Functional characterization of the internal ribosome entry site of eIF4G mRNA
Functional characterization of the internal ribosome entry site of eIF4G mRNA
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DOI：
10.1074/jbc.273.9.5006
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发表时间：
1998-02-27
影响因子：
4.8
通讯作者：
Rhoads, RE
中科院分区：
文献类型：
--
作者：
Gan, WN;La Celle, M;Rhoads, RE
eIF4 基团起始因子是帽依赖性翻译起始所必需的。小核糖核酸病毒感染哺乳动物细胞会导致这些因子之一 eIF4G 的蛋白水解裂解，这严重限制了帽依赖性启动，但允许帽独立启动从小核糖核酸病毒 RNA 的内部核糖体进入位点 (IRES) 进行。 eIF4G mRNA 5'-非翻译区的前 357 个核苷酸 (nt) 也包含 IRES，使用双顺反子构建体在 K562 细胞中表达，我们现在已经表明，5'-非翻译区的渐进删除可以具有刺激或抑制作用。此外，101 nt 片段表现出完整的 IRES 活性，81 nt 片段表现出可检测的 IRES 活性。 3' 末端的聚嘧啶束 (PPT) 对于内部启动至关重要，这是小核糖核酸病毒 IRES 的特征，但迄今为止研究的其他宿主细胞 IRES 则没有这种特性。 IRES 活性不需要超过 357 nt 的序列。当在 PPT 上游引入时，框外 AUG 对 IRES 驱动的荧光素酶表达没有影响，但在 PPT 和 nt 369 处的真实起始密码子之间的位点引入时，表达显着降低。这些结果表明核糖体亚基在 PPT 处或附近进入，然后向下游扫描起始密码子。
The eIF4 group initiation factors are required for cap-dependent translation initiation. Infection of mammalian cells by picornaviruses results in proteolytic cleavage of one of these factors, eIF4G, which severely restricts cap-dependent initiation but permits cap independent initiation to proceed from an internal ribosome entry site (IRES) in picornaviral RNAs. The first 357 nucleotides (nt) of the 5'-untranslated region of eIF4G mRNA also contains an IRES, Using bicistronic constructs for expression in K562 cells, we have now shown that progressive deletions of the 5'-untranslated region can have either stimulatory or inhibitory effects. Furthermore, a 101-nt segment exhibits full IRES activity, and an 81-nt segment exhibits detectable IRES activity. A polypyrimidine tract (PPT) at the 3' terminus is essential for internal initiation, a property which is characteristic of picornaviral IRESs but not the other host cellular IRESs studied to date. IRES activity does not require sequences beyond 357 nt. Out-of-frame AUGs have no effect on IRES-driven luciferase expression when introduced upstream of the PPT but markedly decrease expression when introduced at sites between the PPT and the authentic initiation codon at nt 369. These results suggest that the ribosomal subunit enters at or near the PPT and then scans downstream for the initiation codon.