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Blocking Zebrafish MicroRNAs with Morpholinos

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Morpholino Oligomers

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1565))

Abstract

Antisense morpholino oligonucleotides have been commonly used in zebrafish to inhibit mRNA function, either by inhibiting pre-mRNA splicing or by blocking translation initiation. Even with the advent of genome editing by CRISP/Cas9 technology, morpholinos provide a useful and rapid tool to knockdown gene expression. This is especially true when dealing with multiple alleles and large gene families where genetic redundancy can complicate knockout of all family members. miRNAs are small noncoding RNAs that are often encoded in gene families and can display extensive genetic redundancy. This redundancy, plus their small size which can limit targeting by CRISPR/Cas9, makes morpholino-based strategies particularly attractive for inhibition of miRNA function. We provide the rationale, background, and methods to inhibit miRNA function with antisense morpholinos during early development and in the adult retina in zebrafish.

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References

  1. Flynt AS, Lai EC (2008) Biological principles of microRNA-mediated regulation: shared themes amid diversity. Nat Rev Genet 9(11):831–842

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T (2001) Identification of novel genes coding for small expressed RNAs. Science 294(5543):853–858

    Article  CAS  PubMed  Google Scholar 

  3. Lee RC, Feinbaum RL, Ambros V (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75(5):843–854

    Article  CAS  PubMed  Google Scholar 

  4. Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright AJ (2006) miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res 34(Suppl 1):D140

    Article  CAS  PubMed  Google Scholar 

  5. Flynt AS, Thatcher EJ, Burkewitz K, Li N, Liu Y, Patton JG (2009) miR-8 microRNAs regulate the response to osmotic stress in zebrafish embryos. J Cell Biol 185(1):115–127. doi:10.1083/jcb.200807026

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Flynt AS, Li N, Thatcher EJ, Solnica-Krezel L, Patton JG (2007) Zebrafish miR-214 modulates Hedgehog signaling to specify muscle cell fate. Nat Genet 39(2):259

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Farh KK-H, Grimson A, Jan C, Lewis BP, Johnston WK, Lim LP, Burge CB, Bartel DP (2005) The widespread impact of mammalian microRNAs on mRNA repression and evolution. Science 310(5755):1817–1821. doi:10.1126/science.1121158

    Article  CAS  PubMed  Google Scholar 

  8. Krutzfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M, Stoffel M (2005) Silencing of microRNAs in vivo with 'antagomirs'. Nature 438(7068):685

    Article  PubMed  Google Scholar 

  9. Kloosterman WP, Wienholds E, Ketting RF, Plasterk RHA (2004) Substrate requirements for let-7 function in the developing zebrafish embryo. Nucleic Acids Res 32(21):6284–6291

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Chen YW, Song S, Weng R, Verma P, Kugler JM, Buescher M, Rouam S, Cohen SM (2014) Systematic study of Drosophila microRNA functions using a collection of targeted knockout mutations. 31(6):784-800

    Google Scholar 

  11. Horwich MD, Zamore PD (2008) Design and delivery of antisense oligonucleotides to block microRNA function in cultured Drosophila and human cells. Nat Protoc 3(10):1537–1549

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Choi W-Y, Giraldez AJ, Schier AF (2007) Target protectors reveal dampening and balancing of nodal agonist and antagonist by miR-430. Science 318(5848):271–274. doi:10.1126/science.1147535

    Article  CAS  PubMed  Google Scholar 

  13. Kloosterman WP, Lagendijk AK, Ketting RF, JD M, RHA P (2007) Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development. PLoS Biol 5(8):e203

    Article  PubMed  PubMed Central  Google Scholar 

  14. Grabher C, Payne EM, Johnston AB, Bolli N, Lechman E, Dick JE, Kanki JP, Look AT (2011) Zebrafish microRNA-126 determines hematopoietic cell fate through c-Myb. Leukemia 25(3):506–514

    Article  CAS  PubMed  Google Scholar 

  15. Mishima Y, Abreu-Goodger C, Staton AA, Stahlhut C, Shou C, Cheng C, Gerstein M, Enright AJ, Giraldez AJ (2009) Zebrafish miR-1 and miR-133 shape muscle gene expression and regulate sarcomeric actin organization. Genes Dev 23(5):619–632

