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Detection of Slicer Activity by Immunopurified Plant ARGONAUTE1

  • Laura Arribas-Hernández
  • Maria Louisa Vigh
  • Peter BrodersenEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1932)

Abstract

Small RNA-guided endonucleolysis (“slicing”) of target mRNA is the signature biochemical activity underlying many RNA silencing phenomena. The catalytic slicer activity resides in Argonaute (AGO) proteins. Here, we present two protocols to detect microRNA-guided slicer activity of AGO1 immunopurified from Arabidopsis tissues. The first uses radioactive, cap-labeled RNA substrates produced by in vitro transcription of RNA fragments corresponding to endogenous target sites flanked by 100–200 nucleotides of target sequence. The second protocol uses similarly designed but shorter (around 50 nt) fluorescently labeled RNA. Advantages and disadvantages of the two setups are also discussed.

Key words

Arabidopsis Argonaute miRNA Endonucleolysis Slicing In vitro assay 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Laura Arribas-Hernández
    • 1
  • Maria Louisa Vigh
    • 1
  • Peter Brodersen
    • 1
    Email author
  1. 1.Department of BiologyUniversity of CopenhagenCopenhagenDenmark

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