Pelechano Vicent, Wilkening Stefan, Järvelin Aino Inkeri, Tekkedil Manu M, Steinmetz Lars M
Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
Methods Enzymol. 2012;513:271-96. doi: 10.1016/B978-0-12-391938-0.00012-4.
Alternative polyadenylation site usage gives rise to variation in 3' ends of transcripts in diverse organisms ranging from yeast to human. Accurate mapping of polyadenylation sites of transcripts is of major biological importance, since the length of the 3'UTR can have a strong influence on transcript stability, localization, and translation. However, reads generated using total mRNA sequencing mostly lack the very 3' end of transcripts. Here, we present a method that allows simultaneous analysis of alternative 3' ends and transcriptome dynamics at high throughput. By using transcripts produced in vitro, the high precision of end mapping during the protocol can be controlled. This method is illustrated here for budding yeast. However, this method can be applied to any natural or artificially polyadenylated RNA.
可变聚腺苷酸化位点的使用导致了从酵母到人类等多种生物体中转录本3'端的变化。转录本聚腺苷酸化位点的精确映射具有重要的生物学意义，因为3'非翻译区（3'UTR）的长度会对转录本的稳定性、定位和翻译产生强烈影响。然而，使用总mRNA测序产生的读数大多缺少转录本的最3'端。在此，我们提出一种方法，该方法能够在高通量条件下同时分析可变3'端和转录组动态。通过使用体外产生的转录本，可以控制实验过程中端映射的高精度。本文以芽殖酵母为例对该方法进行了说明。然而，该方法可应用于任何天然或人工聚腺苷酸化的RNA。
