Abstract
RATE-seq is a 4-thiouracil (4-tU)-based method that enables the in vivo measurement of transcriptome-wide RNA degradation rates. 4-tU is an analog of uracil that is rapidly incorporated into newly synthesized RNA and facilitates the conjugation of a biotinylated molecule containing a reactive thiol group. The biotinylated RNA can then be fractionated from the unlabeled RNA with streptavidin magnetic beads. By adding 4-tU to a culture of cells growing in steady-state conditions, fractionating the labeled population of RNA at multiple time points following 4-tU addition, and quantifying the abundance of newly transcribed RNAs using RNAseq, it is possible to estimate the degradation rates of all transcripts in a single experiment. The analysis of the RATE-seq data entails normalization of RNAseq libraries to thiolated RNA spike-ins and nonlinear model fitting to estimate the degradation rate constant for each RNA species.
References
Wiesner RJ, Zak R (1991) Quantitative approaches for studying gene expression. Am J Phys 260(4 Pt 1):L179–L188
Wang Y, Liu CL, Storey JD, Tibshirani RJ, Herschlag D, Brown PO (2002) Precision and functional specificity in mRNA decay. Proc Natl Acad Sci U S A 99:5860–5865. https://doi.org/10.1073/pnas.092538799
Grigull J, Mnaimneh S, Pootoolal J, Robinson MD, Hughes TR (2004) Genome-wide analysis of mRNA stability using transcription inhibitors and microarrays reveals posttranscriptional control of ribosome biogenesis factors. Mol Cell Biol 24:5534–5547. https://doi.org/10.1128/MCB.24.12.5534-5547.2004
Munchel SE, Shultzaberger RK, Takizawa N, Weis K (2014) Dynamic profiling of mRNA turnover reveals gene-specific and system-wide regulation of mRNA decay. Mol Biol Cell 22:2787–2795. https://doi.org/10.1091/mbc.E11-01-0028
Miller C, Schwalb B, Maier K, Schulz D, Dümcke S, Zacher B, Mayer A, Sydow J, Marcinowski L, Dölken L, Martin DE, Tresch A, Cramer P (2011) Dynamic transcriptome analysis measures rates of mRNA synthesis and decay in yeast. Mol Syst Biol 7:58. https://doi.org/10.1038/msb.2010.112
Neymotin B, Athanasiadou R, Gresham D (2014) Determination of in vivo RNA kinetics using RATE-seq. RNA 20:1645–1652. https://doi.org/10.1261/rna.045104.114
Duffy EE, Rutenberg-Schoenberg M, Stark CD, Kitchen RR, Gerstein MB, Simon MD (2015) Tracking distinct RNA populations using efficient and reversible covalent chemistry. Molecular Cell 59(5)858–866
Greenberg JR (1972) High stability of messenger RNA in growing cultured cells. Nature 240:102–104
Kim CH, Warner JR (1983) Messenger RNA for ribosomal proteins in yeast. J Mol Biol 165:79–89
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Abdul-Rahman, F., Gresham, D. (2018). Determining mRNA Decay Rates Using RNA Approach to Equilibrium Sequencing (RATE-Seq). 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_2
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DOI: https://doi.org/10.1007/978-1-4939-7540-2_2
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