ArticleJournal of visualized experiments : JoVE2018
Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity.
Article in Journal of visualized experiments : JoVE, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 6 papers.
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Who cites it
6 citing papers in PubMed.
- Hydroxyurea and inactivation of checkpoint kinase MEC1 inhibit transcription termination and pre-mRNA cleavage at polyadenylation sites in budding yeast.Scientific reports · 2023Article
- SAGA-Dependent Histone H2Bub1 Deubiquitination Is Essential for Cellular Ubiquitin Balance during Embryonic Development.International journal of molecular sciences · 2022Review
- Single-molecule mapping of replisome progression.Molecular cell · 2022Article
- Architectural Mediator subunits are differentially essential for global transcription in Saccharomyces cerevisiae.Genetics · 2021Article
- Acute stress drives global repression through two independent RNA polymerase II stalling events in Saccharomyces.Cell reports · 2021Article
- Biomolecules of 2-Thiouracil, 4-Thiouracil and 2,4-Dithiouracil: A DFT Study of the Hydration, Molecular Docking and Effect in DNA:RNAMicrohelixes.International journal of molecular sciences · 2019Article
Corrections and comments
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Authors and funding
2 authors.
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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.
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