Skip to main content
SpringerLink
Log in

Knockdown of Nuclear lncRNAs by Locked Nucleic Acid (LNA) Gapmers in Nephron Progenitor Cells

  • Protocol
  • First Online:
RNA-Chromatin Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2161))

Abstract

Despite recent advance in our understanding on the role of long noncoding RNAs (lncRNAs), the function of the vast majority of lncRNAs remains poorly understood. To characterize the function of lncRNAs, knockdown studies are essential. However, the conventional silencing methods for mRNA, such as RNA interference (RNAi), may not be as efficient against lncRNAs, partly due to the mismatch of the localization of lncRNAs and RNAi machinery. To circumvent such limitation, a new technique has recently been developed, i.e., locked nucleic acid (LNA) gapmers. This system utilizes RNase H that distributes evenly in both nucleus and cytoplasm and is expected to knock down lncRNAs of interest more consistently regardless of their localization in the cell. In this chapter, we describe the procedure with tips to silence lncRNAs by LNA gapmers, by using mouse nephron progenitor cells as an example.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Jarroux J, Morillon A, Pinskaya M (2017) History, discovery, and classification of lncRNAs. Adv Exp Med Biol 1008:1–46. https://doi.org/10.1007/978-981-10-5203-3_1

    Article  CAS  PubMed  Google Scholar 

  2. Hombach S, Kretz M (2016) Non-coding RNAs: classification, biology and functioning. Adv Exp Med Biol 937:3–17. https://doi.org/10.1007/978-3-319-42059-2_1

    Article  CAS  PubMed  Google Scholar 

  3. Brosius J, Raabe CA (2016) What is an RNA? A top layer for RNA classification. RNA Biol 13:140–144. https://doi.org/10.1080/15476286.2015.1128064

    Article  PubMed  PubMed Central  Google Scholar 

  4. Hutchinson JN, Ensminger AW, Clemson CM, Lynch CR, Lawrence JB, Chess A (2007) A screen for nuclear transcripts identifies two linked noncoding RNAs associated with SC35 splicing domains. BMC Genomics 8:39. https://doi.org/10.1186/1471-2164-8-39

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Brown CJ, Hendrich BD, Rupert JL, Lafrenière RG, Xing Y, Lawrence J, Willard HF (1992) The human XIST gene: analysis of a 17 kb inactive X-specific RNA that contains conserved repeats and is highly localized within the nucleus. Cell 71:527–542. https://doi.org/10.1016/0092-8674(92)90520-m

    Article  CAS  PubMed  Google Scholar 

  6. Ivey KN, Srivastava D (2015) microRNAs as developmental regulators. Cold spring Harb Perspect biol 7:a008144. https://doi.org/10.1101/cshperspect.a008144

  7. Quinodoz S, Guttman M (2014) Long noncoding RNAs: an emerging link between gene regulation and nuclear organization. Trends Cell Biol 24:651–663. https://doi.org/10.1016/j.tcb.2014.08.009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Sarropoulos I, Marin R, Cardoso-Moreira M, Kaessmann H (2019) Developmental dynamics of lncRNAs across mammalian organs and species. Nature 571:510–514. https://doi.org/10.1038/s41586-019-1341-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Delás MJ, Hannon GJ (2017) lncRNAs in development and disease: from functions to mechanisms. Open Biol 7. https://doi.org/10.1098/rsob.170121

  10. Duret L, Chureau C, Samain S, Weissenbach J, Avner P (2006) The Xist RNA gene evolved in eutherians by pseudogenization of a protein-coding gene. Science 312:1653–1655. https://doi.org/10.1126/science.1126316

    Article  CAS  PubMed  Google Scholar 

  11. Sachs M, Onodera C, Blaschke K, Ebata KT, Song JS, Ramalho-Santos M (2013) Bivalent chromatin marks developmental regulatory genes in the mouse embryonic germline in vivo. Cell Rep 3:1777–1784. https://doi.org/10.1016/j.celrep.2013.04.032

