Summary
Recent studies have revealed that Argonaute proteins are crucial components of the RNA-induced silencing complexes (RISCs) that direct both small interfering RNA (siRNA)- and microRNA (miRNA)-mediated gene silencing. Full complementarity between the small RNA and its target messenger RNA (mRNA) results in RISC-mediated cleavage (“Slicing”) of the target mRNA. A subset of Argonaute proteins directly contributes to the target cleavage (“Slicer”) activity of the RISC. We describe (in vitro) Slicer assays using endogenous Argonaute protein immunopurified from animal cells and recombinant Argonaute protein produced in and purified from Escherichia coli.
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Acknowledgments
K. M. is a research fellow supported by the Japan Society for the Promotion of Science (JSPS). This work was supported by grants to M. C. S. and H. S. from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the JSPS, and the New Energy and Industrial Technology Development Organization. M.C.S. is also supported by CREST from Japan Science and Technology Agency.
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Miyoshi, K., Uejima, H., Nagami-Okada, T., Siomi, H., Siomi, M.C. (2008). In vitro RNA Cleavage Assay for Argonaute-Family Proteins. In: Barik, S. (eds) RNAi. Methods in Molecular Biology™, vol 442. Humana Press. https://doi.org/10.1007/978-1-59745-191-8_3
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DOI: https://doi.org/10.1007/978-1-59745-191-8_3
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