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The RIPper Case: Identification of RNA-Binding Protein Targets by RNA Immunoprecipitation

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Plant Circadian Networks

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1158))

Abstract

Control at the posttranscriptional level emerges as an important layer of regulation in the circadian timing system. RNA-binding proteins that specifically interact with cis-regulatory motifs within pre-mRNAs are key elements of this regulation. While the ability to interact with RNA in vitro has been demonstrated for numerous Arabidopsis RNA-binding proteins, a full understanding of posttranscriptional networks controlled by an RNA-binding protein requires the identification of its immediate in vivo targets. Here we describe differential RNA immunoprecipitation in transgenic Arabidopsis thaliana plants expressing RNA-binding protein variants epitope-tagged with green fluorescent protein. To control for RNAs that nonspecifically co-purify with the RNA-binding protein, transgenic plants are generated with a mutated version of the RNA-binding protein that is not capable of binding to its target RNAs. The RNA-binding protein variants are expressed under the control of their authentic promoter and cis-regulatory motifs. Incubation of the plants with formaldehyde in vivo cross-links the proteins to their RNA targets. A whole-cell extract is then prepared and subjected to immunoprecipitation with an antibody against the GFP tag and to mock precipitation with an antibody against the unrelated red fluorescent protein. The RNAs coprecipitating with the proteins are eluted from the immunoprecipitate and identified via reverse transcription-PCR.

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Acknowledgements

We thank Kristina Neudorf for expert technical assistance. This work was supported by the DFG (STA 653).

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Authors and Affiliations

  1. Department of Molecular Cell Physiology, University of Bielefeld, Universitätsstraße 25, 33615, Bielefeld, Germany

    Tino Köster, Meike Haas & Dorothee Staiger

Authors
  1. Tino Köster
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  2. Meike Haas
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  3. Dorothee Staiger
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Correspondence to Dorothee Staiger .

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Editors and Affiliations

  1. Molecular Cell Physiology, University of Bielefeld, Germany

    Dorothee Staiger

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Köster, T., Haas, M., Staiger, D. (2014). The RIPper Case: Identification of RNA-Binding Protein Targets by RNA Immunoprecipitation. In: Staiger, D. (eds) Plant Circadian Networks. Methods in Molecular Biology, vol 1158. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0700-7_7

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  • DOI: https://doi.org/10.1007/978-1-4939-0700-7_7

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0699-4

  • Online ISBN: 978-1-4939-0700-7

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