Skip to main content
SpringerLink
Log in

The Use of Small Noncoding RNAs to Silence Transcription in Human Cells

  • Protocol
  • First Online:
RNA Interference Techniques

Part of the book series: Neuromethods ((NM,volume 58))

  • 822 Accesses

Abstract

In human cells, small noncoding RNAs have been shown to possess an ability to exert regulatory control of gene expression when they are generated to target a gene promoter specifically. Mechanistically, small RNA-directed transcriptional gene suppression functions by targeting of epigenetic modifications, such as histone and DNA methylation, to a targeted promoter locus, the result of which is the restriction of the action of RNA polymerase II and gene transcription specifically at the targeted gene promoter (56, 57). In human cells, this activity requires a transcriptional silencing complex that consists of Argonaute 1 (Ago-1) (54, 55), DNA methyltransferase 3A (47, 48, 56), and histone deacetylase 1 (48, 49) as well as a low-copy promoter-associated RNA that is expressed and spans the RNA-targeted loci (57). Importantly, as this mode of RNA-directed gene regulation functions through targeted silent state epigenetic changes, it has the potential to be a longer lasting if not permanent mode of gene silencing that may also be refractory to compensatory mutations as the targeting is at the level of chromatin and not at the genes mRNA level.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
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

References

  1. Montgomery MK, S. Xu, A. Fire. RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans. Proceedings of the National Academy of Sciences 1998;95:15502–7.

    Google Scholar 

  2. Nishikura K. A short primer on RNAi: RNA-directed RNA polymerase acts as a key catalyst. Cell 2001;107:415–8.

    Article  PubMed  CAS  Google Scholar 

  3. Sharp PA. RNA interference. Genes and Development 2001;15:485–90.

    Article  PubMed  CAS  Google Scholar 

  4. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001;411:494–8.

    Article  PubMed  CAS  Google Scholar 

  5. Fire A, S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver, C.C. Mello. Potent and specific genetic interference by double stranded RNA in Caenorhabditis elegans. Nature 1998;391:806–11.

    Google Scholar 

  6. Hutvagner G, Zamore PD. RNAi: nature abhors a double-strand. Curr Opin Genet Dev 2002;12:225–32.

    Article  PubMed  CAS  Google Scholar 

  7. Tijsterman M, Ketting RF, Plasterk RH. The genetics of RNA silencing. Annu Rev Genet 2002;36:489–519.

    Article  PubMed  CAS  Google Scholar 

  8. Pal-Bhadra M, U. Bhadra, J. A. Birchler. RNAi related mechanisms affect both transcriptional and posttranscriptional transgene silencing in drosophila. Molecular Cell 2002;9:315–27.

    Google Scholar 

  9. Sijen T, I. Vign, A. Rebocho, R. Blokland, D. Roelofs, J. Mol, and J. Kooter. Transcriptional and posttranscriptional gene silencing are mechansitically related. Current Biology 2001;11:436–40.

    Google Scholar 

  10. Langlois MA, Boniface C, Wang G, et al. Cytoplasmic and nuclear retained DMPK mRNAs are targets for RNA interference in myotonic dystrophy cells. J Biol Chem 2005;280:16949–54.

    Article  PubMed  CAS  Google Scholar 

  11. Robb GB, Brown KM, Khurana J, Rana TM. Specific and potent RNAi in the nucleus of human cells. Nat Struct Mol Biol 2005;12:133–7.

    Article  PubMed  CAS  Google Scholar 

  12. Lin SL, Chang DC, Ying SY. Isolation and identification of gene-specific microRNAs. Methods Mol Biol 2006;342:313–20.

    PubMed  CAS  Google Scholar 

  13. Lin SL, Ying SY. Gene silencing in vitro and in vivo using intronic microRNAs. Methods Mol Biol 2006;342:295–312.

    PubMed  CAS  Google Scholar 

  14. Hannon GJ. RNA interference. Nature 2002;418:244–51.

    Article  PubMed  CAS  Google Scholar 

  15. Leung AK, Sharp PA. Function and localization of microRNAs in mammalian cells. Cold Spring Harb Symp Quant Biol 2006;71:29–38.

