Advertisement

Gene Silencing In Vitro and In Vivo Using Intronic MicroRNAs

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1733)

Abstract

MicroRNAs (miRNAs), small single-stranded regulatory RNAs capable of interfering with intracellular messenger RNAs (mRNAs) that contain either complete or partial complementarity, are useful for the design of new therapies against cancer polymorphism and viral mutation. Numerous miRNAs have been reported to induce RNA interference (RNAi), a post-transcriptional gene-silencing mechanism. Recent evidence also indicates that they are involved in the transcriptional regulation of genome activities. They were first discovered in Caenorhabditis elegans as native RNA fragments that modulate a wide range of genetic regulatory pathways during embryonic development, and are now recognized as small gene silencers transcribed from the noncoding regions of a genome. In humans, nearly 97% of the genome is noncoding DNA, which varies from one individual to another, and changes in these sequences are frequently noted to manifest in clinical and circumstantial malfunction; for example, type 2 myotonic dystrophy and fragile X syndrome were found to be associated with miRNAs derived from introns. Intronic miRNA is a new class of miRNAs derived from the processing of non-protein-coding regions of gene transcripts. The intronic miRNAs differ uniquely from previously described intergenic miRNAs in the requirement of RNA polymerase (Pol)-II and spliceosomal components for its biogenesis. Several kinds of intronic miRNAs have been identified in C. elegans, mouse, and human cells; however, their functions and applications have not been reported. Here, we show for the first time that intron-derived miRNA is not only able to induce RNAi in mammalian cells but also in fish, chicken embryos, and adult mice cells, demonstrating the evolutionary preservation of this gene regulation system in vivo. These miRNA-mediated animal models provide artificial means to reproduce the mechanisms of miRNA-induced disease in vivo and will shed further light on miRNA-related therapies.

Key words

MicroRNA (miRNA) RNA interference (RNAi) RNA polymerase type II (Pol II) RNA splicing Intron RNA-induced gene-silencing complex (RISC) Gene silencing in vivo 

