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Use of target protector morpholinos to analyze the physiological roles of specific miRNA-mRNA pairs in vivo

Nature Protocols volume 6, pages 2035–2049 (2011)Cite this article

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Abstract

MicroRNAs (miRNAs) regulate gene expression by pairing with complementary sequences in the 3′ untranslated regions (UTRs) of transcripts. Although the molecular mechanism underlying miRNA biogenesis and activity is becoming better understood, determining the physiological role of the interaction of an miRNA with its target remains a challenge. A number of methods have been developed to inhibit individual miRNAs, but it can be difficult to determine which specific targets are responsible for any observed phenotypes. To address this problem, we use target protector (TP) morpholinos that interfere with a single miRNA-mRNA pair by binding specifically to the miRNA target sequence in the 3′ UTR. In this protocol, we detail the steps for identifying miRNA targets, validating their regulation and using TPs to interrogate their function in zebrafish. Depending on the biological context, this set of experiments can be completed in 6–8 weeks.

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Figure 1: Interfering with miRNA processing can help to reveal the functions of miRNAs.
Figure 2: Several different reporters can be used for validating miRNA targets.
Figure 3: Calibration of the injection needle.
Figure 4: Reporters demonstrating that the sdf1a 3′ UTR confers repression by miR-430.
Figure 5: Use of the sdf1a-TP reveals a role for miR-430 targeting in germ cell migration.

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References

  1. Bartel, D.P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297 (2004).

    Article  CAS  PubMed  Google Scholar 

  2. Ambros, V. The functions of animal microRNAs. Nature 431, 350–355 (2004).

    Article  CAS  PubMed  Google Scholar 

  3. Staton, A.A. & Giraldez, A.J. MicroRNAs in development and disease. Encyclopedia of Life Sciences 1–10 (2008).

  4. Lewis, B.P., Shih, I.H., Jones-Rhoades, M.W., Bartel, D.P. & Burge, C.B. Prediction of mammalian microRNA targets. Cell 115, 787–798 (2003).

    Article  CAS  PubMed  Google Scholar 

  5. Landgraf, P. et al. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 129, 1401–1414 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Lim, L.P. et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433, 769–773 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Lewis, B.P., Burge, C.B. & Bartel, D.P. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15–20 (2005).

    Article  CAS  PubMed  Google Scholar 

  8. Friedman, R.C., Farh, K.K., Burge, C.B. & Bartel, D.P. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res. 19, 92–105 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Choi, W.-Y., Giraldez, A.J. & Schier, A.F. Target protectors reveal dampening and balancing of nodal agonist and antagonist by miR-430. Science 318, 271–274 (2007).

    Article  CAS  PubMed  Google Scholar 

  10. Staton, A.A., Knaut, H. & Giraldez, A.J. miRNA regulation of Sdf1 chemokine signaling provides genetic robustness to germ cell migration. Nat. Genet. 43, 204–211 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Lee, R.C., Feinbaum, R.L. & Ambros, V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75, 843–854 (1993).

    Article  CAS  PubMed  Google Scholar 

  12. Reinhart, B.J. et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403, 901–906 (2000).

    Article  CAS  PubMed  Google Scholar 

  13. Ventura, A. et al. Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters. Cell 132, 875–886 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Rasmussen, K.D. et al. The miR-144/451 locus is required for erythroid homeostasis. J. Exp. Med. 207, 1351–1358 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Sokol, N.S. & Ambros, V. Mesodermally expressed Drosophila microRNA-1 is regulated by Twist and is required in muscles during larval growth. Genes Dev. 19, 2343–2354 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Teleman, A.A. & Cohen, S.M. Drosophila lacking microRNA miR-278 are defective in energy homeostasis. Genes Dev. 20, 417–422 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Bushati, N., Stark, A., Brennecke, J. & Cohen, S.M. Temporal reciprocity of miRNAs and their targets during the maternal-to-zygotic transition in Drosophila. Curr. Biol. 18, 501–506 (2008).