    Article  PubMed  CAS  Google Scholar 

  16. Liu J, Valencia-Sanchez MA, Hannon GJ, Parker R. MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies. Nat Cell Biol 2005;7:719–23.

    Article  PubMed  CAS  Google Scholar 

  17. Matzke MA, Birchler JA. RNAi-mediated pathways in the nucleus. Nat Rev Genet 2005;6:24–35.

    Article  PubMed  CAS  Google Scholar 

  18. Egger G, Liang G, Aparicio A, Jones PA. Epigenetics in human disease and prospects for epigenetic therapy. Nature 2004;429:457–63.

    Article  PubMed  CAS  Google Scholar 

  19. Jackson AL, Bartz SR, Schelter J, et al. Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol 2003;21:635–7.

    Article  PubMed  CAS  Google Scholar 

  20. Weber M, Hellmann I, Stadler MB, et al. Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome. Nat Genet 2007;39:457–66.

    Article  PubMed  CAS  Google Scholar 

  21. Okano M, Bell DW, Haber DA, Li E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 1999;99:247–57.

    Article  PubMed  CAS  Google Scholar 

  22. Morris KV, Chan SW, Jacobsen SE, Looney DJ. Small interfering RNA-induced transcriptional gene silencing in human cells. Science 2004;305:1289–92.

    Article  PubMed  CAS  Google Scholar 

  23. Jenuwein T, Allis CD. Translating the histone code. Science 2001;293:1074–80.

    Article  PubMed  CAS  Google Scholar 

  24. Strahl BD, Allis CD. The language of covalent histone modifications Nature 2000;403:41–5.

    CAS  Google Scholar 

  25. Cao R, Wang L, Wang H, et al. Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science 2002;298:1039–43.

    Article  PubMed  CAS  Google Scholar 

  26. Cao X, Jacobsen SE. Role of the arabidopsis DRM methyltransferases in de novo DNA methylation and gene silencing. Curr Biol 2002;12:1138–44.

    Article  PubMed  CAS  Google Scholar 

  27. Cao R, Zhang Y. The functions of E(Z)/EZH2-mediated methylation of lysine 27 in histone H3. Curr Opin Genet Dev 2004;14:155–64.

    Article  PubMed  CAS  Google Scholar 

  28. Hawkins PG, Morris KV. RNA and transcriptional modulation of gene expression. Cell Cycle 2008;7:602–7.

    Article  PubMed  CAS  Google Scholar 

  29. Matzke MA, M. Primig, J. Trnovsky, and A.J.M. Matzke. Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants. The EMBO Journal 1989;8:643–9.

    Google Scholar 

  30. Wassenegger M, M.W. Graham, M.D. Wang. RNA-directed de novo methylation of genomic sequences in plants. Cell 1994;76:567–76.

    Google Scholar 

  31. Mette MF, W. Aufsatz, J. Van der Winden, A.J.M. Matzke, and M.A. Matzke. Transcriptional silencing and promoter ­methylation triggered by double-stranded RNA. The EMBO Journal 2000;19:5194–201.

    Google Scholar 

  32. Lippman Z, May B, Yordan C, Singer T, Martienssen R. Distinct Mechanisms Determine Transposon Inheritance and Methylation via Small Interfering RNA and Histone Modification. PLoS Biol 2003;1:E67.

    Article  PubMed  Google Scholar 

  33. Zilberman D, Cao X, Jacobsen SE. ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methylation. Science 2003;299:716–9.

    Article  PubMed  CAS  Google Scholar 

  34. Morris KV. siRNA-mediated transcriptional gene silencing: the potential mechanism and a possible role in the histone code. Cell Mol Life Sci 2005;62:3057–66.

    Article  PubMed  CAS  Google Scholar 

  35. Volpe TA, C. Kidner, I.M. Hall, G. Teng, S.I.S. Grewal, R. A. Martienssen. Regulation of Heterchromatic Silencing and Histone H3 Lysine-9 Methylation by RNAi. Science 2002;297:1833–7.

