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Analysis of Small RNA-Guided Endonuclease Activity in Endogenous Piwi Protein Complexes from Mouse Testes

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PIWI-Interacting RNAs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1093))

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Abstract

Small RNAs associate with members of the Argonaute family to function in gene regulation, transposon control, and creation of silent chromatin domains. In this partnership, small RNAs act as guides for the bound Argonaute and other associated proteins. Complementary base pairing of small RNAs to target nucleic acid molecules allow specificity for the small RNA-mediated functions. One key activity of some Argonaute protein family members is their small RNA-guided endonuclease activity called Slicer action. Here we describe a protocol that can be used to probe slicer activity in endogenous Piwi complexes isolated from mouse testes.

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Acknowledgments

We gratefully acknowledge help and advice from Pietro Spinelli, Zhaolin Yang, Elisa Cora, Bruno Rodino, and Sara Silva in developing the assay protocol. We thank Kuan-Ming Chen, Radha Raman Pandey, and David Homolka for their critical comments on the manuscript. Help from Magdalena Wojtas for preparation of the figures is acknowledged. Work in the Pillai lab is supported by a European Research Council Starting Grant (pisilence) from the European Union to R.S.P.

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Authors and Affiliations

  1. EMBL Grenoble Outstation, Grenoble, Cedex, France

    Michael Reuter & Ramesh S. Pillai

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  1. Michael Reuter
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  2. Ramesh S. Pillai
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Editors and Affiliations

  1. The University of Tokyo Dept of Biophysics & Biochemistry, Tokyo, Japan

    Mikiko C. Siomi

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Reuter, M., Pillai, R.S. (2014). Analysis of Small RNA-Guided Endonuclease Activity in Endogenous Piwi Protein Complexes from Mouse Testes. In: Siomi, M. (eds) PIWI-Interacting RNAs. Methods in Molecular Biology, vol 1093. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-694-8_9

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  • DOI: https://doi.org/10.1007/978-1-62703-694-8_9

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-693-1

  • Online ISBN: 978-1-62703-694-8

  • eBook Packages: Springer Protocols

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