Argonaute pull-down and RISC analysis using 2'-O-methylated oligonucleotides affinity matrices

Methods Mol Biol. 2011;725:233-49. doi: 10.1007/978-1-61779-046-1_16.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Caenorhabditis elegans / genetics
  • Caenorhabditis elegans / metabolism
  • Caenorhabditis elegans Proteins / metabolism*
  • Methylation
  • MicroRNAs / genetics
  • MicroRNAs / metabolism
  • Molecular Biology / methods*
  • Oligonucleotides / genetics*
  • Oligonucleotides / metabolism*
  • RNA-Binding Proteins / metabolism*
  • RNA-Induced Silencing Complex / metabolism*

Substances

  • ALG-1 protein, C elegans
  • Caenorhabditis elegans Proteins
  • Let-7 microRNA, Drosophila
  • MicroRNAs
  • Oligonucleotides
  • RNA-Binding Proteins
  • RNA-Induced Silencing Complex