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J Vis Exp. 2018 Oct 22;(140). doi: 10.3791/57982.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity.

Author information

1
Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France; Centre National de la Recherche Scientifique, Illkirch, France; Institut National de la Santé et de la Recherche Médicale, Illkirch, France; Université de Strasbourg.
2
Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France; Centre National de la Recherche Scientifique, Illkirch, France; Institut National de la Santé et de la Recherche Médicale, Illkirch, France; Université de Strasbourg; devys@igbmc.fr.

Abstract

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

PMID:
30394386
DOI:
10.3791/57982

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