Tethered-function analysis reveals that elF4E can recruit ribosomes independent of its binding to the cap structure
Tethered-function analysis reveals that elF4E can recruit ribosomes independent of its binding to the cap structure
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DOI：
10.1017/s1355838201000577
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发表时间：
2001-01-01
期刊：
影响因子：
4.5
通讯作者：
Hentze, MW
中科院分区：
文献类型：
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作者：
De Gregorio, E;Baron, J;Hentze, MW
帽结合复合体elF4F在翻译起始阶段参与核糖体募集，由三个亚基组成：elF4E、-4G和-4A。m(7)GpppN帽结合亚基elF4E结合elF4G的n端区域，进而通过elF4G的中心和c端区域与elF4A接触。我们之前已经证明，通过在转染的HeLa细胞中使用系链功能方法，elF4G与mRNA的结合足以驱动高效翻译（De Gregorio等人，EMBO杂志，1994,18:4865-4874）。在这里，我们利用这种方法来评估哪些elF4F的其他亚单位可以发挥这个功能。elF4AI或突变形式的eIF4E被融合到lambda噬菌体抗菌素N的rna结合域，产生嵌合蛋白lambda 4A， lambda 4E-(102)（取消帽结合）和lambda 4E-(73-102)（与帽和elF4G的结合受损）。融合蛋白通过与顺反电子间隙中特定的lambda -N结合位点（boxB）相互作用，被定向到双反电子报告mRNA。我们发现lambda 4E-(102)，但双突变体lambda 4E-(73-102)和lambda 4A都不足以促进下游基因的翻译。共免疫沉淀分析证实，所有lambda -融合蛋白都能够与适当的内源性eIF4F亚基相互作用。这些结果表明，elF4E和elF4G可以独立于与帽结构的物理连接来驱动核糖体招募。尽管它与内源性elF4G相互作用，但lambda 4A不表现出这种特性，因此elF4A似乎为elF4F提供了一种必要的辅助功能，这可能需要它能够循环进出该复合体。
The cap-binding complex elF4F is involved in ribosome recruitment during the initiation phase of translation and is composed of three subunits: elF4E, -4G, and -4A. The m(7)GpppN cap-binding subunit elF4E binds the N-terminal region of elF4G, which in turn contacts elF4A through its central and C-terminal regions. We have previously shown, through a tethered-function approach in transfected HeLa cells, that the binding of elF4G to an mRNA Is sufficient to drive productive translation (De Gregorio et al., EMBO J, 1999, 18:4865-4874). Here we exploit this approach to assess which of the other subunits of elF4F can exert this function. elF4AI or mutant forms of eIF4E were fused to the RNA-binding domain of the lambda phage antiterminator protein N to generate the chimeric proteins lambda 4A, lambda 4E-(102) (abolished cap binding), and lambda 4E-(73-102) (impaired binding to both, the cap and elF4G). The fusion proteins were directed to a bicistronic reporter mRNA by means of interaction with a specific lambda -N binding site (boxB) in the intercistronic space. We show that lambda 4E-(102), but neither the double mutant lambda 4E-(73-102) nor lambda 4A, suffices to promote translation of the downstream gene in this assay. Coimmunoprecipitation analyses confirmed that all lambda -fusion proteins are capable of interacting with the appropriate endogenous eIF4F subunits. These results reveal that elF4E, as well as elF4G, can drive ribosome recruitment independent of a physical link to the cap structure. in spite of its interaction with endogenous elF4G, lambda 4A does not display this property, elF4A thus appears to supply an essential auxiliary function to elF4F that may require its ability to cycle into and out of this complex.