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

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Trypanosomatids

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

Abstract

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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References

  1. Clayton C (2013) The regulation of trypanosome gene expression by RNA-binding proteins. PLoS Pathog 9:e1003680

    Article  Google Scholar 

  2. Lueong S, Merce C, Fischer B, Hoheisel JD, Erben ED (2016) Gene expression regulatory networks in Trypanosoma brucei: insights into the role of the mRNA-binding proteome. Mol Microbiol 100:457–471

    Article  CAS  Google Scholar 

  3. Erben ED (2018) High-throughput methods for dissection of trypanosome gene regulatory networks. Curr Genomics 19:78–86

    Article  CAS  Google Scholar 

  4. Wurst M, Seliger B, Jha BA, Klein C, Queiroz R, Clayton C (2012) Expression of the RNA recognition motif protein RBP10 promotes a bloodstream-form transcript pattern in Trypanosoma brucei. Mol Microbiol 83:1048–1063

    Article  CAS  Google Scholar 

  5. Mugo E, Clayton C (2017) Expression of the RNA-binding protein RBP10 promotes the bloodstream-form differentiation state in Trypanosoma brucei. PLoS Pathog 13:e1006560

    Article  Google Scholar 

  6. Erben ED, Fadda A, Lueong S, Hoheisel JD, Clayton C (2014) A genome-wide tethering screen reveals novel potential post-transcriptional regulators in Trypanosoma brucei. PLoS Pathog 10:e1004178

    Article  Google Scholar 

  7. Delhi P, Queiroz R, Inchaustegui D, Carrington M, Clayton C (2011) Is there a classical nonsense-mediated decay pathway in trypanosomes? PLoS One 6:e25112

    Article  CAS  Google Scholar 

  8. Droll D, Minia I, Fadda A, Singh A, Stewart M, Queiroz R, Clayton C (2013) Post-transcriptional regulation of the trypanosome heat shock response by a zinc finger protein. PLoS Pathog 9:e1003286

    Article  CAS  Google Scholar 

  9. Chakraborty C, Clayton C (2018) Stress susceptibility in Trypanosoma brucei lacking the RNA-binding protein ZC3H30. PLoS Negl Trop Dis 12:e0006835

    Article  CAS  Google Scholar 

  10. Ouna BA, Stewart M, Helbig C, Clayton C (2012) The Trypanosoma brucei CCCH zinc finger proteins ZC3H12 and ZC3H13. Mol Biochem Parasitol 183:184–188

    Article  CAS  Google Scholar 

  11. Farber V, Erben E, Sharma S, Stoecklin G, Clayton C (2013) Trypanosome CNOT10 is essential for the integrity of the NOT deadenylase complex and for degradation of many mRNAs. Nucleic Acids Res 41:1211–1222

    Article  Google Scholar 

  12. Singh A, Minia I, Droll D, Fadda A, Clayton C, Erben E (2014) Trypanosome MKT1 and the RNA-binding protein ZC3H11: interactions and potential roles in post-transcriptional regulatory networks. Nucleic Acids Res 42:4652–4668

    Article  CAS  Google Scholar 

  13. Coller JM, Gray NK, Wickens MP (1998) mRNA stabilization by poly(A) binding protein is independent of poly(A) and requires translation. Genes Dev 12:3226–3235

    Article  CAS  Google Scholar 

  14. Coller J, Wickens M (2002) Tethered function assays using 3′ untranslated regions. Methods 26:142–150

    Article  CAS  Google Scholar 

  15. Austin RJ, Xia T, Ren J, Takahashi TT, Roberts RW (2002) Designed arginine-rich RNA-binding peptides with picomolar affinity. J Am Chem Soc 124:10966–10967

    Article  CAS  Google Scholar 

  16. Coller J, Wickens M (2007) Tethered function assays: an adaptable approach to study RNA regulatory proteins. Methods Enzymol 429:299–321

    Article  CAS  Google Scholar 

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Acknowledgments

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. https://doi.org/10.1007/978-1-0716-0294-2_18

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  • DOI: https://doi.org/10.1007/978-1-0716-0294-2_18

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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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