Novel fluorescence-based screen to identify small synthetic internal ribosome entry site elements.
Novel fluorescence-based screen to identify small synthetic internal ribosome entry site elements.
复制标题
新型荧光筛选，用于识别小型合成内部核糖体进入位点元素。
DOI：
10.1128/mcb.21.8.2826-2837.2001
复制
发表时间：
2001
影响因子：
5.3
通讯作者：
Dasgupta,A
中科院分区：
文献类型：
--
作者：
Venkatesan,A;Dasgupta,A
我们报告了一种新的基于荧光蛋白的筛选，以确定体内合成的小的内部核糖体进入位点(IRES)元件。在细菌中扩增出编码增强型蓝色和绿色荧光蛋白(EBFP和EGFP)的双顺反子质粒文库，并通过原生质体融合将其导入哺乳动物细胞。使用EBFP和EGFP报告基因以及荧光激活的细胞分选技术分离获得功能IRES元件的细胞，并鉴定了几个小的IRES元件。这些元件中的两个在体外和体内都被证明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。虽然合成的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，但这两个合成的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元件通过利用一些与PV IRES相同的因素来指导翻译，从而与PV IRES介导的翻译竞争。讨论了这种基于荧光蛋白的筛选在鉴定具有更高活性的IRES元件以及在探索IRES介导的翻译机制方面的作用。
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.