Nagaike Takashi, Manley James L
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba-shi, Ibaraki, Japan.
Methods Mol Biol. 2014;1125:65-74. doi: 10.1007/978-1-62703-971-0_6.
In vitro assays have provided a valuable tool to study the mechanism of 3' processing of eukaryotic mRNA precursors and have contributed a great deal to the identification of factors that carry out and regulate 3' processing. Previously, we have shown that transcriptional activators directly enhance polyadenylation by utilizing in vitro transcription-coupled polyadenylation with the prototypical transcription activator GAL4-VP16. In this chapter, we describe a detailed protocol for this assay, which will be useful in examining potential roles for other transcription-related factors in 3' processing and other questions related to the coupling of transcription and mRNA polyadenylation.
体外实验为研究真核生物mRNA前体3'加工机制提供了一种有价值的工具，并为鉴定参与和调节3'加工的因子做出了巨大贡献。此前，我们已经表明转录激活因子通过利用与典型转录激活因子GAL4-VP16的体外转录偶联多聚腺苷酸化直接增强多聚腺苷酸化。在本章中，我们描述了该实验的详细方案，这将有助于研究其他转录相关因子在3'加工中的潜在作用以及与转录和mRNA多聚腺苷酸化偶联相关的其他问题。