References

  1. 1.
    Ambros V (2004) The functions of animal microRNAs. Nature 350:431–355Google Scholar
  2. 2.
    Nelson P, Kiriakidou M, Sharma A, Maniataki E, Mourelatos Z (2003) The microRNA world: small is mighty. Trends Biochem Sci 28:534–539CrossRefPubMedGoogle Scholar
  3. 3.
    Ying SY, Lin SL (2005) Intronic microRNA (miRNA). Biochem Biophys Res Commun 326:515–520CrossRefPubMedGoogle Scholar
  4. 4.
    Lin SL, Ying SY (2004) Novel RNAi therapy – intron-derived microRNA drugs. Drug Design Rev 1:247–255CrossRefGoogle Scholar
  5. 5.
    Tuschl T, Borkhardt A (2002) Small interfering RNAs: a revolutionary tool for the analysis of gene function and gene therapy. Mol Interv 2:158–167CrossRefPubMedGoogle Scholar
  6. 6.
    Ambros V (1989) A hierarchy of regulatory genes controls a larva regulatory specificity, the notion that target-site recogni- to-adult developmental switch in C. elegans. Cell 57:49–57CrossRefPubMedGoogle Scholar
  7. 7.
    Hall IM, Shankaranarayana GD, Noma K, Ayoub N, Cohen A, Grewal SI (2002) Establishment and maintenance of a heterochromatin domain. Science 297:2232–2237CrossRefPubMedGoogle Scholar
  8. 8.
    Llave C, Xie Z, Kasschau KD, Carrington JC (2002) Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science 297:2053–2056CrossRefPubMedGoogle Scholar
  9. 9.
    Rhoades MW, Reinhart BJ, Lim LP, Burge CB, Bartel B, Bartel DP (2002) Prediction of plant microRNA targets. Cell 110:513–520CrossRefPubMedGoogle Scholar
  10. 10.
    Lee RC, Feibaum RL, Ambros V (1993) The C. elegans heterochromic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75:843–854CrossRefPubMedGoogle Scholar
  11. 11.
    Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, Horvitz HR, Ruvkun G (2000) The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403:901–906CrossRefPubMedGoogle Scholar
  12. 12.
    Lau NC, Lim LP, Weinstein EG, Bartel DP (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294:858–862CrossRefPubMedGoogle Scholar
  13. 13.
    Brennecke J, Hipfner DR, Stark A, Russell RB, Cohen SM (2003) Bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell 113:25–36CrossRefPubMedGoogle Scholar
  14. 14.
    Xu P, Vernooy SY, Guo M, Hay BA (2003) The Drosophila microRNA Mir-14 suppresses cell death and is required for normal fat metabolism. Curr Biol 13:790–795CrossRefPubMedGoogle Scholar
  15. 15.
    Lagos-Quintana M, Rauhut R, Meyer J, Borkhardt A, Tuschl T (2003) New microRNAs from mouse and human. RNA 9:175–179CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Mourelatos Z, Dostie J, Paushkin S, Sharma A, Charroux B, Abel L, Rappsilber J, Mann M, Dreyfuss G (2002) miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs. Genes Dev 16:720–728CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Zeng Y, Wagner EJ, Cullen BR (2002) Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol Cell 9:1327–1333CrossRefPubMedGoogle Scholar
  18. 18.
    Zeng Y, Yi R, Cullen BR (2003) MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. Proc Natl Acad Sci U S A 100:9779–9784CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Carthew RW (2001) Gene silencing by double-stranded RNA. Curr Opin Cell Biol 13:244–248CrossRefPubMedGoogle Scholar
  20. 20.
    Lin SL, Chang D, Wu DY, Ying SY (2003) A novel RNA splicing-mediated gene silencing mechanism potential for genome evolution. Biochem Biophys Res Commun 310:754–760CrossRefPubMedGoogle Scholar
  21. 21.
    Lin SL, Chang D, Ying SY (2005) Asymmetry of intronic pre-miRNA structures in functional RISC assembly. Gene 356:32–38CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Ying SY, Lin SL (2004) Intron-derived microRNAs–fine tuning of gene functions. Gene 342:25–28CrossRefPubMedGoogle Scholar
  23. 23.
    Kramer A (1996) The structure and function of proteins involved in mammalian pre-mRNA splicing. Annu Rev Biochem 65:367–409CrossRefPubMedGoogle Scholar
  24. 24.
    Clement JQ, Qian L, Kaplinsky N, Wilkinson MF (1999) The stability and fate of a spliced intron from vertebrate cells. RNA 5:206–220CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Nott A, Meislin SH, Moore MJ (2003) A quantitative analysis of intron effects on mammalian gene expression. RNA 9:607–617CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Lee Y, Kim M, Han J, Yeom KH, Lee S, Baek SH, Kim VN (2004) MicroRNA genes are transcribed by RNA polymerase II. EMBO J 23:4051–4060CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, Lee J, Provost P, Radmark O, Kim S, Kim VN (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature 425:415–419CrossRefPubMedGoogle Scholar
  28. 28.
    Lund E, Guttinger S, Calado A, Dahlberg JE, Kutay U (2004) Nuclear export of microRNA precursors. Science 303:95–98CrossRefPubMedGoogle Scholar
  29. 29.
    Yi R, Qin Y, Macara IG, Cullen BR (2003) Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev 17:3011–3016CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, Zamore PD (2003) Asymmetry in the assembly of the RNAi enzyme complex. Cell 115:199–208CrossRefPubMedGoogle Scholar
  31. 31.
    Khvorova A, Reynolds A, Jayasena SD (2003) Functional siRNAs and miRNAs exhibit strand bias. Cell 115:209–216CrossRefPubMedGoogle Scholar
  32. 32.
    Lee YS, Nakahara K, Pham JW, Kim K, He Z, Sontheimer EJ, Carthew RW (2004) Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell 117:69–81CrossRefPubMedGoogle Scholar
  33. 33.
    Tang G (2005) siRNA and miRNA: an insight into RISCs. Trends Biochem Sci 30:106–114CrossRefPubMedGoogle Scholar
  34. 34.
    Rodriguez A, Griffiths-Jones S, Ashurst JL, Bradley A (2004) Identification of mammalian microRNA host genes and transcription units. Genome Res 14:1902–1910CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Ambros V, Lee RC, Lavanway A, Williams PT, Jewell D (2003) MicroRNAs and other tiny endogenous RNAs in C. elegans. Curr Biol 13:807–818CrossRefPubMedGoogle Scholar
  36. 36.
    Liquori CL, Ricker K, Moseley ML, Jacobsen JF, Kress W, Naylor SL, Day JW, Ranum LPW (2001) Myotinic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9. Science 293:864–867CrossRefPubMedGoogle Scholar
  37. 37.
    Jin P, Alisch RS, Warren ST (2004) RNA and microRNAs in fragile X mental retardation. Nat Cell Biol 6:1048–1053CrossRefPubMedGoogle Scholar
  38. 38.
    Eberhart DE, Malter HE, Feng Y, Warren ST (1996) The fragile X mental retardation protein is a ribonucleoprotein containing both nuclear localization and nuclear export signals. Hum Mol Genet 5:1083–1091CrossRefPubMedGoogle Scholar
  39. 39.
    Lin SL, Ying SY (2004) New drug design for gene therapy – taking advantage of Introns. Lett Drug Design Discov 1:256–262CrossRefGoogle Scholar
  40. 40.
    Zhang G, Taneja KL, Singer RH, Green MR (1994) Localization of pre-mRNA splicing in mammalian nuclei. Nature 372:809–812CrossRefPubMedGoogle Scholar
  41. 41.
    Ghosh S, Garcia-Blanco MA (2000) Coupled in vitro synthesis and splicing of RNA polymerase II transcripts. RNA 6:1325–1334CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD (1998) How cells respond to interferons. Annu Rev Biochem 67:227–264CrossRefPubMedGoogle Scholar
  43. 43.
    Sledz CA, Holko M, de Veer MJ, Silverman RH, Williams BR (2003) Activation of the interferon system by short-interfering RNAs. Nat Cell Biol 5:834–839CrossRefPubMedGoogle Scholar
  44. 44.
    Boden D, Pusch O, Silbermann R, Lee F, Tucker L, Ramratnam B (2004) Enhanced gene silencing of HIV-1 specific siRNA using microRNA designed hairpins. Nucleic Acid Res 32:1154–1158CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Division of Regenerative MedicineWJWU & LYNN Institute for Stem Cell ResearchSanta Fe SpringsUSA
  2. 2.Department of Integrative Anatomical Sciences, Keck School of MedicineUniversity of Southern CaliforniaLos AngelesUSA

Personalised recommendations