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Choi PS, Zakhary L, Choi W-Y, Caron S, Alvarez-Saavedra E, Miska EA, McManus M, Harfe B, Giraldez AJ, Horvitz RH, Schier AF, Dulac C (2008) Members of the miRNA-200 family regulate olfactory neurogenesis. Neuron 57(1):41–55

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Nicoli S, Standley C, Walker P, Hurlstone A, Fogarty KE, Lawson ND (2010) MicroRNA-mediated integration of haemodynamics and Vegf signalling during angiogenesis. Nature 464(7292):1196–1200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Bill BR, Petzold AM, Clark KJ, Schimmenti LA, Ekker SC (2009) A primer for Morpholino use in zebrafish. Zebrafish 6 (1):69-77. doi:10.1089/zeb.2008.0555

  19. Lee Y, Jeon K, Lee JT, Kim S, Kim VN (2002) MicroRNA maturation: stepwise processing and subcellular localization. EMBO J 21(17):4663–4670

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Rodriguez A, Griffiths-Jones S, Ashurst JL, Bradley A (2004) Identification of mammalian microRNA host genes and transcription units. Genome Res 14(10A):1902–1910

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Ruby JG, Stark A, Johnston WK, Kellis M, Bartel DP, Lai EC (2007) Evolution, biogenesis, expression, and target predictions of a substantially expanded set of Drosophila microRNAs. Genome Res 17(12):1850–1864

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Denli AM, Tops BB, Plasterk RH, Ketting RF, Hannon GJ (2004) Processing of primary microRNAs by the Microprocessor complex. Nature 432(7014):231–235

    Article  CAS  PubMed  Google Scholar 

  23. Ketting RF, Fischer S, Bernstein E, Sijen T, Hannon GJ, Plasterk R (2001) Dicer functions in RNA interfence and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev 15:2654–2659

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, Song JJ, Hammond SM, Joshua-Tor L, Hannon GJ (2004) Argonaute2 is the catalytic engine of mammalian RNAi. Science 305(5689):1437–1441

    Article  CAS  PubMed  Google Scholar 

  25. Lai EC (2002) Micro RNAs are complementary to 3' UTR sequence motifs that mediate negative post-transcriptional regulation. Nat Genet 30(4):363–364

    Article  CAS  PubMed  Google Scholar 

  26. Doench JG, Petersen CP, Sharp PA (2003) siRNAs can function as miRNAs. Genes Dev 17(4):438–442

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Rehwinkel JAN, Behm-Ansmant I, Gatfield D, Izaurralde E (2005) A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing. RNA 11(11):1640–1647

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Giraldez AJ, Mishima Y, Rihel J, Grocock RJ, Van Dongen S, Inoue K, Enright AJ, Schier AF (2006) Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs. Science 312(5770):75–79

    Article  CAS  PubMed  Google Scholar 

  29. Eisen JS, Smith JC (2008) Controlling morpholino experiments: don't stop making antisense. Development 135(10):1735–1743

    Article  CAS  PubMed  Google Scholar 

  30. Bedell VM, Westcot SE, Ekker SC (2011) Lessons from morpholino-based screening in zebrafish. Brief Funct Genomics 10(4):181–188. doi:10.1093/bfgp/elr021

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Lewis BP, Burge CB, Bartel DP (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120(1):15–20. doi:10.1016/j.cell.2004.12.035

    Article  CAS  PubMed  Google Scholar 

  32. Chen PY, Manninga H, Slanchev K, Chien M, Russo JJ, Ju J, Sheridan R, John B, Marks DS, Gaidatzis D, Sander C, Zavolan M, Tuschl T (2005) The developmental miRNA profiles of zebrafish as determined by small RNA cloning. Genes Dev 19(11):1288–1293

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Kumar A, ML WAF-T, Ml T, RJ F-M, Rj M, Fau-Lefevre C (2012) Lefevre C miRNA_targets: a database for miRNA target predictions in coding and non-coding regions of mRNAs. Genomics 100(6):352–356

