Isolation and Identification of Gene-Specific MicroRNAs

  • Shi-Lung Lin
  • Donald C. Chang
  • Shao-Yao Ying
Part of the Methods in Molecular Biology™ book series (MIMB, volume 342)

Abstract

Prediction of microRNA (miRNA) candidates using computer programming has identified hundreds and hundreds of genomic hairpin sequences, of which, the functions remain to be determined. Because direct transfection of hairpin-like miRNA precursors (pre)-miRNAs in mammalian cells is not always sufficient to trigger effective RNA-induced gene-silencing complex (RISC) assembly, a key step for RNA interference (RNAi)-related gene silencing, we developed an intronic miRNA-expressing system to overcome this problem, and successfully increased the efficiency and effectiveness of miRNA-associated RNAi induction in vitro and in vivo. By insertion of a hairpin-like pre-miRNA structure into the intron region of a gene, this intronic miRNA biogenesis system has been found to depend on a coupled interaction of nascent precursor messenger RNA transcription and intron excision within a specific nuclear region proximal to genomic perichromatin fibrils. The intronic miRNA was transcribed by RNA type II polymerases, coexpressed with a primary gene transcript, and excised out of its encoding gene transcript by intracellular RNA splicing and processing mechanisms. Currently, some ribonuclease III endonucleases have been found to be involved in the processing of spliced introns and probably facilitating the intronic miRNA maturation. Using this miRNA-expressing system, we have shown for the first time that the intron-derived miRNAs were able to induce strong RNAi effects in not only human and mouse cells but also zebrafish, chicken embryos, and adult mice. Based on the strand complementarity between the designed miRNA and its target gene sequence, we have also developed a miRNA isolation protocol to purify and identify the mature miRNAs generated by the intronic miRNA-expressing system. Several intronic miRNA identities and structures are currently confirmed to be active in vitro and in vivo. According to this proof-of-principle method, we now have the knowledge to design pre-miRNA inserts that are more efficient and effective for the intronic miRNA-expressing system.

Key Words

MicroRNA (miRNA) biogenesis gene cloning RNA interference (RNAi) RNA-induced gene-silencing complex (RISC) asymmetric assembly zebrafish 

References

  1. 1.
    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
  2. 2.
    Liquori, C. L., Ricker, K., Moseley, M. L., et al. (2001) Myotinic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9. Science 293, 864–867.CrossRefPubMedGoogle Scholar
  3. 3.
    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
  4. 4.
    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
  5. 5.
    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
  6. 6.
    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. Com-mun. 310, 754–760.CrossRefGoogle Scholar
  7. 7.
    Lin, S. L. and Ying, S. Y. (2004) Novel RNAi therapy-intron-derived microRNA drugs. Drug Design Reviews 1, 247–255.CrossRefGoogle Scholar
  8. 8.
    Verri, T., Argenton, F., Tomanin, R., et al. (1997) The bacteriophage T7 binary system activates transient transgene expression in zebrafish (Danio rerio) embryos. Biochem. Bio-phys. Res. Commun. 237, 492–495.CrossRefGoogle Scholar
  9. 9.
    Tourriere, H., Chebli, K., and Tazi, J. (2002) mRNA degradation machines in eukaryotic cells. Biochimie 84, 821–837.CrossRefPubMedGoogle Scholar
  10. 10.
    Wilusz, C. J. and Wilusz, J. (2004) Bringing the role of mRNA decay in the control of gene expression into focus. Trends Genet. 20, 491–497.CrossRefPubMedGoogle Scholar
  11. 11.
    Lee, Y., Ahn, C., Han, J., et al. (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415–419.CrossRefPubMedGoogle Scholar
  12. 12.
    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

  • Shi-Lung Lin
    • 1
  • Donald C. Chang
    • 1
  • Shao-Yao Ying
    • 1
  1. 1.Department of Cell and Neurobiology, Keck School of MedicineUniversity of Southern CaliforniaLos Angeles

Personalised recommendations