Skip to main content
SpringerLink
Log in

RNA Interference: Story and Mechanisms

  • Protocol
  • First Online:
Design and Delivery of SiRNA Therapeutics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2282))

Abstract

The discovery that gene expression can be silenced by exogenously introduced double-stranded RNAs into cells unveiled a hidden level of gene regulation by a variety of small RNA pathways, which are involved in regulating endogenous gene expression, defending against virus infections, and protecting the genome from invading transposons, both at the posttranscriptional and epigenetic levels. All endogenous RNA interference pathways share a conserved effector complex, which contains at least an argonaute protein and a short single-stranded RNA. Such argonaute-RNA complexes can repress the transcription of genes, target mRNA for site-specific cleavage, or block mRNA translation into proteins. This review outlines the history of RNAi discovery, function, and mechanisms of action. For comparison, it also touches on CRISPR interference.

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 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
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. Napoli C, Lemieux C, Jorgensen R (1990) Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2:279–289

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Romano N, Macino G (1992) Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol Microbiol 6(22):3343–3353

    Article  CAS  PubMed  Google Scholar 

  3. Guo S, Kemphues KJ (1995) Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 81:611–620

    Article  CAS  PubMed  Google Scholar 

  4. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811

    Article  CAS  PubMed  Google Scholar 

  5. Zamore PD, Tuschl T, Sharp PA, Bartel DP (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101:25–33

    Article  CAS  PubMed  Google Scholar 

  6. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494–498

    Article  CAS  PubMed  Google Scholar 

  7. Sioud M, Sørensen DR (2003) Cationic liposome-mediated delivery of siRNAs in adult mice. Biochem Biophys Res Commun 312:1220–1225

    Article  CAS  PubMed  Google Scholar 

  8. Sioud M (2005) Induction of inflammatory cytokines and interferon responses by double-stranded and single-stranded siRNAs is sequence-dependent and requires endosomal localization. J Mol Biol 348:1079–1090

    Article  CAS  PubMed  Google Scholar 

  9. Judge AD, Sood V, Shaw JR, Fank D, Mcclintock K, Maclachlan I (2005) Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat Biotechnol 23:457–462

    Article  CAS  PubMed  Google Scholar 

  10. Sioud M (2006) Single-stranded small interfering RNAs are more immunostimulatory than their double-stranded counterparts: a central role for 2′-hydroxyl uridines in immune responses. Eur J Immunol 36:1222–1230

    Article  CAS  PubMed  Google Scholar 

  11. Diebold SS, Massacrier C, Akira S, Paturel C, Morel Y, Reis e Sousa, C. (2006) Nucleic acid agonists for Toll-like receptor 7 are defined by the presence of uridine ribonucleotides. Eur J Immunol 36:3256–3267

    Article  CAS  PubMed  Google Scholar 

  12. Cekaite L, Furset G, Hovig E, Sioud M (2007) Gene expression analysis in blood cells in response to unmodified and 2′-modified siRNAs reveals TLR-dependent and independent effects. J Mol Biol 365:90–108

    Article  CAS  PubMed  Google Scholar 

  13. Schubert MS, Cedrone E, Neun B, Behlke MA, Dobrovolskaia MA (2018) Chemical modification of CRISPR gRNAs eliminate type I interferon responses in human peripheral blood mononuclear cells. J Cytokine Biol 3:121

    Article  PubMed  PubMed Central  Google Scholar 

  14. Sioud M, Furset G, Cekaite L (2007) Suppression of immunostimulatory siRNA-driven innate immune activation by 2′-modified RNAs. Biochem Biophys Res Commun 361:122–126

    Article  CAS  PubMed  Google Scholar 

  15. Robbins M, Judge A, Liang L, McClintock K, Yaworski E, MacLachlan I (2007) 2′-O-methyl-modified RNAs act as TLR7 antagonists. Mol Ther 15(9):1663–1669

    Article  CAS  PubMed  Google Scholar 

  16. Hamilton AJ, Baulcombe DC (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286:950–952

    Article  CAS  PubMed  Google Scholar 

  17. Yan KS, Yan S, Farooq A, Han A, Zeng L, Zhou MM (2003) Structure and conserved RNA binding of the PAZ domain. Nature 426:468–474

