genomics, 9, 253.PubMedCrossRefGoogle Scholar
  23. 23.
    Landgraf, P., Rusu, M., Sheridan, R. et al. (2007) A mammalian microRNA expression atlas based on small RNA library sequencing. Cell, 129, 1401–1414.PubMedCrossRefGoogle Scholar
  24. 24.
    Berezikov, E., Guryev, V., van de Belt, J. et al. (2005) Phylogenetic shadowing and computational identification of human microRNA genes. Cell, 120, 21–24.PubMedCrossRefGoogle Scholar
  25. 25.
    Giraldez, A.J., Mishima, Y., Rihel, J. et al. (2006) Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs. Science, 312, 75–79.PubMedCrossRefGoogle Scholar
  26. 26.
    Lim, L.P., Lau, N.C., Garrett-Engele, P. et al. (2005) Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature, 433, 769–773.PubMedCrossRefGoogle Scholar
  27. 27.
    Cohen, S.M. and Brennecke, J. (2006) Developmental biology. Mixed messages in early development. Science, 312, 65–66.PubMedCrossRefGoogle Scholar
  28. 28.
    Begemann, G. (2008) MicroRNAs and RNA interference in zebrafish development. Zebrafish, 5, 111–119.PubMedCrossRefGoogle Scholar
  29. 29.
    Kloosterman, W.P., Lagendijk, A.K., Ketting, R.F. et al. (2007) Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development. PLoS Biol, 5, e203.PubMedCrossRefGoogle Scholar
  30. 30.
    Choi, W.Y., Giraldez, A.J., and Schier, A.F. (2007) Target protectors reveal dampening and balancing of Nodal agonist and antagonist by miR-430. Science, 318, 271–274.PubMedCrossRefGoogle Scholar
  31. 31.
    Lents, N.H. and Baldassare, J.J. (2006) RNA interference takes flight: a new RNAi screen reveals cell cycle regulators in Drosophila cells. Trends Endocrinol Metab, 17, 173–174.PubMedCrossRefGoogle Scholar
  32. 32.
    Coumoul, X. and Deng, C.X. (2006) RNAi in mice: a promising approach to decipher gene functions in vivo. Biochimie, 88, 637–643.PubMedCrossRefGoogle Scholar
  33. 33.
    Cullen, L.M. and Arndt, G.M. (2005) Genome-wide screening for gene function using RNAi in mammalian cells. Immunol Cell Biol, 83, 217–223.PubMedCrossRefGoogle Scholar
  34. 34.
    Paddison, P.J. (2008) RNA interference in mammalian cell systems. Curr Top Microbiol Immunol, 320, 1–19.PubMedCrossRefGoogle Scholar
  35. 35.
    Nguyen, T., Menocal, E.M., Harborth, J. et al. (2008) RNAi therapeutics: an update on delivery. Curr Opin Mol Ther, 10, 158–167.PubMedGoogle Scholar
  36. 36.
    Durcan, N., Murphy, C., and Cryan, S.A. (2008) Inhalable siRNA: potential as a therapeutic agent in the lungs. Mol Pharm, 5, 559–566.PubMedCrossRefGoogle Scholar
  37. 37.
    Wargelius, A., Ellingsen, S., and Fjose, A. (1999) Double-stranded RNA induces specific developmental defects in zebrafish embryos. Biochem Biophys Res Commun, 263, 156–161.PubMedCrossRefGoogle Scholar
  38. 38.
    Gruber, J., Manninga, H., Tuschl, T. et al. (2005) Specific RNAi mediated gene knockdown in zebrafish cell lines. RNA Biol, 2, 101–105.PubMedCrossRefGoogle Scholar
  39. 39.
    Zhao, X.F., Fjose, A., Larsen, N. et al. (2008) Treatment with small interfering RNA affects the microRNA pathway and causes unspecific defects in zebrafish embryos. FEBS J, 275, 2177–2184.PubMedCrossRefGoogle Scholar
  40. 40.
    Kloosterman, W.P., Steiner, F.A., Berezikov, E. et al. (2006) Cloning and expression of new microRNAs from zebrafish. Nucleic Acids Res, 34, 2558–2569.PubMedCrossRefGoogle Scholar
  41. 41.
    Chen, P.Y., Manninga, H., Slanchev, K. et al. (2005) The developmental miRNA profiles of zebrafish as determined by small RNA cloning. Genes Dev, 19, 1288–1293.PubMedCrossRefGoogle Scholar
  42. 42.
    Lim, L.P., Lau, N.C., Weinstein, E.G. et al. (2003) The microRNAs of Caenorhabditis elegans. Genes Dev, 17, 991–1008.PubMedCrossRefGoogle Scholar
  43. 43.
    Eisen, J.S. and Smith, J.C. (2008) Controlling morpholino experiments: don’t stop making antisense. Development, 135, 1735–1743.PubMedCrossRefGoogle Scholar
  44. 44.
    Chen, E. and Ekker, S.C. (2004) Zebrafish as a genomics research model. Curr Pharm Biotechnol, 5, 409–413.PubMedCrossRefGoogle Scholar
  45. 45.