    Article  CAS  PubMed  Google Scholar 

  18. Karres, J.S., Hilgers, V., Carrera, I., Treisman, J. & Cohen, S.M. The conserved microRNA MiR-8 tunes atrophin levels to prevent neurodegeneration in Drosophila. Cell 131, 136–145 (2007).

    Article  CAS  PubMed  Google Scholar 

  19. Nasevicius, A. & Ekker, S.C. Effective targeted gene 'knockdown' in zebrafish. Nat. Genet. 26, 216–220 (2000).

    Article  CAS  PubMed  Google Scholar 

  20. Moulton, J.D. & Yan, Y.-L. Using morpholinos to control gene expression. Curr. Protoc. Mol. Biol. 83, 26.28.21–26.28.29 (2008).

    Google Scholar 

  21. Kloosterman, W.P., Lagendijk, A.K., Ketting, R.F., Moulton, J.D. & Plasterk, R.H. Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development. PLoS Biol. 5, e203 (2007).

    PubMed  PubMed Central  Google Scholar 

  22. Woltering, J.M. & Durston, A.J. MiR-10 represses HoxB1a and HoxB3 in zebrafish. PLoS ONE 3, e1396 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Pase, L. et al. miR-451 regulates zebrafish erythroid maturation in vivo via its target gata2. Blood 113, 1794–1804 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Conte, I. et al. miR-204 is required for lens and retinal development via Meis2 targeting. Proc. Natl Acad. Sci. USA 107, 15491–15496 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Horwich, M.D. & Zamore, P.D. Design and delivery of antisense oligonucleotides to block microRNA function in cultured Drosophila and human cells. Nat. Protoc. 3, 1537–1549 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Krutzfeldt, J. et al. Silencing of microRNAs in vivo with 'antagomirs'. Nature 438, 685–689 (2005).

    Article  PubMed  Google Scholar 

  27. Ebert, M.S., Neilson, J.R. & Sharp, P.A. MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat. Meth. 4, 721–726 (2007).

    Article  CAS  Google Scholar 

  28. Ebert, M.S. & Sharp, P.A. MicroRNA sponges: progress and possibilities. RNA 16, 2043–2050 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Loya, C.M., Lu, C.S., Van Vactor, D. & Fulga, T.A. Transgenic microRNA inhibition with spatiotemporal specificity in intact organisms. Nat. Meth. 6, 897–903 (2009).

    Article  CAS  Google Scholar 

  30. Long, J.M. & Lahiri, D.K. MicroRNA-101 downregulates Alzheimer's amyloid-β precursor protein levels in human cell cultures and is differentially expressed. Biochem. Biophys. Res. Commun. 404, 889–895 (2011).

    Article  CAS  PubMed  Google Scholar 

  31. Rosa, A., Spagnoli, F.M. & Brivanlou, A.H. The miR-430/427/302 family controls mesendodermal fate specification via species-specific target selection. Dev. Cell 16, 517–527 (2009).

    Article  CAS  PubMed  Google Scholar 

  32. Bonev, B., Pisco, A. & Papalopulu, N. MicroRNA-9 reveals regional diversity of neural progenitors along the anterior-posterior axis. Dev. Cell 20, 19–32 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Gehrke, S., Imai, Y., Sokol, N. & Lu, B. Pathogenic LRRK2 negatively regulates microRNA-mediated translational repression. Nature 466, 637–641 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Summerton, J. & Weller, D. Morpholino antisense oligomers: design, preparation, and properties. Antisense Nucleic Acid Drug Dev. 7, 187–195 (1997).

