López de Quinto S, Lafuente E, Martínez-Salas E
Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Cantoblanco, Madrid, Spain.
RNA. 2001 Sep;7(9):1213-26. doi: 10.1017/s1355838201010433.
Translation initiation promoted by picornavirus internal ribosome entry site (IRES) elements is dependent on the association of specific IRES sequences to the initiation factor eIF4G. However the RNA determinants interacting with other components of the translational machinery are still unknown. In this study, we have identified novel RNA-protein interactions between the foot-and-mouth disease virus (FMDV) IRES and three translation initiation factors. A doublet of 116/110 kDa that crosslinked to the FMDV IRES is a component of eIF3. We show here that domain 5 holds the preferential binding site for eIF3, although this complex initiation factor can establish multiple contacts with the IRES structure. We have also identified the phylogenetically conserved hairpin of domain 5 as the RNA motif responsible for eIF4B interaction. Mutation of this stem-loop structure abrogated eIF4B, but not eIF3, binding to the IRES. Remarkably, IRES mutants severely affected in their interaction with eIF4B showed a mild reduction in IRES activity when tested in the context of a bicistronic expression vector in transfected cells. Finally, we provide evidence of the interaction of eIF4GII with FMDV IRES, the RNA determinants for this interaction being shared with its functional homolog eIF4GI. The FMDV Lb protease generated a C-terminal fragment of eIF4GII that binds to the IRES as efficiently as the intact protein. Competition experiments showed that titration of eIF4B or p110/116 interaction with the FMDV IRES required a large excess of competitor relative to eIF4G, strongly suggesting that eIF4G-IRES interaction is a limiting factor to titrate the IRES. Comparative analysis of the activity of IRES mutants affected in domains 4 and 5 regarding their pattern of RNA-protein complex formation demonstrates that while binding of eIF4B with the FMDV IRES is dispensable, interaction of eIF4G is a central feature of the activity of this element.
微小核糖核酸病毒内部核糖体进入位点（IRES）元件所促进的翻译起始依赖于特定IRES序列与起始因子eIF4G的结合。然而，与翻译机制其他组分相互作用的RNA决定簇仍不清楚。在本研究中，我们鉴定了口蹄疫病毒（FMDV）IRES与三种翻译起始因子之间新的RNA-蛋白质相互作用。与FMDV IRES交联的116/110 kDa双峰是eIF3的一个组分。我们在此表明，结构域5拥有eIF3的优先结合位点，尽管这个复合起始因子能与IRES结构建立多个接触点。我们还鉴定了结构域5中系统发育保守的发夹结构作为负责与eIF4B相互作用的RNA基序。该茎环结构的突变消除了eIF4B与IRES的结合，但未消除eIF3与IRES的结合。值得注意的是，在与eIF4B的相互作用中受到严重影响的IRES突变体，当在转染细胞中的双顺反子表达载体背景下进行测试时，其IRES活性仅轻微降低。最后，我们提供了eIF4GII与FMDV IRES相互作用的证据，这种相互作用的RNA决定簇与其功能同源物eIF4GI共享。FMDV Lb蛋白酶产生了eIF4GII的一个C末端片段，其与IRES的结合效率与完整蛋白相同。竞争实验表明，相对于eIF4G，滴定eIF4B或p110/116与FMDV IRES的相互作用需要大量过量的竞争者，这强烈表明eIF4G-IRES相互作用是滴定IRES的一个限制因素。对在结构域4和5中受到影响的IRES突变体关于其RNA-蛋白质复合物形成模式的活性进行比较分析表明，虽然eIF4B与FMDV IRES的结合是可有可无的，但eIF4G的相互作用是该元件活性的一个核心特征。
