Purification of Arabidopsis Argonaute Complexes and Associated Small RNAs

  • Yijun Qi
  • Shijun Mi
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 592)

Abstract

Argonaute (AGO) proteins recruit small RNAs to form effector complexes of RNA interference (RNAi), collectively termed RNA-induced silencing complexes (RISCs). Here, we describe detailed protocols for the purification of AGO complexes and their associated small RNAs, using Arabidopsis AGO1 as an example.

Key words

RNAi Argonaute microRNA Purification 

Notes

Acknowledgments

This work was supported by the Ministry of Science and Technology of China.

References

  1. 1.
    Hannon GJ (2002) RNA interference. Nature 418(6894):244–251CrossRefPubMedGoogle Scholar
  2. 2.
    Meister G, Tuschl T (2004) Mechanisms of gene silencing by double-stranded RNA. Nature 431(7006):343–349CrossRefPubMedGoogle Scholar
  3. 3.
    Vaucheret H et al (2004) The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Genes Dev 18(10):1187–1197CrossRefPubMedGoogle Scholar
  4. 4.
    Morel JB et al (2002) Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in post-transcriptional gene silencing and virus resistance. Plant Cell 14(3):629–639CrossRefPubMedGoogle Scholar
  5. 5.
    Baumberger N, Baulcombe DC (2005) Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs. Proc Natl Acad Sci U S A 102:11928–11933CrossRefPubMedGoogle Scholar
  6. 6.
    Qi Y, Denli AM, Hannon GJ (2005) Biochemical specialization within Arabidopsis RNA silencing pathways. Mol Cell 19:421–428CrossRefPubMedGoogle Scholar
  7. 7.
    Qi Y et al (2006) Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed DNA methylation. Nature 443(7114):1008–1012CrossRefPubMedGoogle Scholar
  8. 8.
    Zilberman D, Cao X, Jacobsen SE (2003) ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methylation. Science 299(5607):716–719CrossRefPubMedGoogle Scholar
  9. 9.
    Zilberman D et al (2004) Role of Arabidopsis ARGONAUTE4 in RNA-directed DNA methylation triggered by inverted repeats. Curr Biol 14(13):1214–1220CrossRefPubMedGoogle Scholar
  10. 10.
    Zheng X et al (2007) Role of Arabidopsis AGO6 in siRNA accumulation. DNA methylation and transcriptional gene silencing. Embo J 26(6):1691–1701CrossRefPubMedGoogle Scholar
  11. 11.
    Sambrook J, Fritsch EF, Maniatis T (eds) (1989) Molecular cloning, a laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor, New YorkGoogle Scholar
  12. 12.
    Motamedi MR et al (2004) Two RNAi complexes, RITS and RDRC, physically interact and localize to noncoding centromeric RNAs. Cell 119(6):789–802CrossRefPubMedGoogle Scholar
  13. 13.
    Dalmay T et al (2000) Potato virus X amplicons in arabidopsis mediate genetic and epigenetic gene silencing. Plant Cell 12(3):369–379CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Yijun Qi
    • 1
  • Shijun Mi
    • 1
  1. 1.National Institute of Biological SciencesBeijingChina

Personalised recommendations