Chapter title |
Argonaute pull-down and RISC analysis using 2'-O-methylated oligonucleotides affinity matrices.
|
---|---|
Chapter number | 16 |
Book title |
Argonaute Proteins
|
Published in |
Methods in molecular biology, January 2011
|
DOI | 10.1007/978-1-61779-046-1_16 |
Pubmed ID | |
Book ISBNs |
978-1-61779-045-4, 978-1-61779-046-1
|
Authors |
Guillaume Jannot, Alejandro Vasquez-Rifo, Martin J. Simard, Jannot, Guillaume, Vasquez-Rifo, Alejandro, Simard, Martin J. |
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. |
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