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The Tethering Assay: A Simple Method for the Characterization of mRNA-Fate Regulators

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


In trypanosomatids, posttranscriptional controls are very important in regulation of individual gene expression. These are achieved through combinatorial sets of RNA-binding proteins (RBPs) which recognize RNA regulatory motifs or regions of secondary structure within RNAs. To analyze the potential functional impact of an RBP on their mRNA targets, we have applied a robust technique called tethering assay. In this method, the protein under study is attached to an mRNA reporter through an artificial RNA–protein interaction. Therefore, the functional activity of a protein can be analyzed independently of its intrinsic ability to bind to RNA. By making use of a cell line expressing a chloramphenicol acetyltransferase (CAT) reporter mRNA, we have characterized dozens of novel mRNA-fate regulators in cultured Trypanosoma brucei. After induction of the candidate fusion protein, the effect on the reporter expression is determined by a rapid CAT assay. The protocol is simple and typically takes one working day for analysis of a single protein and controls. In this chapter, we provide a description of materials and methods for the tethering method and should allow the assay to be successfully deployed in any laboratory with minimal user training.

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We thank Christine Clayton for her continuous support and all former and current CC lab members who have contributed to improve this and many other techniques.

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Correspondence to Esteban D. Erben .

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Mugo, E., Erben, E.D. (2020). The Tethering Assay: A Simple Method for the Characterization of mRNA-Fate Regulators. In: Michels, P., Ginger, M., Zilberstein, D. (eds) Trypanosomatids. Methods in Molecular Biology, vol 2116. Humana, New York, NY.

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0293-5

  • Online ISBN: 978-1-0716-0294-2

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