    Google Scholar 

  36. Irvine DV, Zaratiegui M, Tolia NH, et al. Argonaute slicing is required for heterochromatic silencing and spreading. Science 2006;313:1134–7.

    Article  PubMed  CAS  Google Scholar 

  37. Kato H, Goto DB, Martienssen RA, Urano T, Furukawa K, Murakami Y. RNA polymerase II is required for RNAi-dependent heterochromatin assembly. Science 2005;309:467–9.

    Article  PubMed  CAS  Google Scholar 

  38. Romano N, Macino G. Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol Microbiol 1992;6:3343–53.

    Article  PubMed  CAS  Google Scholar 

  39. Chicas A, Forrest EC, Sepich S, Cogoni C, Macino G. Small interfering RNAs that trigger posttranscriptional gene silencing are not required for the histone H3 Lys9 methylation necessary for transgenic tandem repeat stabilization in Neurospora crassa. Mol Cell Biol 2005;25:3793–801.

    Article  PubMed  CAS  Google Scholar 

  40. Fagard M, Boutet S, Morel JB, Bellini C, Vaucheret H. AGO1, QDE-2, and RDE-1 are related proteins required for post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference in animals. Proc Natl Acad Sci U S A 2000;97:11650–4.

    Article  PubMed  CAS  Google Scholar 

  41. Grishok A, Pasquinelli AE, Conte D, et al. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 2001;106:23–34.

    CAS  Google Scholar 

  42. Tabara H, Sarkissian M, Kelly WG, et al. The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 1999;99:123–32.

    Google Scholar 

  43. Dernburg AF, Zalevsky J, Colaiacovo MP, Villeneuve AM. Transgene-mediated ­cosuppression in the C. elegans germ line. Genes Dev 2000;14:1578–83.

    Google Scholar 

  44. Ketting RF, Plasterk RH. A genetic link between co-suppression and RNA interference in C. elegans. Nature 2000;404:296–8.

    Google Scholar 

  45. Grishok A, Sinskey JL, Sharp PA. Transcriptional silencing of a transgene by RNAi in the soma of C. elegans. Genes Dev 2005;19:683–96.

    CAS  Google Scholar 

  46. Vastenhouw NL, Brunschwig K, Okihara KL, Muller F, Tijsterman M, Plasterk RH. Gene expression: long-term gene silencing by RNAi. Nature 2006;442:882.

    Article  PubMed  CAS  Google Scholar 

  47. Hawkins PG, Santoso S, Adams C, Anest V, Morris KV. Promoter targeted small RNAs induce long-term transcriptional gene silencing in human cells. Nucleic Acids Res 2009;37:2984–95.

    Article  PubMed  CAS  Google Scholar 

  48. Turner AM, De La Cruz J, Morris KV. Mobilization-competent Lentiviral Vector-mediated Sustained Transcriptional Modulation of HIV-1 Expression. Mol Ther 2009;17:360–8.

    Article  PubMed  CAS  Google Scholar 

  49. Suzuki K, Juelich T, Lim H, Ishida T, Watanebe T, Cooper DA, et al. Closed chromatin architecture is induced by an RNA duplex targeting the HIV-1 promoter region. J Biol Chem 2008;283:23353–63.

    Article  PubMed  CAS  Google Scholar 

  50. Castanotto D, Tommasi S, Li M, et al. Short hairpin RNA-directed cytosine (CpG) methylation of the RASSF1A gene promoter in HeLa cells. Mol Ther 2005;12:179–83.

    Article  PubMed  CAS  Google Scholar 

  51. Buhler M, Mohn F, Stalder L, Muhlemann O. Transcriptional silencing of nonsense codon-containing immunoglobulin minigenes. Mol Cell 2005;18:307–17.

    Article  PubMed  Google Scholar 

  52. Janowski BA, K.E. Huffman, J.C. Schwartz, R. Ram, D. Hardy, D.S. Shames, J.D. Minna, D.R. Corey. Inhibiting gene expression at transcription start sites in chromosomal DNA with antigene RNAs. Nature Chemical Biology 2005;1:210–5.

