Cullen Bryan R
HowardHughes Medical Institute and Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA.
Methods Mol Biol. 2004;257:85-92. doi: 10.1385/1-59259-750-5:085.
This chapter describes a simple method for the analysis of nuclear messenger RNA (mRNA) export in human cells in culture. The assay described relies on the observation that mRNA molecules containing an intron are generally retained in the nucleus until splicing is completed. Upon sequestration of the cat indicator gene in a single intron located 5' to an mRNA cap site, CAT protein expression becomes dependent on the specific recruitment of a nuclear RNA export factor to the unspliced cat RNA via an inserted RNA binding site. This site can be a natural, high affinity RNA target for the nuclear export factor or alternately the export factor can be tethered to the unspliced cat mRNA by fusion to a heterologous RNA binding domain.
本章描述了一种用于分析培养的人类细胞中核信使核糖核酸（mRNA）输出的简单方法。所述测定法基于这样的观察结果：含有内含子的mRNA分子通常会保留在细胞核中，直到剪接完成。当将氯霉素乙酰转移酶（CAT）指示基因隔离在位于mRNA帽位点5'端的单个内含子中时，CAT蛋白的表达就依赖于通过插入的RNA结合位点将核RNA输出因子特异性募集到未剪接的cat RNA上。该位点可以是核输出因子的天然高亲和力RNA靶标，或者也可以通过与异源RNA结合域融合，将输出因子拴系到未剪接的cat mRNA上。
