Jünemann Christiane, Song Yutong, Bassili Gergis, Goergen Dagmar, Henke Jura, Niepmann Michael
Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University Giessen, Friedrichstrasse 24, 35392 Giessen, Germany.
J Biol Chem. 2007 Jan 5;282(1):132-41. doi: 10.1074/jbc.M608750200. Epub 2006 Nov 8.
Certain viral and cellular mRNAs initiate translation cap-independently at internal ribosome entry site (IRES) elements. Picornavirus IRES elements are widely used in dicistronic or multicistronic vectors in gene therapy, virus replicon systems, and analysis of IRES function. In such vectors, expression of the upstream gene often serves as internal control to standardize the readings of IRES-driven downstream reporter activity. Picornaviral IRES elements translate optimally at up to 120 mM K(+) concentration, whereas genes used as upstream reporters usually have lower salt optima when present in monocistronic mRNAs. However, here we show that such reporter genes are efficiently translated at higher K(+) concentrations when placed upstream of a functional picornavirus IRES. This translation enhancement occurs in cis, is independent of the nature of the first reporter and of second reporter translation, and is conferred by the IRESs of picornaviruses but not of hepatitis C virus. A defective picornavirus IRES with a deletion killing IRES activity but leaving the binding site for initiation factor eIF4G intact retains translation enhancement activity. Translation enhancement on a capped mRNA is disabled by m(7)GDP. In addition, the C-terminal fragment of eIF4G can confer translation enhancement also on uncapped mRNA. We conclude that whenever eIF4F has been captured to a dicistronic mRNA by binding to a picornavirus IRES via its eIF4G moiety, it can be provided in cis to the 5'-end of the RNA and there stimulate translation initiation, either by binding to the cap nucleotide using its eIF4E moiety or by binding to the RNA cap-independently using its eIF4G moiety.
某些病毒和细胞mRNA在内部核糖体进入位点（IRES）元件处独立于帽结构起始翻译。微小核糖核酸病毒IRES元件广泛用于基因治疗的双顺反子或多顺反子载体、病毒复制子系统以及IRES功能分析。在这类载体中，上游基因的表达常作为内部对照，以标准化IRES驱动的下游报告基因活性的读数。微小核糖核酸病毒IRES元件在高达120 mM K(+)浓度下能实现最佳翻译，而用作上游报告基因的基因在单顺反子mRNA中时通常具有较低的盐最佳浓度。然而，我们在此表明，当这些报告基因置于功能性微小核糖核酸病毒IRES上游时，在较高K(+)浓度下能有效翻译。这种翻译增强是顺式发生的，与第一个报告基因的性质以及第二个报告基因的翻译无关，由微小核糖核酸病毒的IRES而非丙型肝炎病毒的IRES赋予。一个有缺陷的微小核糖核酸病毒IRES，其缺失导致IRES活性丧失但起始因子eIF4G的结合位点保持完整，仍保留翻译增强活性。m(7)GDP会使加帽mRNA上的翻译增强失效。此外，eIF4G 的C末端片段也能在无帽mRNA上赋予翻译增强作用。我们得出结论，只要eIF4F通过其eIF4G部分与微小核糖核酸病毒IRES结合而被捕获到双顺反子mRNA上，它就能顺式提供给RNA的5'端，并在那里刺激翻译起始，要么通过其eIF4E部分与帽核苷酸结合，要么通过其eIF4G部分独立于RNA帽结合。
