Interaction of the eIF4G initiation factor with the aphthovirus IRES is essential for internal translation initiation in vivo
Interaction of the eIF4G initiation factor with the aphthovirus IRES is essential for internal translation initiation in vivo
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DOI：
10.1017/s1355838200000753
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发表时间：
2000-10-01
期刊：
影响因子：
4.5
通讯作者：
Martínez-Salas, E
中科院分区：
文献类型：
--
作者：
De Quinto, SL;Martínez-Salas, E
人们对内部核糖体进入位点（IRES）元件招募翻译机制的策略知之甚少。在这项研究中，我们表明 eIF4G 翻译起始因子与口蹄疫病毒 (FMDV) IRES 序列的蛋白质-RNA 相互作用是活细胞中内部翻译起始的关键决定因素。此外，我们使用来自 FMDV 易感细胞提取物的天然蛋白质，鉴定了 eIF4G-RNA 功能相互作用所需的核苷酸。结构域 4 碱基的保守内部 AA 环的取代导致体外 eIF4G-RNA 相互作用和体内 IRES 依赖性翻译起始的强烈损害。相反，内部 AA 环附近的取代不会损害 IRES 活性，但保留了与 eIF4G 相互作用的能力。直接 UV 交联以及竞争测定表明 IRES 的结构域 1-2、3 和 5 对这种相互作用没有贡献。与此一致的是，单独与结构域 4 的结合与与全长 IRES 的结合一样有效。 eIF4G 的 C 端片段经 FMDV Lb 蛋白酶蛋白水解处理，足以与 IRES 或其结构域 4 单独相互作用。此外，我们在此表明，eIF4B 起始因子与 IRES 的结合需要结构域 5 序列。此外，在没有 eIF4B-IRES 结合的情况下检测到 eIF4G-IRES 相互作用，这表明两种起始因子都与 IRES 的 3' 区域相互作用，但使用不同的残基。在转染细胞中，eIF4G-RNA 相互作用与 IRES 活性之间的强相关性表明，eIF4G 在 IRES 依赖性起始过程中充当了招募翻译机制的接头。
The strategies developed by internal ribosome entry site (IRES) elements to recruit the translational machinery are poorly understood. In this study we show that protein-RNA interaction of the eIF4G translation initiation factor with sequences of the foot-and-mouth disease virus (FMDV) IRES is a key determinant of internal translation initiation in living cells. Moreover, we have identified the nucleotides required for eIF4G-RNA functional interaction, using native proteins from FMDV-susceptible cell extracts. Substitutions in the conserved internal AA loop of the base of domain 4 led to strong impairment of both eIF4G-RNA interaction in vitro and IRES-dependent translation initiation in vivo. Conversely, substitutions in the vicinity of the internal AA loop that did not impair IRES activity retained their ability to interact with eIF4G. Direct UV-crosslinking as well as competition assays indicated that domains 1-2, 3, and 5 of the IRES did not contribute to this interaction, In agreement with this, binding to domain 4 alone was as efficient as to-the full-length IRES. The C-terminal fragment of eIF4G, proteolytically processed by the FMDV Lb protease, was sufficient:to interact with the IRES or to its domain 4 alone, Additionally, we show here that binding of the eIF4B initiation factor to the IRES required domain 5 sequences. Moreover, eIF4G-IRES interaction was detected in the absence of eIF4B-IRES binding, suggesting that both initiation factors interact with the 3' region of the IRES but use different residues. The strong correlation found between eIF4G-RNA interaction and IRES activity in transfected cells suggests that eIF4G acts as a linker to recruit the translational machinery in IRES-dependent initiation.