    Article  CAS  PubMed  Google Scholar 

  34. Grimson A, Farh KK-H, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP (2007) MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol Cell 27(1):91

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Li N, Flynt AS, Kim HR, Solnica-Krezel L, Patton JG (2008) Dispatched Homolog 2 is targeted by miR-214 through a combination of three weak microRNA recognition sites. Nucleic Acids Res 36(13):4277–4285

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Wei C, Salichos L, Wittgrove CM, Rokas A, Patton JG (2012) Transcriptome-wide analysis of small RNA expression in early zebrafish development. RNA 18(5):915–929

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Yao Y, Ma L, Jia Q, Deng W, Liu Z, Zhang Y, Ren J, Xue Y, Jia H, Yang Q (2014) Systematic characterization of small RNAome during zebrafish early developmental stages. BMC Genomics 15(1):1–17

    Article  Google Scholar 

  38. Giraldez AJ, Cinalli RM, Glasner ME, Enright AJ, Thomson JM, Baskerville S, Hammond SM, Bartel DP, Schier AF (2005) MicroRNAs regulate brain morphogenesis in zebrafish. Science 308(5723):833–838

    Article  CAS  PubMed  Google Scholar 

  39. Wienholds E, Kloosterman WP, Miska E, Alvarez-Saavedra E, Berezikov E, de Bruijn E, Horvitz HR, Kauppinen S, Plasterk RHA (2005) MicroRNA expression in zebrafish embryonic development. Science 309(5732):310–311

    Article  CAS  PubMed  Google Scholar 

  40. Thatcher EJ, Paydar I, Anderson KK, Patton JG (2008) Regulation of zebrafish fin regeneration by microRNAs. Proc Natl Acad Sci 105(47):18384–18389

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JRJ, Joung JK (2013) Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 31(3):227–229. doi:10.1038/nbt.2501

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Sertori R, Trengove M, Basheer F, Ward AC, Liongue C (2015) Genome editing in zebrafish: a practical overview. Brief Funct Genomics 15(4):322–330

    Article  PubMed  Google Scholar 

  43. Weinholds E, Koudijs MJ, van Eeden F, Cuppen E, Plasterk R (2003) The microRNA-producing enzyme Dicer1 is essential for zebrafish development. Nat Genet 35(3):217–218

    Article  Google Scholar 

  44. Kok Fatma O, Shin M, Ni C-W, Gupta A, Grosse Ann S, van Impel A, Kirchmaier Bettina C, Peterson-Maduro J, Kourkoulis G, Male I, DeSantis DF, Sheppard-Tindell S, Ebarasi L, Betsholtz C, Schulte-Merker S, Wolfe Scot A, Lawson Nathan D (2015) Reverse genetic screening reveals poor correlation between morpholino-induced and mutant phenotypes in zebrafish. Dev Cell 32(1):97–108. doi:10.1016/j.devcel.2014.11.018

    Article  CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Biological Sciences, University of Southern Mississippi, Hattiesburg, MS, USA

    Alex Sutton Flynt

  2. Department of Biological Sciences, Vanderbilt University, 2325 Stevenson Center, Box 1820 Station B, Nashville, TN, 37235, USA

    Mahesh Rao & James G. Patton

Authors
  1. Alex Sutton Flynt
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  2. Mahesh Rao
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  3. James G. Patton
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Corresponding author

Correspondence to James G. Patton .

Editor information

Editors and Affiliations

  1. Department of Biomedical Sciences College of Veterinary Medicine, Oregon State University, Corvallis, Oregon, USA

    Hong M. Moulton

  2. Gene Tools, LLC, Philomath, Oregon, USA

    Jon D. Moulton

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Flynt, A.S., Rao, M., Patton, J.G. (2017). Blocking Zebrafish MicroRNAs with Morpholinos. In: Moulton, H., Moulton, J. (eds) Morpholino Oligomers. Methods in Molecular Biology, vol 1565. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6817-6_6

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  • DOI: https://doi.org/10.1007/978-1-4939-6817-6_6

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6815-2

  • Online ISBN: 978-1-4939-6817-6

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