    Article  CAS  PubMed  Google Scholar 

  35. Doyon, Y. et al. Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat. Biotechnol. 26, 702–708 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Meng, X., Noyes, M.B., Zhu, L.J., Lawson, N.D. & Wolfe, S.A. Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat. Biotechnol. 26, 695–701 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Witkos, T.M., Koscianska, E. & Krzyzosiak, W.J. Practical aspects of microRNA target prediction. Curr. Mol. Med. 11, 93–109 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Hammell, M. Computational methods to identify miRNA targets. Semin. Cell Dev. Biol. 21, 738–744 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Wienholds, E. et al. MicroRNA expression in zebrafish embryonic development. Science 309, 310–311 (2005).

    Article  CAS  PubMed  Google Scholar 

  40. Grimson, A. et al. MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol. Cell 27, 91–105 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Kiriakidou, M. et al. A combined computational-experimental approach predicts human microRNA targets. Genes Dev. 18, 1165–1178 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Didiano, D. & Hobert, O. Perfect seed pairing is not a generally reliable predictor for miRNA-target interactions. Nat. Struct. Mol. Biol. 13, 849–851 (2006).

    Article  CAS  PubMed  Google Scholar 

  43. Brennecke, J., Stark, A., Russell, R.B. & Cohen, S.M. Principles of microRNA-target recognition. PLoS Biol. 3, e85 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Shin, C. et al. Expanding the microRNA targeting code: functional sites with centered pairing. Mol. Cell 38, 789–802 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Giraldez, A.J. et al. Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs. Science 312, 75–79 (2006).

    Article  CAS  PubMed  Google Scholar 

  46. Mishima, Y. et al. Zebrafish miR-1 and miR-133 shape muscle gene expression and regulate sarcomeric actin organization. Genes Dev. 23, 619–632 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Shkumatava, A., Stark, A., Sive, H. & Bartel, D.P. Coherent but overlapping expression of microRNAs and their targets during vertebrate development. Genes Dev. 23, 466–481 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Baek, D. et al. The impact of microRNAs on protein output. Nature 455, 64–71 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Selbach, M. et al. Widespread changes in protein synthesis induced by microRNAs. Nature 455, 58–63 (2008).

    Article  CAS  PubMed  Google Scholar 

  50. Zhang, L. et al. Systematic identification of C. elegans miRISC proteins, miRNAs, and mRNA targets by their interactions with GW182 proteins AIN-1 and AIN-2. Mol. Cell 28, 598–613 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Karginov, F.V. et al. A biochemical approach to identifying microRNA targets. Proc. Natl Acad. Sci. USA 104, 19291–19296 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Landthaler, M. et al. Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs. RNA 14, 2580–2596 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Beitzinger, M., Peters, L., Zhu, J.Y., Kremmer, E. & Meister, G. Identification of human microRNA targets from isolated argonaute protein complexes. RNA Biol. 4, 76–84 (2007).

    Article  CAS  PubMed  Google Scholar 

  54. Hendrickson, D.G., Hogan, D.J., Herschlag, D., Ferrell, J.E. & Brown, P.O. Systematic identification of mRNAs recruited to argonaute 2 by specific microRNAs and corresponding changes in transcript abundance. PLoS ONE 3, e2126 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  55. Hong, X., Hammell, M., Ambros, V. & Cohen, S.M. Immunopurification of Ago1 miRNPs selects for a distinct class of microRNA targets. Proc. Natl Acad. Sci. USA 106, 15085–15090 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Chi, S.W., Zang, J.B., Mele, A. & Darnell, R.B. Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature 460, 479–486 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Zisoulis, D.G. et al. Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans. Nat. Struct. Mol. Biol. 17, 173–179 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Cormack, B. Directed mutagenesis using the polymerase chain reaction. Curr. Protoc. Mol. Biol. 8.5.1–8.5.10 (2001).

  59. Brennecke, J., Hipfner, D.R., Stark, A., Russell, R.B. & Cohen, S.M. bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell 113, 25–36 (2003).

    Article  CAS  PubMed  Google Scholar 

  60. Mansfield, J.H. et al. MicroRNA-responsive 'sensor' transgenes uncover Hox-like and other developmentally regulated patterns of vertebrate microRNA expression. Nat. Genet. 36, 1079–1083 (2004).

