Abstract
MicroRNAs (miRNAs) are short non-coding RNAs transcribed from intergenic or intronic sequences as long precursors that are sequentially processed by the endonucleases Drosha and Dicer into short double-stranded sequences. It is clear that miRNAs play essential roles in gene expression, development, and cell fate specification in animals. However, one of the barriers of miRNA research is how to find the target genes. In this study, we have developed a rapid and effective method to isolate miRNA target genes in vivo. MicroRNA was synthesized in vitro and labeled by biotin. After transfected into cells, the miRNA/mRNA complexes were isolated by streptavidin-coated magnetic beads. hsa-miR155 was taken as model to validate this method, which is a very important modulator in tumor development. It is useful for validation of targets predicted in silico, and, potentially, for discovery of previously uncharacterized targets.
Similar content being viewed by others
References
Garzon, R., Calin, G. A., & Croce, C. M. (2009). MicroRNAs in cancer. Annual Review of Medicine, 60, 167–179.
Cai, X., Hagedorn, C. H., & Cullen, B. R. (2004). Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. RNA, 10, 1957–1966.
Denli, A. M., Tops, B. B., Plasterk, R. H., Ketting, R. F., & Hannon, G. J. (2004). Processing of primary microRNAs by the Microprocessor complex. Nature, 432, 231–235.
Gregory, R. I., Yan, K. P., Amuthan, G., Chendrimada, T., Doratotaj, B., Cooch, N., et al. (2004). The Microprocessor complex mediates the genesis of microRNAs. Nature, 432, 235–240.
Lee, Y., Ahn, C., Han, J., Choi, H., Kim, J., Yim, J., et al. (2003). The nuclear RNase III Drosha initiates microRNA processing. Nature, 425, 415–419.
Han, J., Lee, Y., Yeom, K. H., Nam, J. W., Heo, I., Rhee, J. K., et al. (2006). Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell, 125, 887–901.
Lund, E., Guttinger, S., Calado, A., Dahlberg, J. E., & Kutay, U. (2004). Nuclear export of microRNA precursors. Science, 303, 95–98.
Hutvagner, G., McLachlan, J., Pasquinelli, A. E., Balint, E., Tuschl, T., & Zamore, P. D. (2001). A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science, 293, 834–838.
Salzman, D. W., Shubert-Coleman, J., & Furneaux, H. (2007). P68 RNA helicase unwinds the human let-7 microRNA precursor duplex and is required for let-7-directed silencing of gene expression. Journal of Biological Chemistry, 282, 32773–32779.
Khvorova, A., Reynolds, A., & Jayasena, S. D. (2003). Functional siRNAs and miRNAs exhibit strand bias. Cell, 115, 209–216.
Schwarz, D. S., Hutvagner, G., Du, T., Xu, Z., Aronin, N., & Zamore, P. D. (2003). Asymmetry in the assembly of the RNAi enzyme complex. Cell, 115, 199–208.
Sen, G. L., & Blau, H. M. (2005). Argonaute 2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies. Nature Cell Biology, 7, 633–636.
Orom, U. A., Nielsen, F. C., & Lund, A. H. (2008). MicroRNA-10a binds the 5′UTR of ribosomal protein mRNAs and enhances their translation. Molecular Cell, 30, 460–471.
Khraiwesh, B., Arif, M. A., Seumel, G. I., Ossowski, S., Weigel, D., Reski, R., et al. (2010). Transcriptional control of gene expression by microRNAs. Cell, 140, 111–122.
Stadler, B. M., & Ruohola-Baker, H. (2008). Small RNAs: Keeping stem cells in line. Cell, 132, 563–566.
Enright, A. J., John, B., Gaul, U., Tuschl, T., Sander, C., & Marks, D. S. (2003). MicroRNA targets in Drosophila. Genome Biology, 5, R1.
Lewis, B. P., Shih, I. H., Jones-Rhoades, M. W., Bartel, D. P., & Burge, C. B. (2003). Prediction of mammalian microRNA targets. Cell, 115, 787–798.
Rehmsmeier, M., Steffen, P., Hochsmann, M., & Giegerich, R. (2004). Fast and effective prediction of microRNA/target duplexes. RNA, 10, 1507–1517.
Krek, A., Grun, D., Poy, M. N., Wolf, R., Rosenberg, L., Epstein, E. J., et al. (2005). Combinatorial microRNA target predictions. Nature Genetics, 37, 495–500.
Burgler, C., & Macdonald, P. M. (2005). Prediction and verification of microRNA targets by MovingTargets, a highly adaptable prediction method. BMC Genomics, 6, 88.
Ambros, V., Bartel, B., Bartel, D. P., Burge, C. B., Carrington, J. C., Chen, X., et al. (2003). A uniform system for microRNA annotation. RNA, 9, 277–279.
Karginov, F. V., Conaco, C., Xuan, Z., Schmidt, B. H., Parker, J. S., Mandel, G., et al. (2007). A biochemical approach to identifying microRNA targets. Proceedings of the National Academy of Sciences of the United States of America, 104, 19291–19296.
Tan, L. P., Seinen, E., Duns, G., de Jong, D., Sibon, O. C., Poppema, S., et al. (2009). A high throughput experimental approach to identify miRNA targets in human cells. Nucleic Acids Research, 37, e137.
Orom, U. A., & Lund, A. H. (2007). Isolation of microRNA targets using biotinylated synthetic microRNAs. Methods, 43, 162–165.
Hsu, R. J., Yang, H. J., & Tsai, H. J. (2009). Labeled microRNA pull-down assay system: An experimental approach for high-throughput identification of microRNA-target mRNAs. Nucleic Acids Research, 37, e77.
Nonne, N., Ameyar-Zazoua, M., Souidi, M., & Harel-Bellan, A. (2010). Tandem affinity purification of miRNA target mRNAs (TAP-Tar). Nucleic Acids Research, 38, e20.
Faraoni, I., Antonetti, F. R., Cardone, J., & Bonmassar, E. (2009). miR-155 gene: A typical multifunctional microRNA. Biochimica et Biophysica Acta, 1792, 497–505.
Ma, J. B., Ye, K., & Patel, D. J. (2004). Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain. Nature, 429, 318–322.
Parker, J. S., Roe, S. M., & Barford, D. (2005). Structural insights into mRNA recognition from a PIWI domain-siRNA guide complex. Nature, 434, 663–666.
Wang, Y., Juranek, S., Li, H., Sheng, G., Tuschl, T., & Patel, D. J. (2008). Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex. Nature, 456, 921–926.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Zheng, W., Zou, HW., Tan, YG. et al. Identification of MicroRNA Target Genes in Vivo. Mol Biotechnol 47, 200–204 (2011). https://doi.org/10.1007/s12033-010-9329-7
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12033-010-9329-7