Simultaneous Measurement of Transcriptional and Post-transcriptional Parameters by 3' end RNA-seq

Cellular RNA levels are determined by transcription and decay rates, which are fundamental in understanding gene expression regulation. Measurement of these two parameters is usually performed independently, complicating analysis and introducing methodological biases that hamper direct comparison. Here, we present a simple approach of concurrent sequencing of S. cerevisiae polyA+ and polyA- RNA 3' ends to simultaneously estimate total RNA levels, transcription and decay rates from the same RNA sample. The transcription data generated correlate well with reported estimates and also reveal local RNA polymerase stalling and termination sites with high precision. Although the method by design uses brief metabolic labeling of newly synthesized RNA with 4-thiouridine, the results demonstrate that transcription estimates can also be gained from unlabeled RNA samples. These findings underscore the potential of the approach, which should be generally applicable to study a range of biological questions in diverse organisms.细胞内RNA水平由转录速率与降解速率共同决定，二者是解析基因表达调控机制的核心基础。长期以来，对这两类参数的检测通常独立开展，这不仅加剧了分析复杂度，还会引入方法学偏差，阻碍不同实验间的直接比较。本研究提出一种简便方法，可同时对酿酒酵母（Saccharomyces cerevisiae）的聚腺苷酸化阳性（polyA+）与聚腺苷酸化阴性（polyA-）RNA的3'端进行测序，进而从同一份RNA样本中同步估算总RNA水平、转录速率与降解速率。所获得的转录数据与已发表的估算结果具有良好的相关性，同时还能以极高精度识别RNA聚合酶的局部停滞位点与终止位点。尽管该方法的设计初衷是利用4-硫尿苷（4-thiouridine）对新合成RNA进行短时代谢标记，但研究结果表明，未经过标记的RNA样本同样可用于获取转录速率的估算值。上述发现凸显了该方法的应用潜力，其有望广泛适用于探究不同物种中的各类生物学问题。