    Google Scholar 

  53. Suzuki K, T. Shijuuku, T. Fukamachi, J. Zaunders, G. Guillemin, D. Cooper, and A. Kelleher. Prolonged transcriptional silencing and CpG methylation induced by siRNAs targeted to the HIV-1 promoter region. Journal of RNAi and Gene Silencing 2005;1:66–78.

    Google Scholar 

  54. Janowski BA, Huffman KE, Schwartz JC, et al. Involvement of AGO1 and AGO2 in mammalian transcriptional silencing. Nat Struct Mol Biol 2006.

    Google Scholar 

  55. Kim DH, Villeneuve LM, Morris KV, Rossi JJ. Argonaute-1 directs siRNA-mediated transcriptional gene silencing in human cells. Nat Struct Mol Biol 2006;13:793–7.

    Article  PubMed  CAS  Google Scholar 

  56. Weinberg MS, L.M. Villeneuve, A. Ehsani, M. Amarzguioui, L. Aagaard, Z. Chen, A.D. Riggs, J.J. Rossi, and K.V. Morris. The antisense strand of small interfering RNAs directs histone methylation and transcriptional gene silencing in human cells. RNA 2005;12.

    Google Scholar 

  57. Han J, D. Kim, and K.V. Morris. Promoter-associated RNA is required for RNA-directed transcriptional gene silencing in human cells. PNAS 2007;104.

    Google Scholar 

  58. Amaral PP, Dinger ME, Mercer TR, Mattick JS. The eukaryotic genome as an RNA machine. Science 2008;319:1787–9.

    Article  PubMed  CAS  Google Scholar 

  59. Murali S, K. Pulukuri, and J.S. Rao. Small Interfering RNA-directed reversal of Urokinase plasminogen activator demethylation inhibits prostate tumor growth and metastasis. Cancer Reserach 2007;67:6637–46.

    Google Scholar 

  60. Ting AH, Schuebel KE, Herman JG, Baylin SB. Short double-stranded RNA induces transcriptional gene silencing in human cancer cells in the absence of DNA methylation. Nat Genet 2005;37:906–10.

    Article  PubMed  CAS  Google Scholar 

  61. Zhang M, H. Ou, Y.H. Shen, J. Wang, J. Wang, J. Coselli, X.L. Wang. Regulation of endothelial nitric oxide synthase by small RNA. PNAS 2005;102:16967–72.

    Google Scholar 

  62. Kim JW, Zhang YH, Zern MA, Rossi JJ, Wu J. Short hairpin RNA causes the methylation of transforming growth factor-beta receptor II promoter and silencing of the target gene in rat hepatic stellate cells. Biochem Biophys Res Commun 2007;359:292–7.

    Article  PubMed  CAS  Google Scholar 

  63. Kim DH, Longo M, Han Y, Lundberg P, Cantin E, Rossi JJ. Interferon induction by siRNAs and ssRNAs synthesized by phage polymerase. Nat Biotechnol 2004;22:321–5.

    Article  PubMed  CAS  Google Scholar 

  64. Ebralidze AK, Guibal FC, Steidl U, et al. PU.1 expression is modulated by the balance of functional sense and antisense RNAs regulated by a shared cis-regulatory element. Genes Dev 2008;22:2085–92.

    Google Scholar 

  65. Schwartz JC, Younger ST, Nguyen NB, et al. Antisense transcripts are targets for activating small RNAs. Nat Struct Mol Biol 2008.

    Google Scholar 

  66. Yu W, D. Gius, P. Onyango, K. Muldoon-Jacobs, J. Karp, A.P. Feinberg, H. Cui. Epigenetic silencing of tumour suppressor gene p15 by its antisense RNA. Nature 2008;451:202–6.

    Google Scholar 

  67. Hawkins P, S. Santoso, C. Adams, V. Anest, and K.V. Morris. Promoter targeted small RNAs modulate long term stable transcriptional gene silencing in human cells. RNA 2008;(in review, July 2008).

