Abstract
Recent studies have shown that the microRNA (miRNA) pathway is an evolutionarily conserved endogenous pathway that is important for normal development. Mature miRNAs are excised from precursors in a stepwise process and subsequently incorporated into an RNA-induced silencing complex (RISC), which mediates either cleavage of the target messenger RNA (mRNA) or translational repression, depending on the complementarity between the miRNA and its target mRNA. In this chapter, we describe in vitro precursor (pre)-miRNA processing assays using Drosophila Schneider-2 (S2) cell lysates and immunopurified materials.
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References
Ambros, V. (2004) The functions of animal microRNAs. Nature 431, 350–355.
Bartel, D. P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297.
Baulcombe, D. (2004) RNA silencing in plants. Nature 431, 356–363.
Kim, V. N. (2005) MicroRNA biogenesis: coordinated cropping and dicing. Nature Rev. Mol. Cell Biol. 6, 376–385.
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., Jeon, K., Lee, J. T., Kim, S., and Kim, V. N. (2002) MicroRNA maturation: stepwise processing and subcellular localization. EMBO J. 21, 4663–4670.
Denli, A. M., Tops, B. B., Plasterk, R. H., Ketting, R. F., and Hannon, G. J. (2004) Processing of primary microRNAs by the Microprocessor complex. Nature 432, 231–235.
Gregory, R. I., Yan, K. P., Amuthan, G., et al. (2004). The Microprocessor complex medi-ates the genesis of microRNAs. Nature 432, 235–240.
Han, J., Lee, Y., Yeom, K. H., Kim, Y. K., Jin, H., and Kim, V. N. (2005) The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev. 18, 3016–3027.
Landthaler, M., Yalcin, A., and Tuschl, T. (2004) The human DiGeorge syndrome critical region gene 8 and its D. melanogaster homolog are required for miRNA biogenesis. Curr. Biol. 15, 2162–2167.
Lee, Y., Ahn, C., Han, J., et al. (2003). The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415–419.
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.
Bohnsack, M. T., Czaplinski, K., and Gorlich, D. (2004) Exportin 5 is a RanGTP-depen-dent dsRNA-biding protein that mediates nuclear export of pre-miRNAs. RNA 10, 185–191.
Lund, E., Guttinger, S., Calado, A., Dahlberg, J. E., and Kutay, U. (2004) Nuclear export of microRNA precursors. Science 303, 95–98.
Meister, G. and Tuschl, T. (2004) Mechanisms of gene silencing by double-stranded RNA. Nature 431, 343–349.
Sontheimer, E. J. (2005) Assembly and function of RNA silencing complexes. Nature Rev. Mol. Cell Biol. 6, 127–138.
Tomari, Y. and Zamore, P. D. (2005) Perspective: machines for RNAi. Genes Dev. 19, 517–529.
Ishizuka, A., Siomi, M. C., and Siomi, H. (2002) A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins. Genes Dev. 16, 2497–2508.
Caudy, A. A., Myers, M., Hannon, G. J., and Hammond, S. M. (2002) Fragile X-related protein and VIG associate with the RNA interference machinery. Genes Dev. 16, 2491–2496.
Liu, J., Carmell, M. A., Rivas, F. V., et al. (2004) Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437–1441.
Meister, G., Landthaler, M., Patkaniowska, A., Dorsett, Y., Teng, G., and Tuschl, T. (2004) Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol. Cell 15, 185–197.
Tabara, H., Yigit, E., Siomi, H., and Mello, C. C. (2002) The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans. Cell 109, 861–871.
Lee, Y. S., Nakahara, 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.
Pham, J. W., Pellino, J. L., Lee, Y. S., Carthew, R. W., and Sontheimer, E. J. (2004) A Dicer-2-dependent 80s complex cleaves targeted mRNAs during RNAi in Drosophila. Cell 117, 83–94.
Saito, K., Ishizuka, A., Siomi, H., and Siomi, M. C. (2005) Processing of pre-microRNAs by the Dicer-1-Loquacious complex in Drosophila cells. PLoS Biol. 3, e235.
Forstemann, K., Tomari, Y., Du, T., et al. (2005) Normal microRNA maturation by Dicer-1 and germ-line stem cell maintenance requires the double-stranded RNA-binding domain protein, Loquacious. PLoS Biol. 3, e236.
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.
Towers, P. R. and Sattelle, D. B. (2002) A Drosophila melanogaster cell line (S2) facilitates post-genome functional analysis of receptors and ion channels. Bioessays 24, 1066–1073.
Boutros, M., Kiger, A. A., Armknecht, S., et al. (2004) Genome-wide RNAi analysis of growth and viability in Drosophila cells. Science 303, 832–835.
Siomi, M. C. and Siomi, H. (2005) Identification of components of RNAi pathways using the tandem affinity purification method. Methods Mol. Biol. 309, 1–9.
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Ishizuka, A., Saito, K., Siomi, M.C., Siomi, H. (2006). In Vitro Precursor MicroRNA Processing Assays Using Drosophila Schneider-2 Cell Lysates. In: Ying, SY. (eds) MicroRNA Protocols. Methods in Molecular Biology™, vol 342. Humana Press. https://doi.org/10.1385/1-59745-123-1:277
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DOI: https://doi.org/10.1385/1-59745-123-1:277
Publisher Name: Humana Press
Print ISBN: 978-1-58829-581-1
Online ISBN: 978-1-59745-123-9
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