Venkatesan A, Dasgupta A
Molecular Biology Institute, University of California, Los Angeles 90095, USA.
Mol Cell Biol. 2001 Apr;21(8):2826-37. doi: 10.1128/MCB.21.8.2826-2837.2001.
We report here a novel fluorescent protein-based screen to identify small, synthetic internal ribosome entry site (IRES) elements in vivo. A library of bicistronic plasmids encoding the enhanced blue and green fluorescent proteins (EBFP and EGFP) separated by randomized 50-nucleotide-long sequences was amplified in bacteria and delivered into mammalian cells via protoplast fusion. Cells that received functional IRES elements were isolated using the EBFP and EGFP reporters and fluorescence-activated cell sorting, and several small IRES elements were identified. Two of these elements were subsequently shown to possess IRES activity comparable to that of a variant of the encephalomyocarditis virus IRES element in a context-independent manner both in vitro and in vivo, and these elements functioned in multiple cell types. Although no sequence or structural homology was apparent between the synthetic IRES elements and known viral and cellular IRES elements, the two synthetic IRES elements specifically blocked poliovirus (PV) IRES-mediated translation in vitro. Competitive protein-binding experiments suggested that these IRES elements compete with PV IRES-mediated translation by utilizing some of the same factors as the PV IRES to direct translation. The utility of this fluorescent protein-based screen in identifying IRES elements with improved activity as well as in probing the mechanism of IRES-mediated translation is discussed.
我们在此报告一种基于新型荧光蛋白的筛选方法，用于在体内鉴定小型合成内部核糖体进入位点（IRES）元件。一个双顺反子质粒文库，其编码由随机的50个核苷酸长序列隔开的增强型蓝色和绿色荧光蛋白（EBFP和EGFP），在细菌中扩增后通过原生质体融合导入哺乳动物细胞。利用EBFP和EGFP报告基因以及荧光激活细胞分选技术分离出接受功能性IRES元件的细胞，并鉴定出了几个小型IRES元件。随后证明其中两个元件在体外和体内均以不依赖上下文的方式具有与脑心肌炎病毒IRES元件变体相当的IRES活性，并且这些元件在多种细胞类型中发挥作用。尽管合成IRES元件与已知的病毒和细胞IRES元件之间没有明显的序列或结构同源性，但这两个合成IRES元件在体外特异性地阻断了脊髓灰质炎病毒（PV）IRES介导的翻译。竞争性蛋白质结合实验表明，这些IRES元件通过利用与PV IRES相同的一些因子来指导翻译，从而与PV IRES介导的翻译竞争。本文讨论了这种基于荧光蛋白的筛选方法在鉴定具有更高活性的IRES元件以及探究IRES介导的翻译机制方面的实用性。
