Assaying nuclear messenger RNA export in human cells.
Assaying nuclear messenger RNA export in human cells.
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DOI：
10.1385/1-59259-750-5:085
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发表时间：
2004-01-01
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影响因子：
--
通讯作者：
Cullen, Bryan R
中科院分区：
文献类型：
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作者：
Cullen, Bryan R
本章描述了一种分析培养的人类细胞中核信使RNA(MRNA)输出的简单方法。所描述的分析依赖于观察到含有内含子的mRNA分子通常保留在细胞核中，直到剪接完成。当CAT指示基因在位于5‘端的单个内含子中被隔离到mRNA帽部位时，CAT蛋白的表达依赖于核RNA输出因子通过插入的RNA结合位点特异性地募集到未剪接的CAT RNA中。这个位点可以是核输出因子的天然高亲和力RNA靶点，或者也可以通过融合到异源RNA结合域将输出因子拴在未剪接的CAT mRNA上。
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.