Abstract
The intronless β-globin reporter, whose mRNA is intrinsically unstable due to the lack of introns, is a useful tool to study RNA stability elements in a heterologous transcript. Insertion of a stability element leads to the accumulation of intronless β-globin mRNA that can be visualized by conventional Northern blot analyses. In this chapter, we explain how to perform the β-globin reporter assay using the ENE (expression and nuclear retention element), a triple-helix-forming RNA stability element that protects reporter mRNA from 3′– 5′ decay. A list of considerations is included for the use of ENEs as a tool to stabilize other RNAs. In this chapter, we provide a brief description of how to insert an ENE sequence into the 3′-untranslated region of an intronless β-globin reporter plasmid using basic cloning technology. Then, we provide a detailed protocol for quantitative measurements of steady-state levels of β-globin mRNA. This entails the transient transfection of mammalian cells with β-globin reporter plasmids, isolation of total cellular RNA, and detection of reporter mRNA via Northern blot. This methodology can be applied for the study of any nuclear RNA stability element using the intronless β-globin reporter.
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Acknowledgments
We thank Mei-Di Shu and Kazimierz Tycowski for critical review of the manuscript, Angela Miccinello for editorial work, and all Steitz lab members for thoughtful discussions. This work was supported by NIH grants GM111430 (J.A.B.) and GM026154 (J.A.S.). J.A.S. is an investigator of the Howard Hughes Medical Institute.
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Brown, J.A., Steitz, J.A. (2016). Intronless β-Globin Reporter: A Tool for Studying Nuclear RNA Stability Elements. In: Rhoads, R. (eds) Synthetic mRNA. Methods in Molecular Biology, vol 1428. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3625-0_5
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DOI: https://doi.org/10.1007/978-1-4939-3625-0_5
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