Beilharz Traude H, Preiss Thomas
Molecular Genetics Division, Victor Chang Cardiac Research Institute (VCCRI), Lowy Packer Building, 405 Liverpool Street, Darlinghurst (Sydney), NSW 2010, Australia.
Methods. 2009 Jul;48(3):294-300. doi: 10.1016/j.ymeth.2009.02.003. Epub 2009 Feb 20.
The 3' poly(A) tail has important roles throughout the eukaryotic mRNA life cycle. A characteristic aspect of poly(A) tail function is furthermore that it can be modulated by changes in its length. This is in turn a well-recognised cellular means to regulate both, mRNA translation and stability, and a positive correlation has often been found between the efficiency of mRNA translation and the length of its poly(A) tail. Here we describe methodology to measure mRNA polyadenylation state in a transcriptome-wide manner, using separation of cellular mRNA populations on poly(U) sepharose in combination with microarray analysis of the resulting fractions. We further detail methods for bulk and mRNA-specific poly(A) tail length measurements to monitor the efficiency of initial mRNA separation and to verify candidates selected from the microarray data. Although detailed here for the study of yeast mRNAs, these methods are adaptable to the investigation of any cellular context in which poly(A) tail length control is known or suspected to operate.
3' 聚腺苷酸尾巴在整个真核生物mRNA生命周期中发挥着重要作用。聚腺苷酸尾巴功能的一个显著特点是，它可以通过长度的变化进行调节。这反过来又是一种公认的调节mRNA翻译和稳定性的细胞机制，并且人们经常发现mRNA翻译效率与其聚腺苷酸尾巴长度之间存在正相关。在这里，我们描述了一种以转录组范围的方式测量mRNA聚腺苷酸化状态的方法，该方法利用在聚（U）琼脂糖上分离细胞mRNA群体，并结合对所得组分进行微阵列分析。我们进一步详细介绍了用于大量和mRNA特异性聚腺苷酸尾巴长度测量的方法，以监测初始mRNA分离的效率，并验证从微阵列数据中选择的候选物。尽管这里是针对酵母mRNA的研究进行详细介绍，但这些方法适用于任何已知或怀疑存在聚腺苷酸尾巴长度控制的细胞环境的研究。
