Different modes and potencies of translational repression by sequence-specific RNA-protein interaction at the 5'-UTR.
Different modes and potencies of translational repression by sequence-specific RNA-protein interaction at the 5'-UTR.
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DOI：
10.1093/nar/gkl584
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发表时间：
2006
影响因子：
14.9
通讯作者：
Htun, Han
中科院分区：
文献类型：
--
作者：
Nie, Minghua;Htun, Han
为了确定在5′-非翻译区（5′-UTR）的序列特异性RNA-蛋白质相互作用是否可以有效地抑制哺乳动物细胞中的翻译，开发了双顺反子翻译抑制测定以允许直接评估瞬时转染的活哺乳动物细胞中的RNA-蛋白质相互作用和翻译抑制。通过荧光显微镜监测帽依赖性黄色荧光蛋白（YFP）和内部核糖体进入序列（IRES）依赖性青色荧光蛋白（CFP）翻译的变化。YFP的选择性抑制或YFP和CFP翻译的协同抑制发生，表明RNA结合蛋白通过5′-UTR抑制翻译的两种不同模式。有趣的是，来自枯草芽孢杆菌的单链RNA结合蛋白，色氨酸RNA结合衰减蛋白（TRAP），显示出有效的翻译抑制，这取决于TRAP表达水平和其同源结合位点在双顺反子报告转录本中的位置。作为第一个在哺乳动物细胞中检测的一类蛋白，其通过5′-UTR抑制翻译的效力可能是这类单链RNA结合蛋白的一般特征。最后，基于通过5′-UTR的翻译抑制的单杂交筛选鉴定了支持融合蛋白背景下TRAP的完全翻译抑制以及一系列部分抑制的接头。
To determine whether sequence-specific RNA–protein interaction at the 5′-untranslated region (5′-UTR) can potently repress translation in mammalian cells, a bicistronic translational repression assay was developed to permit direct assessment of RNA–protein interaction and translational repression in transiently transfected living mammalian cells. Changes in cap-dependent yellow fluorescent protein (YFP) and internal ribosome entry sequence (IRES)-dependent cyan fluorescent protein (CFP) translation were monitored by fluorescence microscopy. Selective repression of YFP or coordinate repression of both YFP and CFP translation occurred, indicating two distinct modes by which RNA-binding proteins repress translation through the 5′-UTR. Interestingly, a single-stranded RNA-binding protein from Bacillus subtilis, tryptophan RNA-binding attenuation protein (TRAP), showed potent translational repression, dependent on the level of TRAP expression and position of its cognate binding site within the bicistronic reporter transcript. As the first of its class to be examined in mammalian cells, its potency in repression of translation through the 5′-UTR may be a general feature for this class of single-stranded RNA-binding proteins. Finally, a one-hybrid screen based on translational repression through the 5′-UTR identified linkers supporting full-translational repression as well as a range of partial repression by TRAP within the context of a fusion protein.
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影响因子：
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