Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Edinburgh EH9 3JR, United Kingdom.
RNA. 2010 Dec;16(12):2570-80. doi: 10.1261/rna.2162610. Epub 2010 Oct 25.
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 3'-end cleavage and polyadenylation. This system permits pre-mRNA production, splicing, 3'-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 3'-end cleavage and polyadenylation, that is, cotranscriptionally.
我们描述了在 budding yeast 中以高分辨率获得 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 tetOFF（用于抑制、去抑制和 RNA 降解研究）酵母菌株，一系列报告基因在 tetO7 启动子的控制下整合到基因组中。我们对逆转录和定量实时 PCR（RT-qPCR）方法进行了改编，以允许在群体中每个细胞的平均拷贝数确定 mRNA 的丰度。荧光原位杂交（FISH）测量个别细胞中转录本数量验证了 RT-qPCR 方法，尽管细胞群体中的转录本水平分布广泛，但该方法适用于平均拷贝数的确定。此外，RT-qPCR 用于区分报告转录本剪接的不同步骤的产物，并开发了用于映射和量化 3'-末端切割和聚腺苷酸化的方法。该系统允许以前所未有的动力学细节定量监测前体 mRNA 的产生、剪接、3'-末端成熟和降解，适合数学建模。使用这种方法，我们证明报告转录本在其 3'-末端切割和聚腺苷酸化之前被剪接，即共转录。
