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In Vivo Tethering System to Isolate RNA-Binding Proteins Regulating mRNA Decay in Leishmania

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Trypanosomatids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2116))

Abstract

RNA-binding proteins (RBPs) play key roles in many aspects of RNA metabolism. In Leishmania, a unicellular eukaryote that favors the posttranscriptional mode of regulation for controlling gene expression levels, the function of RBPs becomes even more critical. However, due largely to limited in vivo approaches available for identifying RBPs in these parasites, there have been no significant advances to our understanding of the role these proteins play in posttranscriptional control through binding to cis-acting elements in the 3′ untranslated region (3′UTR) of mRNAs. Here we describe an optimized in vivo RNA tethering approach using the bacteriophage MS2 coat protein combined to immunoprecipitation and mass spectrometry analysis to identify RBPs specifically interacting with 3′UTR short interspersed degenerated retroposon elements (SIDERs). Members of the SIDER2 subfamily were shown previously to promote mRNA degradation through a novel mechanism of mRNA decay. Using this modified MS2 tethering approach, we have identified candidate RBPs specifically interacting with SIDER2 elements and contributing to the decay mechanism.

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Acknowledgments

This work was supported by the Canadian Institutes of Health Research Grant MOP-12182 awarded to B.P.

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Authors and Affiliations

  1. Division of Infectious and Immune Diseases, CHU de Quebec Research Center—Laval University, Québec, QC, Canada

    Hiva Azizi & Barbara Papadopoulou

  2. Department of Microbiology, Infectious Disease and Immunology, Faculty of Medicine, Laval University, Québec, QC, Canada

    Barbara Papadopoulou

Authors
  1. Hiva Azizi
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  2. Barbara Papadopoulou
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Corresponding author

Correspondence to Barbara Papadopoulou .

Editor information

Editors and Affiliations

  1. School of Biological Sciences, University of Edinburgh, Edinburgh, UK

    Paul A. M. Michels

  2. School of Applied Sciences, University of Huddersfield, Huddersfield, UK

    Michael L. Ginger

  3. Faculty of Biology, Technion–Israel Institute of Technology, Haifa, Israel

    Dan Zilberstein

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Azizi, H., Papadopoulou, B. (2020). In Vivo Tethering System to Isolate RNA-Binding Proteins Regulating mRNA Decay in Leishmania. In: Michels, P., Ginger, M., Zilberstein, D. (eds) Trypanosomatids. Methods in Molecular Biology, vol 2116. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0294-2_20

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  • DOI: https://doi.org/10.1007/978-1-0716-0294-2_20

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0293-5

  • Online ISBN: 978-1-0716-0294-2

  • eBook Packages: Springer Protocols

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