    Flynt, A.S., Li, N., Thatcher, E.J. et al. (2007) Zebrafish miR-214 modulates Hedgehog signaling to specify muscle cell fate. Nat Genet, 39, 259–263.PubMedCrossRefGoogle Scholar
  46. 46.
    Valoczi, A., Hornyik, C., Varga, N. et al. (2004) Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes. Nucleic Acids Res, 32, e175.PubMedCrossRefGoogle Scholar
  47. 47.
    Wienholds, E., Kloosterman, W.P., Miska, E. et al. (2005) MicroRNA expression in zebrafish embryonic development. Science, 309, 310–311.PubMedCrossRefGoogle Scholar
  48. 48.
    Ason, B., Darnell, D.K., Wittbrodt, B. et al. (2006) Differences in vertebrate microRNA expression. Proc Natl Acad Sci USA, 103, 14385–14389.PubMedCrossRefGoogle Scholar
  49. 49.
    Wienholds, E., Koudijs, M.J., van Eeden, F.J. et al. (2003) The microRNA-producing enzyme Dicer1 is essential for zebrafish development. Nat Genet, 35, 217–218.PubMedCrossRefGoogle Scholar
  50. 50.
    Bernstein, E., Kim, S.Y., Carmell, M.A. et al. (2003) Dicer is essential for mouse development. Nat Genet, 35, 215–217.PubMedCrossRefGoogle Scholar
  51. 51.
    Giraldez, A.J., Cinalli, R.M., Glasner, M.E. et al. (2005) MicroRNAs regulate brain morphogenesis in zebrafish. Science, 308, 833–838.PubMedCrossRefGoogle Scholar
  52. 52.
    Newport, J. and Kirschner, M. (1982) A major developmental transition in early Xenopus embryos: II. Control of the onset of transcription. Cell, 30, 687–696.PubMedCrossRefGoogle Scholar
  53. 53.
    Watanabe, T., Takeda, A., Mise, K. et al. (2005) Stage-specific expression of microRNAs during Xenopus development. FEBS Lett, 579, 318–324.PubMedCrossRefGoogle Scholar
  54. 54.
    Lykke-Andersen, K., Gilchrist, M.J., Grabarek, J.B. et al. (2008) Maternal Argonaute 2 is essential for early mouse development at the maternal–zygotic transition. Mol Biol Cell, 19, 4383–4392.PubMedCrossRefGoogle Scholar
  55. 55.
    Bushati, N., Stark, A., Brennecke, J. et al. (2008) Temporal reciprocity of miRNAs and their targets during the maternal-to-zygotic transition in Drosophila. Curr Biol, 18, 501–506.PubMedCrossRefGoogle Scholar
  56. 56.
    Pearson, J.C., Lemons, D., and McGinnis, W. (2005) Modulating Hox gene functions during animal body patterning. Nat Rev Genet, 6, 893–904.PubMedCrossRefGoogle Scholar
  57. 57.
    Woltering, J.M. and Durston, A.J. (2008) MiR-10 represses HoxB1a and HoxB3a in zebrafish. PLoS ONE, 3, e1396.PubMedCrossRefGoogle Scholar
  58. 58.
    Tanzer, A., Amemiya, C.T., Kim, C.B. et al. (2005) Evolution of microRNAs located within Hox gene clusters. J Exp Zoolog B Mol Dev Evol, 304, 75–85.CrossRefGoogle Scholar
  59. 59.
    Mansfield, J.H., Harfe, B.D., Nissen, R. et al. (2004) MicroRNA-responsive 'sensor' transgenes uncover Hox-like and other developmentally regulated patterns of vertebrate microRNA expression. Nat Genet, 36, 1079–1083.PubMedCrossRefGoogle Scholar
  60. 60.
    Pase, L., Layton, J.E., Kloosterman, W.P. et al. (2009) miR-451 regulates zebrafish erythroid maturation in vivo via its target gata2. Blood, 113, 1794–1804.PubMedCrossRefGoogle Scholar
  61. 61.
    Yin, V.P., Thomson, J.M., Thummel, R. et al. (2008) Fgf-dependent depletion of microRNA-133 promotes appendage regeneration in zebrafish. Genes Dev, 22, 728–733.PubMedCrossRefGoogle Scholar
  62. 62.
    Morton, S.U., Scherz, P.J., Cordes, K.R. et al. (2008) microRNA-138 modulates cardiac patterning during embryonic development. Proc Natl Acad Sci USA, 105, 17830–17835.PubMedCrossRefGoogle Scholar
  63. 63.
    Li, Y.X., Farrell, M.J., Liu, R. et al. (2000) Double-stranded RNA injection produces null phenotypes in zebrafish. Dev Biol, 217, 394–405.PubMedCrossRefGoogle Scholar
  64. 64.
