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.
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References
Ambros, V. (2004) The functions of animal microRNAs. Nature 350, 431–355.
Nelson, P., Kiriakidou, M., Sharma, A., Maniataki, E., and Mourelatos, Z. (2003) The microRNA world: small is mighty. Trends Biochem. Sci. 28, 534–539.
Ying, S. Y. and Lin, S. L. (2005) Intronic microRNA (miRNA). Biochem. Biophys. Res. Commun. 326, 515–520.
Lin, S. L. and Ying, S. Y. (2004) Novel RNAi therapy-intron-derived microRNA drugs. Drug Design Reviews 1, 247–255.
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.
Ambros, V. (1989) A hierarchy of regulatory genes controls a larva-to-adult developmen-tal switch in C. elegans. Cell 57, 49–57.
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.
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.
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.
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.
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.
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.
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.
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.
Lagos-Quintana, M., Rauhut, R., Meyer, J., Borkhardt, A., and Tuschl, T. (2003) New microRNAs from mouse and human. RNA 9, 175–179.
Mourelatos, Z., Dostie, J., Paushkin, S., et al. (2002) miRNPs: a novel class of ribonucle-oproteins containing numerous microRNAs. Genes Dev. 16, 720–728.
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.
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.
Carthew, R. W. (2001) Gene silencing by double-stranded RNA. Curr. Opin. Cell Biol. 13, 244–248.
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.
Lin, S. L., Chang, D., and Ying, S. Y. (2005) Asymmetry of intronic pre-miRNA structures in functional RISC assembly. Gene 356, 32–38.
Ying, S. Y. and Lin S. L. (2004) Intron-derived microRNAs-fine tuning of gene func-tions. Gene 342, 25–28.
Kramer, A. (1996) The structure and function of proteins involved in mammalian pre-mRNA splicing. Annu. Rev. Biochem. 65, 367–409.
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.
Nott, A., Meislin, S. H., and Moore, M. J. (2003) A quantitative analysis of intron effects on mammalian gene expression. RNA 9, 607–617.
Lee, Y., Kim, M., Han, J., et al. (2004) MicroRNA genes are transcribed by RNA poly-merase II. EMBO J. 23, 4051–4060.
Lee, Y., Ahn, C., Han, J., et al. (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415–419.
Lund, E., Guttinger, S., Calado, A., Dahlberg, J. E., and Kutay, U. (2004) Nuclear export of microRNA precursors. Science 303, 95–98.
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.
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.
Khvorova, A., Reynolds, A., and Jayasena, S. D. (2003) Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209–216.
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.
Tang, G. (2005) siRNA and miRNA: an insight into RISCs. Trends Biochem. Sci. 30, 106-114.
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.
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.
Jin, P., Alisch, R. S., and Warren, S. T. (2004) RNA and microRNAs in fragile X mental retardation. Nat. Cell. Biol. 6, 1048–1053.
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.
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.
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.
Ghosh, S. and Garcia-Blanco, M. A. (2000) Coupled in vitro synthesis and splicing of RNA polymerase II transcripts. RNA 6, 1325–1334.
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.
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.
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.
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Lin, SL., Ying, SY. (2006). Gene Silencing In Vitro and In Vivo Using Intronic MicroRNAs. In: Ying, SY. (eds) MicroRNA Protocols. Methods in Molecular Biology™, vol 342. Humana Press. https://doi.org/10.1385/1-59745-123-1:295
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DOI: https://doi.org/10.1385/1-59745-123-1:295
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Print ISBN: 978-1-58829-581-1
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