Abstract
The RNA-induced silencing complex (RISC) is a programmable gene-silencing machine involved in many aspects of eukaryotic biology. In humans, RISC is programmed or “loaded” with a small-guide RNA by the action of a tri-molecular assembly termed the RISC-loading complex (RLC). The human RLC is composed of the proteins Dicer, TRBP, and Argonaute2 (Ago2). To facilitate structural and biochemical dissection of the RISC-loading process, we have developed a system for the in vitro reconstitution of the human RLC. Here, we describe in detail methods for the expression and purification of recombinant Dicer, TRBP, and Ago2 and protocols for the assembly of RLCs and RLC subcomplexes. We also describe several simple assays to observe the biochemical activities of the assembled protein complexes.
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References
Filipowicz, W. (2005) RNAi: the nuts and bolts of the RISC machine. Cell 122, 17–20.
Gregory, R.I., Chendrimada, T.P., Cooch, N., & Shiekhattar, R. (2005) Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. Cell 123, 631–640.
Maniataki, E. & Mourelatos, Z. (2005) A human, ATP-independent, RISC assembly machine fueled by pre-miRNA. Genes Dev. 19, 2979–2990.
Tomari, Y., Matranga, C., Haley, B., Martinez, N., & Zamore, P.D. (2004) A protein sensor for siRNA asymmetry. Science 306, 1377–1380.
Meister, G. et al. (2004) Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol. Cell 15, 185–197.
Liu, J. et al. (2004) Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437–1441.
Song, J.J., Smith, S.K., Hannon, G.J. & Joshua-Tor, L. (2004) Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305, 1434–1437.
Rivas, F.V., Tolia, N.H., Song, J.J., Aragon, J.P., Liu, J., Hannon, G.J., & Joshua-Tor, L. (2005) Purified Argonaute2 and an siRNA form recombinant human RISC. Nature Struct. Mol. Biol. 12, 340–349.
MacRae, I.J., Ma, E., Zhou, M., Robinson, C.V., Doudna, J.A. (2008) In vitro reconstitution of the human RISC-loading complex. Proc Natl Acad Sci U S A. 105, 512–7.
Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, & Zamore PD (2003) Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208.
Khvorova A, Reynolds A, & Jayasena SD (2003) Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209–216.
Batey RT, Sagar MB, & Doudna JA (2001) Structural and energetic analysis of RNA recognition by a universally conserved protein from the signal recognition particle. J. Mol. Biol. 307, 229–246.
Acknowledgments
We thank the members of the MacRae lab for helpful suggestions. Work on the Human RLC is funded by the NIH (R01 GM086701). I.J.M. is a Pew Scholar in the Biomedical Sciences.
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De, N., MacRae, I.J. (2011). Purification and Assembly of Human Argonaute, Dicer, and TRBP Complexes. 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_8
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DOI: https://doi.org/10.1007/978-1-61779-046-1_8
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Online ISBN: 978-1-61779-046-1
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