    Article  PubMed  CAS  Google Scholar 

  18. Liu Q, Rand TA, Kalidas S, Du F, Kim HE, Smith DP, Wang X (2003) R2d2, a bridge between the initiation and effector steps of the drosophila RNAi pathway. Science 301:1921–1925

    Article  CAS  PubMed  Google Scholar 

  19. Liu X, Jiang F, Kalidas S, Smith D, Liu Q (2006) Dicer-2 and R2D2 coordinately bind siRNA to promote assembly of the siRISC complexes. RNA 12:1514–1520

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Tomari Y, Matranga C, Haley B, Martinez N, Zamore PD (2004) A protein sensor for siRNA asymmetry. Science 306:1377–1380

    Article  CAS  PubMed  Google Scholar 

  21. Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N et al (2003) Asymmetry in the assembly of the RNAi enzyme complex. Cell 115:199–208

    Article  CAS  PubMed  Google Scholar 

  22. Matranga C, Tomari Y, Shin C, Bartel DP, Zamore PD (2005) Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell 123:607–620

    Article  CAS  PubMed  Google Scholar 

  23. Wee LM, Flores-Jasso CF, Salomon WE, Zamore PD (2012) Argonaute divides its RNA guide into domains with distinct functions and RNA-binding properties. Cell 151:1055–1067

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Reyolds A, Leake D, Boese Q, Scaringe S, Marchall WS et al (2004) Rational siRNA design for RNA interference. Nat Biotechnol 22:326–330

    Article  CAS  Google Scholar 

  25. Liu Y, Ye X, Jiang F, Liang C, Chen D, Peng J, Kinch LN, Grishin NV, Liu Q (2009) C3PO, an endoribonuclease that promotes RNAi by facilitating RISC activation. Science 325(5941):750–753

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Bhaskaran M, Mohan M (2014) MicroRNAs: history, biogenesis, and their evolving role in animal development and disease. Vet Pathol 51(4):759–774

    Article  CAS  PubMed  Google Scholar 

  27. Kim VN (2005) MicroRNA biogenesis: coordinated cropping and dicing. Nat Rev Mol Cell Biol 6:376–385

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  29. Lai EC, Tomancak P, Williams RW, Rubin GM (2003) Computational identification of Drosophila microRNA genes. Genome Biol 4:R42

    Article  PubMed  PubMed Central  Google Scholar 

  30. Jones-Rhoades MW, Bartel DP, Bartel B (2006) microRNAs and their regulatory roles in plants. Annu Rev Plant Biol 57:19–53

    Article  CAS  PubMed  Google Scholar 

  31. Cui J, You C, Chen X (2017) The evolution of microRNAs in plants. Curr Opin Plant Biol 35:61–67

    Article  CAS  PubMed  Google Scholar 

  32. Baskerville S, Bartel DP (2005) Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes. RNA 11(3):241–247

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Aravin A, Gaidatzis D, Pfeffer S, Lagos-Quintana M, Landgraf P, Iovino N, Morris P, Brownstein MJ, Kuramochi-Miyagawa S, Nakano T, Chien M, Russo JJ, Ju J, Sheridan R, Sander C, Zavolan M, Tuschl T (2006) A novel class of small RNAs bind to MILI protein in mouse testes. Nature 442:203–207

    Article  CAS  PubMed  Google Scholar 

  34. Aravin AA, Hannon GJ, Brennecke J (2007) The Piwi-piRNA pathway provides an adaptive defense in the transposon arms race. Science 318:761–764

    Article  CAS  PubMed  Google Scholar 

  35. Vagin VV, Sigova A, Li C, Seitz H, Gvozdev V, Zamore PD (2006) A distinct small RNA pathway silences selfish genetic elements in the germline. Science 313:320–324

    Article  CAS  PubMed  Google Scholar 

  36. Ozata DM, Gainetdinov I, Zoch A, O’Carroll D, Zamore PD (2019) PIWI-interacting RNAs: small RNAs with big functions. Nat Rev Genet 20:89–108

    Article  CAS  PubMed  Google Scholar 

  37. Shen H (2009) UAP56 - a key player with surprisingly diverse roles in pre-mRNA splicing and nuclear export. BMB Rep 42(4):185–188