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Gagnon KT, Li L, Chu Y, Janowski BA, Corey DR (2014) RNAi factors are present and active in human cell nuclei. Cell Rep 6:211–221. https://doi.org/10.1016/j.celrep.2013.12.013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zeng Y, Cullen BR (2002) RNA interference in human cells is restricted to the cytoplasm. RNA 8:855–860. https://doi.org/10.1017/s1355838202020071

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Kawasaki H, Taira K (2003) Short hairpin type of dsRNAs that are controlled by tRNA(Val) promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cells. Nucleic Acids Res 31:700–707. https://doi.org/10.1093/nar/gkg158

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Chiu Y-L, Ali A, Chu C-Y, Cao H, Rana TM (2004) Visualizing a correlation between siRNA localization, cellular uptake, and RNAi in living cells. Chem Biol 11:1165–1175. https://doi.org/10.1016/j.chembiol.2004.06.006

    Article  CAS  PubMed  Google Scholar 

  16. Crooke ST (1999) Molecular mechanisms of action of antisense drugs. Biochim Biophys Acta 1489:31–44. https://doi.org/10.1016/s0167-4781(99)00148-7

    Article  CAS  PubMed  Google Scholar 

  17. Ideue T, Hino K, Kitao S, Yokoi T, Hirose T (2009) Efficient oligonucleotide-mediated degradation of nuclear noncoding RNAs in mammalian cultured cells. RNA 15:1578–1587. https://doi.org/10.1261/rna.1657609

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Vickers TA, Koo S, Bennett CF, Crooke ST, Dean NM, Baker BF (2003) Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. A comparative analysis. J Biol Chem 278:7108–7118. https://doi.org/10.1074/jbc.M210326200

    Article  CAS  PubMed  Google Scholar 

  19. Pendergraff HM, Krishnamurthy PM, Debacker AJ, Moazami MP, Sharma VK, Niitsoo L, Yu Y, Tan YN, Haitchi HM, Watts JK (2017) Locked nucleic acid Gapmers and conjugates potently silence ADAM33 , an asthma-associated Metalloprotease with nuclear-localized mRNA. Mol Ther Nucleic Acids 8:158–168. https://doi.org/10.1016/j.omtn.2017.06.012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Amodio N, Stamato MA, Juli G, Morelli E, Fulciniti M, Manzoni M, Taiana E, Agnelli L, Cantafio MEG, Romeo E, Raimondi L, Caracciolo D, Zuccalà V, Rossi M, Neri A, Munshi NC, Tagliaferri P, Tassone P (2018) Drugging the lncRNA MALAT1 via LNA gapmeR ASO inhibits gene expression of proteasome subunits and triggers anti-multiple myeloma activity. Leukemia 32:1948–1957. https://doi.org/10.1038/s41375-018-0067-3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Fluiter K, Mook ORF, Vreijling J, Langkjær N, Højland T, Wengel J, Baas F (2009) Filling the gap in LNA antisense oligo gapmers: the effects of unlocked nucleic acid (UNA) and 4′-C-hydroxymethyl-DNA modifications on RNase H recruitment and efficacy of an LNA gapmer. Mol BioSyst 5:838. https://doi.org/10.1039/b903922h

    Article  CAS  PubMed  Google Scholar 

  22. Moreno PMD, Ferreira AR, Salvador D, Rodrigues MT, Torrado M, Carvalho ED, Tedebark U, Sousa MM, Amaral IF, Wengel J, Pêgo AP (2018) Hydrogel-assisted antisense LNA Gapmer delivery for in situ gene silencing in spinal cord injury. Mol Ther Nucleic Acids 11:393–406. https://doi.org/10.1016/j.omtn.2018.03.009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Nishikawa M, Kimura H, Yanagawa N, Hamon M, Hauser P, Zhao L, Jo OD, Yanagawa N (2018) An optimal serum-free defined condition for in vitro culture of kidney organoids. Biochem Biophys Res Commun 501:996–1002. https://doi.org/10.1016/j.bbrc.2018.05.098

    Article  CAS  PubMed  Google Scholar 

  24. Souleimanian N, Deleavey GF, Soifer H, Wang S, Tiemann K, Damha MJ, Stein CA (2012) Antisense 2′-Deoxy, 2′-Fluoroarabino nucleic acid (2′F-ANA) oligonucleotides: in vitro Gymnotic silencers of gene expression whose potency is enhanced by fatty acids. Mol Ther Nucleic Acids 1:e43. https://doi.org/10.1038/mtna.2012.35

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Rowan CJ, Sheybani-Deloui S, Rosenblum ND (2017) Origin and function of the renal Stroma in health and disease. Results Probl Cell Differ 60:205–229. https://doi.org/10.1007/978-3-319-51436-9_8

    Article  CAS  PubMed  Google Scholar 

  26. Little MH, McMahon AP (2012) Mammalian kidney development: principles, Progress, and projections. Cold Spring Harb Perspect Biol 4:a008300. https://doi.org/10.1101/cshperspect.a008300

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Brown AC, Muthukrishnan SD, Oxburgh L (2015) A synthetic niche for nephron progenitor cells. Dev Cell 34:229–241. https://doi.org/10.1016/j.devcel.2015.06.021

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Tanigawa S, Taguchi A, Sharma N, Perantoni AO, Nishinakamura R (2016) Selective in vitro propagation of nephron progenitors derived from embryos and pluripotent stem cells. Cell Rep 15:801–813. https://doi.org/10.1016/j.celrep.2016.03.076

    Article  CAS  PubMed  Google Scholar 

  29. Yuri S, Nishikawa M, Yanagawa N, Jo OD, Yanagawa N (2015) Maintenance of mouse nephron progenitor cells in aggregates with gamma-Secretase inhibitor. PLoS One 10:e0129242. https://doi.org/10.1371/journal.pone.0129242

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Li Z, Araoka T, Wu J, Liao H-K, Li M, Lazo M, Zhou B, Sui Y, Wu M-Z, Tamura I, Xia Y, Beyret E, Matsusaka T, Pastan I, Rodriguez Esteban C, Guillen I, Guillen P, Campistol JM, Izpisua Belmonte JC (2016) 3D culture supports long-term expansion of mouse and human nephrogenic progenitors. Cell Stem Cell 19:516–529. https://doi.org/10.1016/j.stem.2016.07.016

    Article  CAS  PubMed  Google Scholar 

  31. Stein CA, Hansen JB, Lai J, Wu S, Voskresenskiy A, Høg A, Worm J, Hedtjärn M, Souleimanian N, Miller P, Soifer HS, Castanotto D, Benimetskaya L, Ørum H, Koch T (2010) Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents. Nucleic Acids Res 38:e3–e3. https://doi.org/10.1093/nar/gkp841

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. School of Engineering, Chemical System Engineering, University of Tokyo, Tokyo, Japan

    Masaki Nishikawa

  2. Medical and Research Services, Greater Los Angeles Veterans Affairs Healthcare System at Sepulveda, North Hills, CA, USA

    Norimoto Yanagawa

  3. David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA

    Norimoto Yanagawa

Authors
  1. Masaki Nishikawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Norimoto Yanagawa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Masaki Nishikawa or Norimoto Yanagawa .

Editor information

Editors and Affiliations

  1. Department for Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark

    Ulf Andersson Vang Ørom

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Nishikawa, M., Yanagawa, N. (2020). Knockdown of Nuclear lncRNAs by Locked Nucleic Acid (LNA) Gapmers in Nephron Progenitor Cells. In: Ørom, U. (eds) RNA-Chromatin Interactions. Methods in Molecular Biology, vol 2161. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0680-3_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0680-3_3

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0679-7

  • Online ISBN: 978-1-0716-0680-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation