Abstract
Small-interfering RNAs (siRNAs) and microRNAs (miRNAs) regulate expression of their target mRNAs via the RNA-induced silencing complex (RISC). A core component of RISC is the Argonaute (Ago) protein, which dictates the RISC function. In Drosophila, miRNAs and siRNAs are generally loaded into Ago1-containing RISC (Ago1-RISC) and Ago2-containing RISC (Ago2-RISC), respectively. We developed a native agarose gel system to directly detect Ago1-RISC, Ago2-RISC, and their precursor complexes. Methods presented here will provide powerful tools to biochemically dissect the RISC assembly pathways.
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Acknowledgments
We thank M. Siomi and H. Siomi (Keio University) for the antibody to Ago1, R. Carthew (Northwestern University) for dcr-2 L811fsX and dcr-2 416X flies. We also thank members of the Tomari laboratory for helpful discussions. This work was supported in part by a Grant-in-Aid for Young Scientists (B) to T.K., and a Grant-in-Aid for Young Scientists (A) and Grant-in-Aid for Scientific Research on Innovative Areas “Functional machinery for non-coding RNAs” to Y.T. from the Japan Ministry of Education, Culture, Sports, Science and Technology, and a Carrier Development Award from The International Human Frontier Science Program Organization to Y.T.
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Kawamata, T., Tomari, Y. (2011). Native Gel Analysis for RISC Assembly. In: Hobman, T., Duchaine, T. (eds) Argonaute Proteins. Methods in Molecular Biology, vol 725. Humana Press. https://doi.org/10.1007/978-1-61779-046-1_7
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DOI: https://doi.org/10.1007/978-1-61779-046-1_7
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