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Plant MicroRNAs pp 295–316Cite as

Detection of Slicer Activity by Immunopurified Plant ARGONAUTE1

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Part of the book series: Methods in Molecular Biology ((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.

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Author information

Authors and Affiliations

  1. Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, Copenhagen, DK-2200, Copenhagen N, Denmark

    Laura Arribas-Hernández, Maria Louisa Vigh & Peter Brodersen

Authors
  1. Laura Arribas-Hernández
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  2. Maria Louisa Vigh
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  3. Peter Brodersen
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Corresponding author

Correspondence to Peter Brodersen .

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Editors and Affiliations

  1. Unidad de Genómica Avanzada, Laboratorio Nacional de Genómica para la Biodiversidad (LANGEBIO), Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Irapuato, Guanajuato, Mexico

    Stefan de Folter

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Arribas-Hernández, L., Vigh, M.L., Brodersen, P. (2019). Detection of Slicer Activity by Immunopurified Plant ARGONAUTE1. In: de Folter, S. (eds) Plant MicroRNAs. Methods in Molecular Biology, vol 1932. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9042-9_22

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  • DOI: https://doi.org/10.1007/978-1-4939-9042-9_22

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9041-2

  • Online ISBN: 978-1-4939-9042-9

  • eBook Packages: Springer Protocols

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