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Isolation and Identification of Gene-Specific MicroRNAs

  • Shi-Lung Lin
  • Donald C. Chang
  • Shao-Yao Ying
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1733)

Abstract

Computer programming has identified hundreds of genomic hairpin sequences, many with functions yet to be determined. Because transfection of hairpin-like microRNA precursors (pre-miRNAs) into mammalian cells is not always sufficient to trigger RNA-induced gene silencing complex (RISC) assembly, a key step for inducing RNA interference (RNAi)-related gene silencing, we have developed an intronic miRNA expression system to overcome this problem by inserting a hairpin-like pre-miRNA structure into the intron region of a gene, and hence successfully increase the efficiency and effectiveness of miRNA-associated RNAi induction in vitro and in vivo. This intronic miRNA biogenesis mechanism has been found to depend on a coupled interaction of nascent messenger RNA transcription and intron excision within a specific nuclear region proximal to genomic perichromatin fibrils. The intronic miRNA so obtained is transcribed by type-II RNA polymerases, coexpressed within a primary gene transcript, and then excised out of the gene transcript by intracellular RNA splicing and processing machineries. After that, ribonuclease III (RNaseIII) endonucleases further process the spliced introns into mature miRNAs. Using this intronic miRNA expression system, we have shown for the first time that the intron-derived miRNAs are able to elicit strong RNAi effects in not only human and mouse cells in vitro but also in zebrafishes, chicken embryos, and adult mice in vivo. We have also developed a miRNA isolation protocol, based on the complementarity between the designed miRNA and its targeted gene sequence, to purify and identify the mature miRNAs generated. As a result, several intronic miRNA identities and structures have been confirmed. According to this proof-of-principle methodology, we now have full knowledge to design various intronic pre-miRNA inserts that are more efficient and effective for inducing specific gene silencing effects in vitro and in vivo.

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 JL, Bradley A (2004) Identification of mammalian microRNA host genes and transcription units. Genome Res 14:1902–1910CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    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
  3. 3.
    Jin P, Alisch RS, Warren ST (2004) RNA and microRNAs in fragile X mental retardation. Nat Cell Biol 6:1048–1053CrossRefPubMedGoogle Scholar
  4. 4.
    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
  5. 5.
    Lin SL, Chang D, Ying SY (2005) Asymmetry of intronic pre-miRNA structures in functional RISC assembly. Gene 365:32–38CrossRefGoogle Scholar
  6. 6.
    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
  7. 7.
    Lin SL, Ying SY (2004) Novel RNAi therapy – intron-derived microRNA drugs. Drug Des Rev 1:247–255Google Scholar
  8. 8.
    Verri T, Argenton F, Tomanin R, Scarpa M, Storelli C, Costa R, Colombo L, Bortolussi M (1997) The bacteriophage T7 binary system activates transient transgene expression in zebrafish (Danio Rerio) embryos. Biochem Biophys Res Commun 237:492–495CrossRefPubMedGoogle Scholar
  9. 9.
    Tourriere H, Chebli K, Tazi J (2002) mRNA degradation machines in eukaryotic cells. Biochimie 84:821–837CrossRefPubMedGoogle Scholar
  10. 10.
    Wilusz CJ, Wilusz J (2004) Bringing the role of mRNA decay in the control of gene expression into focus. Trends Genet 20:491–497CrossRefPubMedGoogle Scholar
  11. 11.
    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
  12. 12.
    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 Acids 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

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