Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity
Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity
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DOI：
10.3791/57982
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发表时间：
2018-10-01
影响因子：
1.2
通讯作者：
Devys, Didier
中科院分区：
文献类型：
--
作者：
Baptista, Tiago;Devys, Didier
RNA聚合酶II转录中的全局缺陷可能被分析稳态RNA的转录组学研究所忽视。事实上，mRNA合成的整体减少已被证明是通过mRNA降解的同时减少来补偿的，以恢复正常的稳态水平。因此，mRNA合成的全基因组定量，独立于mRNA衰变，是RNA聚合酶II转录活性的最佳直接反映。在这里，我们讨论了一种方法，使用非扰动代谢标记的新生RNA在酿酒酵母（S。cerevisiae）。具体地，将细胞与尿嘧啶类似物4-硫尿嘧啶一起培养6分钟，并纯化和定量标记的新转录的RNA以确定所有单个mRNA的合成速率。此外，使用标记的粟酒裂殖酵母细胞作为内标，可以比较不同S.酿酒酵母菌株。使用该方案并用动态动力学模型拟合数据，可以确定相应的mRNA衰减速率。
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.