Detection of Slicer Activity by Immunopurified Plant ARGONAUTE1
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 wordsArabidopsis Argonaute miRNA Endonucleolysis Slicing In vitro assay
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