Abstract
During the last decade, several novel small non-coding RNA pathways have been unveiled, which reach out to many biological processes. Common to all these pathways is the binding of a small RNA molecule to a protein member of the Argonaute family, which forms a minimal core complex called the RNA-induced silencing complex or RISC. The RISC targets mRNAs in a sequence-specific manner, either to induce mRNA cleavage through the intrinsic activity of the Argonaute protein or to abrogate protein synthesis by a mechanism that is still under investigation. We describe here, in details, a method for the affinity chromatography of the let-7 RISC starting from extracts of the nematode Caenorhabditis elegans. Our method exploits the sequence specificity of the RISC and makes use of biotinylated and 2′-O-methylated oligonucleotides to trap and pull-down small RNAs and their associated proteins. Importantly, this technique may easily be adapted to target other small RNAs expressed in different cell types or model organisms. This method provides a useful strategy to identify the proteins associated with the RISC, and hence gain insight in the functions of small RNAs.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Stefani, G., and Slack, F. J. (2008) Small non-coding RNAs in animal development, Nat Rev Mol Cell Biol 9, 219–230.
Hutvagner, G., and Simard, M. J. (2008) Argonaute proteins: key players in RNA silencing, Nat Rev Mol Cell Biol 9, 22–32.
Hutvágner, G., Simard, M. J., Mello, C. C., and Zamore, P. D. (2004) Sequence-specific inhibition of small RNA function, PLoS Biol 2, E98.
Inoue, H., Hayase, Y., Imura, A., Iwai, S., Miura, K., and Ohtsuka, E. (1987) Synthesis and hybridization studies on two complementary nona(2′-O-methyl)ribonucleotides, Nucleic Acids Res 15, 6131–6148.
Fabian, M. R., Mathonnet, G., Sundermeier, T., Mathys, H., Zipprich, J. T., Svitkin, Y. V., Rivas, F., Jinek, M., Wohlschlegel, J., Doudna, J. A., Chen, C. Y., Shyu, A. B., Yates, J. R., 3rd, Hannon, G. J., Filipowicz, W., Duchaine, T. F., and Sonenberg, N. (2009) Mammalian miRNA RISC recruits CAF1 and PABP to affect PABP-dependent deadenylation, Mol Cell 35, 868–880.
Aoki, K., Moriguchi, H., Yoshioka, T., Okawa, K., and Tabara, H. (2007) In vitro analyses of the production and activity of secondary small interfering RNAs in C. elegans, EMBO J 26, 5007–5019.
Yigit, E., Batista, P. J., Bei, Y., Pang, K. M., Chen, C. C., Tolia, N. H., Joshua-Tor, L., Mitani, S., Simard, M. J., and Mello, C. C. (2006) Analysis of the C. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi, Cell 127, 747–757.
Mayr, C., Hemann, M. T., and Bartel, D. P. (2007) Disrupting the pairing between let-7 and Hmga2 enhances oncogenic transformation, Science 315, 1576–1579.
Acknowledgments
We are grateful to the members of our laboratory for comments on this methods manuscript. Nematode strain, N2 was provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR). Our research is funded work by the Canadian Institutes of Health Research (CIHR), the Natural Sciences and Engineering Research Council of Canada, and the Cancer Research Society. M.J.S. is a CIHR New Investigator.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Jannot, G., Vasquez-Rifo, A., Simard, M.J. (2011). Argonaute Pull-Down and RISC Analysis Using 2’-O-Methylated Oligonucleotides Affinity Matrices. 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_16
Download citation
DOI: https://doi.org/10.1007/978-1-61779-046-1_16
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-045-4
Online ISBN: 978-1-61779-046-1
eBook Packages: Springer Protocols