    Article  CAS  PubMed  Google Scholar 

  38. Pane A, Wehr K, Schüpbach T (2007) zucchini and squash encode two putative nucleases required for rasiRNA production in the Drosophila germline. Dev Cell 12(6):851–862

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Kamminga LM, Luteijn MJ, den Broeder MJ, Redl S, Kaaij LJ, Roovers EF, Ladurner P, Berezikov E, Ketting RF (2010) Hen1 is required for oocyte development and piRNA stability in zebrafish. EMBO J 29:3688–3700

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Webster A, Li S, Hur JK, Wachsmuth M, Bois JS, Perkins EM, Patel DJ, Aravin AA (2015) Aub and Ago3 are recruited to nuage through two mechanisms to form a Ping-Pong complex assembled by krimper. Mol Cell 59:564–575

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Wang W, Han BW, Tipping C, Ge DT, Zhang Z, Weng Z, Zamore PD (2015) Slicing and binding by Ago3 or Aub trigger piwi-bound piRNA production by distinct mechanisms. Mol Cell 59:819–830

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Rojas-Ríos P, Simonelig M (2018) piRNAs and PIWI proteins: regulators of gene expression in development and stem cells. Development 145(17):dev161786

    Article  PubMed  CAS  Google Scholar 

  43. Tushir JS, Zamore PD, Zhang Z (2009) SnapShot: mouse piRNAs, PIWI proteins, and the ping-pong cycle. Cell 139:830–830.e1

    Article  CAS  PubMed  Google Scholar 

  44. Liu Y, Dou M, Song X, Dong Y, Liu S, Liu H, Tao J, Li W, Yin X, Xu W (2019) The emerging role of the piRNA/piwi complex in cancer. Mol Cancer 18(1):123

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Sioud M (2007) RNA interference and innate immunity. Adv Drug Deliv Rev 59:153–163

    Article  CAS  PubMed  Google Scholar 

  46. Horvath P, Barrangou R (2010) CRISPR/Cas, the immune system of bacteria and archaea. Science 327:167–170

    Article  CAS  PubMed  Google Scholar 

  47. Barrangou R, Fremaux C, Deveau H et al (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315:1709–1712

    Article  CAS  PubMed  Google Scholar 

  48. Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A (1987) Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol 169:5429–5433

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Terns MP (2018) CRISPR-based technologies: impact of RNA-targeting systems. Mol Cell 72:404–412

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Mali P, Yang L, Esvelt KM et al (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Rahdar M, McMahon MA, Prakash TP, Swayze EE, Bennett CF, Cleveland DW (2015) Synthetic CRISPR RNA-Cas9-guided genome editing in human cells. Proc Natl Acad Sci U S A 112:E7110–E7117

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, Belanto JJ, Verdine V, Cox DBT, Kellner MJ, Regev A, Lander ES, Voytas DF, Ting AY, Zhang F (2017) RNA targeting with CRISPR-Cas13. Nature 550:280–284

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. Hu JH, Miller SM, Geurts MH, Tang W, Chen L, Sun N, Zeina CM, Gao X, Rees HA, Lin Z, Liu DR (2018) Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556:57–63

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from the Norwegian Cancer Society and the Southern and Eastern Norway Regional Health Authority (Helse Sør-Øst).

Author information

Authors and Affiliations

  1. Department of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital-Radiumhospitalet, Oslo, Norway

    Mouldy Sioud

Authors
  1. Mouldy Sioud
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mouldy Sioud .

Editor information

Editors and Affiliations

  1. Department of Oncology, Odense University Hospital Odense, Denmark, Department of Cancer and Inflammation Research, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark

    Henrik J. Ditzel

  2. Department of Cancer and Inflammation Research, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark

    Martina Tuttolomondo

  3. Center for RNA Medicine, Department of Clinical Medicine, Aalborg University, Copenhagen, Denmark

    Sakari Kauppinen

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Sioud, M. (2021). RNA Interference: Story and Mechanisms. In: Ditzel, H.J., Tuttolomondo, M., Kauppinen, S. (eds) Design and Delivery of SiRNA Therapeutics. Methods in Molecular Biology, vol 2282. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1298-9_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1298-9_1

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1297-2

  • Online ISBN: 978-1-0716-1298-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation