RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3′-end processing in Saccharomyces cerevisiae
RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3′-end processing in Saccharomyces cerevisiae
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DOI：
10.1261/rna.2162610
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发表时间：
2010-12-01
期刊：
影响因子：
4.5
通讯作者：
Beggs, Jean D.
中科院分区：
文献类型：
--
作者：
Alexander, Ross D.;Barrass, J. David;Beggs, Jean D.
我们描述了在发芽酵母中获得高分辨率RNA加工的定量描述的方法。作为一个模型基因表达系统，我们构建了Teton(用于诱导研究)和teOFF(用于阻遏、去阻遏和RNA降解研究)酵母菌株，并将一系列报告基因整合到基因组中，这些报告基因是在TetO7启动子的控制下进行的。逆转录和定量实时-聚合酶链式反应(RT-qPCR)方法被用来确定一个群体中每个细胞的平均拷贝数的信使核糖核酸丰度。荧光原位杂交(FISH)对单个细胞中转录数量的测量验证了RT-qPCR方法用于确定平均拷贝数的有效性，尽管转录水平在细胞群体中的广泛分布。此外，利用RT-qPCR来区分报告转录本剪接不同步骤的产物，并建立了定位和定量39端裂解和多聚腺苷化的方法。该系统能够以前所未有的动力学细节对前mRNA的产生、剪接、39端成熟和降解进行定量监测，适合于数学建模。使用这种方法，我们证明了报告转录本在其39端切割和聚腺苷酸化之前被剪接，即共转录。
We describe methods for obtaining a quantitative description of RNA processing at high resolution in budding yeast. As a model gene expression system, we constructed tetON (for induction studies) and tetOFF (for repression, derepression, and RNA degradation studies) yeast strains with a series of reporter genes integrated in the genome under the control of a tetO7 promoter. Reverse transcription and quantitative real-time-PCR (RT-qPCR) methods were adapted to allow the determination of mRNA abundance as the average number of copies per cell in a population. Fluorescence in situ hybridization (FISH) measurements of transcript numbers in individual cells validated the RT-qPCR approach for the average copy-number determination despite the broad distribution of transcript levels within a population of cells. In addition, RT-qPCR was used to distinguish the products of the different steps in splicing of the reporter transcripts, and methods were developed to map and quantify 39-end cleavage and polyadenylation. This system permits pre-mRNA production, splicing, 39-end maturation and degradation to be quantitatively monitored with unprecedented kinetic detail, suitable for mathematical modeling. Using this approach, we demonstrate that reporter transcripts are spliced prior to their 39-end cleavage and polyadenylation, that is, cotranscriptionally.