Advertisement

Gene Silencing In Vitro and In Vivo Using Intronic MicroRNAs

Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 342)

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 posttranscriptional 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 clinical and circumstantial malfunction. 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 nonprotein-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, neither function nor application has 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, 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 
This is a preview of subscription content, log in to check access.

References

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

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  1. 1.Department of Cell and Neurobiology, Keck School of MedicineUniversity of Southern CaliforniaLos Angeles

Personalised recommendations

Cite protocol

Buy options