Detection of slicer activity by immunopurified plant ARGONAUTE1

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearchpeer-review

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.

Original languageEnglish
Title of host publicationPlant MicroRNAs : Methods and Protocols
EditorsStefan de Folter
Number of pages22
PublisherHumana Press
Publication date2019
ISBN (Print)978-1-4939-9041-2
ISBN (Electronic)978-1-4939-9042-9
Publication statusPublished - 2019
SeriesMethods in Molecular Biology

    Research areas

  • Arabidopsis, Argonaute, Endonucleolysis, In vitro assay, miRNA, Slicing

ID: 214511805