    Hsieh, D.J. and Liao, C.F. (2002) Zebrafish M2 muscarinic acetylcholine receptor: cloning, pharmacological characterization, expression patterns and roles in embryonic bradycardia. Br J Pharmacol, 137, 782–792.PubMedCrossRefGoogle Scholar
  65. 65.
    Zhao, Z., Cao, Y., Li, M. et al. (2001) Double-stranded RNA injection produces nonspecific defects in zebrafish. Dev Biol, 229, 215–223.PubMedCrossRefGoogle Scholar
  66. 66.
    Oates, A.C., Bruce, A.E., and Ho, R.K. (2000) Too much interference: injection of double-stranded RNA has nonspecific effects in the zebrafish embryo. Dev Biol, 224, 20–28.PubMedCrossRefGoogle Scholar
  67. 67.
    Heasman, J. (2002) Morpholino oligos: making sense of antisense? Dev Biol, 243, 209–214.PubMedCrossRefGoogle Scholar
  68. 68.
    Ekker, S.C. and Larson, J.D. (2001) Morphant technology in model developmental systems. Genesis, 30, 89–93.PubMedCrossRefGoogle Scholar
  69. 69.
    Nasevicius, A. and Ekker, S.C. (2000) Effective targeted gene 'knockdown' in zebrafish. Nat Genet, 26, 216–220.PubMedCrossRefGoogle Scholar
  70. 70.
    Stark, G.R., Kerr, I.M., Williams, B.R. et al. (1998) How cells respond to interferons. Annu Rev Biochem, 67, 227–264.PubMedCrossRefGoogle Scholar
  71. 71.
    Elbashir, S.M., Harborth, J., Lendeckel, W. et al. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 411, 494–498.PubMedCrossRefGoogle Scholar
  72. 72.
    Kanungo, J., Li, B.S., Zheng, Y. et al. (2006) Cyclin-dependent kinase 5 influences Rohon–Beard neuron survival in zebrafish. J Neurochem, 99, 251–259.PubMedCrossRefGoogle Scholar
  73. 73.
    Liu, W.Y., Wang, Y., Sun, Y.H. et al. (2005) Efficient RNA interference in zebrafish embryos using siRNA synthesized with SP6 RNA polymerase. Dev Growth Differ, 47, 323–331.PubMedCrossRefGoogle Scholar
  74. 74.
    Dodd, A., Chambers, S.P., and Love, D.R. (2004) Short interfering RNA-mediated gene targeting in the zebrafish. FEBS Lett, 561, 89–93.PubMedCrossRefGoogle Scholar
  75. 75.
    Kok, K.H., Ng, M.H., Ching, Y.P. et al. (2007) Human TRBP and PACT directly interact with each other and associate with dicer to facilitate the production of small interfering RNA. J Biol Chem, 282, 17649–17657.PubMedCrossRefGoogle Scholar
  76. 76.
    Haase, A.D., Jaskiewicz, L., Zhang, H. et al. (2005) TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing. EMBO Rep, 6, 961–967.PubMedCrossRefGoogle Scholar
  77. 77.
    Chendrimada, T.P., Gregory, R.I., Kumaraswamy, E. et al. (2005) TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature, 436, 740–744.PubMedCrossRefGoogle Scholar
  78. 78.
    Blidner, R.A., Svoboda, K.R., Hammer, R.P. et al. (2008) Photoinduced RNA interference using DMNPE-caged 2'-deoxy-2'-fluoro substituted nucleic acids in vitro and in vivo. Mol Biosyst, 4, 431–440.PubMedCrossRefGoogle Scholar
  79. 79.
    Hitz, C., Steuber-Buchberger, P., Delic, S. et al. (2009) Generation of shRNA transgenic mice. Methods Mol Biol, 530, 1–29.CrossRefGoogle Scholar
  80. 80.
    Dietzl, G., Chen, D., Schnorrer, F. et al. (2007) A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature, 448, 151–156.PubMedCrossRefGoogle Scholar
  81. 81.
    Emelyanov, A. and Parinov, S. (2008) Mifepristone-inducible LexPR system to drive and control gene expression in transgenic zebrafish. Dev Biol, 320, 113–121.PubMedCrossRefGoogle Scholar
  82. 82.
    Asakawa, K., Suster, M.L., Mizusawa, K. et al. (2008) Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish. Proc Natl Acad Sci USA, 105, 1255–1260.PubMedCrossRefGoogle Scholar
  83. 83.
    Davison, J.M., Akitake, C.M., Goll, M.G. et al. (2007) Transactivation from Gal4-VP16 transgenic insertions for tissue-specific cell labeling and ablation in zebrafish. Dev Biol, 304, 811–824.PubMedCrossRefGoogle Scholar
  84. 84.
    Robu, M.E., Larson, J.D., Nasevicius, A. et al. (2007) p53 activation by knockdown technologies. PLoS Genet, 3, e78.PubMedCrossRefGoogle Scholar
  85. 85.
    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.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Anders Fjose
    • 1
  • Xiao-Feng Zhao
    • 1
  1. 1.Department of Molecular BiologyUniversity of BergenBergenNorway

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