    Article  CAS  PubMed  Google Scholar 

  61. De Pietri Tonelli, D. et al. Single-cell detection of microRNAs in developing vertebrate embryos after acute administration of a dual-fluorescence reporter/sensor plasmid. Biotechniques 41, 727–732 (2006).

    Article  PubMed  Google Scholar 

  62. Thisse, C. & Thisse, B. High-Resolution in situ hybridization to whole-mount zebrafish embryos. Nat. Protoc. 3, 59–69 (2008).

    Article  CAS  PubMed  Google Scholar 

  63. Novak, A. & Ribera, A. Immunocytochemistry as a tool for zebrafish developmental neurobiology. Methods Cell Sci. 25, 79–83 (2003).

    Article  CAS  PubMed  Google Scholar 

  64. Machluf, Y. & Levkowitz, G. Visualization of mRNA expression in the zebrafish embryo. Methods Mol. Biol. 714, 83–102 (2011).

    Article  CAS  PubMed  Google Scholar 

  65. Avanesov, A. & Malicki, J. Approaches to study neurogenesis in the zebrafish retina. Methods Cell Biol. 76 (2004).

  66. Doitsidou, M. et al. Guidance of primordial germ cell migration by the chemokine SDF-1. Cell 111, 647–659 (2002).

    Article  CAS  PubMed  Google Scholar 

  67. Boldajipour, B., Mahabaleshwar, H. & Kardash, E. Control of chemokine-guided cell migration by ligand sequestration. Cell 132, 463–473 (2008).

    Article  CAS  PubMed  Google Scholar 

  68. Giraldez, A.J. et al. MicroRNAs regulate brain morphogenesis in zebrafish. Science 308, 833–838 (2005).

    Article  CAS  PubMed  Google Scholar 

  69. Rembold, M., Lahiri, K., Foulkes, N.S. & Wittbrodt, J. Transgenesis in fish: efficient selection of transgenic fish by co-injection with a fluorescent reporter construct. Nat. Protoc. 1, 1133–1139 (2006).

    Article  CAS  PubMed  Google Scholar 

  70. Maeder, M.L., Thibodeau-Beganny, S., Sander, J.D., Voytas, D.F. & Joung, J.K. Oligomerized pool engineering (OPEN): an 'open-source' protocol for making customized zinc-finger arrays. Nat. Protoc. 4, 1471–1501 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Westerfield, M. The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio rerio) 5th edn. (Univ. of Oregon Press, 2007).

  72. Fisher, S. et al. Evaluating the biological relevance of putative enhancers using Tol2 transposon-mediated transgenesis in zebrafish. Nat. Protoc. 1, 1297–1305 (2006).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We acknowledge W.-Y. Choi and A. Schier, who together with A.J.G., developed the target protector technology. This work was supported by a National Research Service Award–US National institutes of Health (NIH)/National Institute of General Medical Sciences T32 GM007223 Training Grant (A.A.S.); and by NIH grants R01GM081602-05, the Yale Scholar program and the Pew Scholars Program in Biomedical Sciences (A.J.G.).

AUTHOR CONTRIBUTIONS

A.A.S. and A.J.G. designed experiments and interpreted results. A.A.S. performed all experiments and prepared the manuscript with input from A.J.G.

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  1. Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, USA

    Alison A Staton & Antonio J Giraldez

  2. Yale Stem Cell Center, Yale University School of Medicine, New Haven, Connecticut, USA

    Antonio J Giraldez

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Correspondence to Antonio J Giraldez.

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A.J.G. is an author on a patent to use TP technology to block the regulation of target mRNAs.

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Staton, A., Giraldez, A. Use of target protector morpholinos to analyze the physiological roles of specific miRNA-mRNA pairs in vivo. Nat Protoc 6, 2035–2049 (2011). https://doi.org/10.1038/nprot.2011.423

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