    Google Scholar 

  68. Turner A-M, J. De La Cruz, and K.V. Morris. Mobilization competent lentiviral vector mediated sustained transcriptional modulation of HIV-1 expression. Molecular Therapy 2008;In re-review (July 2008).

    Google Scholar 

  69. Morris KV, Gilbert J, Wong-Staal F, Gasmi M, Looney DJ. Transduction of cell lines and primary cells by FIV-packaged HIV vectors. Mol Ther 2004;10:181–90.

    Article  PubMed  CAS  Google Scholar 

  70. Makinen PI, Koponen JK, Karkkainen AM, et al. Stable RNA interference: comparison of U6 and H1 promoters in endothelial cells and in mouse brain. J Gene Med 2006;8:433–41.

    Article  PubMed  CAS  Google Scholar 

  71. Rumi M, Ishihara S, Aziz M, et al. RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector. Biochem Biophys Res Commun 2006;339:540–7.

    Article  PubMed  CAS  Google Scholar 

  72. Barichievy S, Saayman S, Von Eije KJ, Morris KV, Arbuthnot P, Weinberg MS. The Inhibitory Efficacy of RNA POL III-Expressed Long Hairpin RNAs Targeted to Untranslated Regions of the HIV-1 5’ Long Terminal Repeat. Oligonucleotides 2007.

    Google Scholar 

  73. Saayman S, Barichievy S, Capovilla A, Morris KV, Arbuthnot P, Weinberg MS. The efficacy of generating three independent anti-HIV-1 siRNAs from a single U6 RNA Pol III-expressed long hairpin RNA. PLoS ONE 2008;3:e2602.

    Article  PubMed  Google Scholar 

  74. Li M, Rossi JJ. Lentiviral vector delivery of siRNA and shRNA encoding genes into cultured and primary hematopoietic cells. Methods Mol Biol 2005;309:261–72.

    PubMed  CAS  Google Scholar 

  75. Aagaard L, Amarzguioui M, Sun G, et al. A facile lentiviral vector system for expression of doxycycline-inducible shRNAs: knockdown of the pre-miRNA processing enzyme Drosha. Mol Ther 2007;15:938–45.

    PubMed  CAS  Google Scholar 

  76. Zhou J, Li H, Li S, Zaia J, Rossi JJ. Novel Dual Inhibitory Function Aptamer-siRNA Delivery System for HIV-1 Therapy. Mol Ther 2008;16:1481–9.

    Article  PubMed  CAS  Google Scholar 

  77. Compagno D, Merle C, Morin A, et al. SIRNA-directed in vivo silencing of androgen receptor inhibits the growth of castration-resistant prostate carcinomas. PLoS ONE 2007;2:e1006.

    Article  PubMed  Google Scholar 

  78. Morris KV. Therapeutic potential of siRNA-mediated transcriptional gene silencing. Biotechniques 2006;Suppl:7–13.

    Google Scholar 

  79. Jeffery L, Nakielny S. Components of the DNA methylation system of chromatin control are RNA-binding proteins. J Biol Chem 2004;279:49479–87.

    Article  PubMed  CAS  Google Scholar 

  80. Katayama S, Tomaru Y, Kasukawa T, et al. Antisense transcription in the mammalian transcriptome. Science 2005;309:1564–6.

    Article  PubMed  Google Scholar 

  81. Ogawa Y, Sun BK, Lee JT. Intersection of the RNA interference and X-inactivation pathways. Science 2008;320:1336–41.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA

    Kevin V. Morris

Authors
  1. Kevin V. Morris
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kevin V. Morris .

Editor information

Editors and Affiliations

  1. Research Institute, Center for Gene Therapy, Nationwide Children’s Hospital, Children's Drive 700, Columbus, 43205, Ohio, USA

    Scott Q. Harper

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Morris, K.V. (2011). The Use of Small Noncoding RNAs to Silence Transcription in Human Cells. In: Harper, S. (eds) RNA Interference Techniques. Neuromethods, vol 58. Humana Press. https://doi.org/10.1007/978-1-61779-114-7_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-114-7_3

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-113-0

  • Online ISBN: 978-1-61779-114-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation