Abstract
Cellular mRNA levels are determined by the competing forces of transcription and decay. A wide array of cellular mRNA decay pathways carry out RNA turnover either on a constitutive basis or in response to changing cellular conditions. Here, we outline a method to investigate mRNA decay that employs RNAi knockdown of known or putative decay factors in commercially available Tet-off cell systems. Reporter mRNAs of interest are expressed under the control of a tetracycline-regulated promoter, allowing pulse-chase mRNA decay assays to be conducted. Levels of reporter and constitutively expressed control RNAs throughout the decay assay time course are detected by traditional northern blot analysis and used to calculate mRNA half-lives. We describe the utility of this approach to study nonsense-mediated mRNA decay substrates and factors, but it can be readily adapted to investigate key mechanistic features that dictate the specificity and functions of any mRNA decay pathway.
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References
Chen CA, Shyu AB (2016) Emerging themes in regulation of global mRNA turnover in cis. Trends Biochem Sci. https://doi.org/10.1016/j.tibs.2016.08.014
Palumbo MC, Farina L, Paci P (2015) Kinetics effects and modeling of mRNA turnover. Wiley Interdiscip Rev RNA 6(3):327–336. https://doi.org/10.1002/wrna.1277
Karousis ED, Nasif S, Muhlemann O (2016) Nonsense-mediated mRNA decay: novel mechanistic insights and biological impact. Wiley Interdiscip Rev RNA 7(5):661–682. https://doi.org/10.1002/wrna.1357
Baker SL, Hogg JR (2017) A system for coordinated analysis of translational readthrough and nonsense-mediated mRNA decay. PLoS One 12(3):e0173980. https://doi.org/10.1371/journal.pone.0173980
Tang X, Zhu Y, Baker SL, Bowler MW, Chen BJ, Chen C, Hogg JR, Goff SP, Song H (2016) Structural basis of suppression of host translation termination by Moloney Murine Leukemia Virus. Nat Commun 7:12070. https://doi.org/10.1038/ncomms12070
Lykke-Andersen J, Shu MD, Steitz JA (2000) Human Upf proteins target an mRNA for nonsense-mediated decay when bound downstream of a termination codon. Cell 103(7):1121–1131
Mendell JT, ap Rhys CM, Dietz HC (2002) Separable roles for rent1/hUpf1 in altered splicing and decay of nonsense transcripts. Science 298(5592):419–422. https://doi.org/10.1126/science.1074428
Acknowledgments
This work was supported by the Intramural Research Program, National Institutes of Health, National Heart, Lung, and Blood Institute. We thank Zhiyun Ge, Aparna Kishor, and Stacey Baker for troubleshooting aspects of this protocol.
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Baird, T.D., Hogg, J. (2018). Using Tet-Off Cells and RNAi Knockdown to Assay mRNA Decay. In: Lamandé, S. (eds) mRNA Decay. Methods in Molecular Biology, vol 1720. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7540-2_12
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DOI: https://doi.org/10.1007/978-1-4939-7540-2_12
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7539-6
Online ISBN: 978-1-4939-7540-2
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