Toward the mechanism of eIF4F-mediated ribosomal attachment to mammalian capped mRNAs.
Toward the mechanism of eIF4F-mediated ribosomal attachment to mammalian capped mRNAs.
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朝着EIF4F介导的核糖体附着在哺乳动物上限的mRNA上的机理。
DOI：
10.1101/gad.282418.116
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发表时间：
2016-07-01
影响因子：
10.5
通讯作者：
Pestova TV
中科院分区：
文献类型：
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作者：
Kumar P;Hellen CU;Pestova TV
Kumar等研究了mRNA进入40S核糖体亚基mRNA结合通道的机制。利用体外重构的翻译系统，他们的研究结果支持eIF4E - eif4g - eif3 - 40S相互作用将eIF4E置于40S亚基的前沿，mRNA被插入mRNA结合通道中，Met-tRNAiMet可以从第一个核苷酸开始检查它。哺乳动物带帽mRNA的核糖体附着是通过帽-真核起始因子4E (eIF4E) -eIF4G-eIF3-40S相互作用链实现的，但mRNA进入40S亚基mRNA结合通道的机制尚不清楚。为了研究这一过程，我们使用重组的翻译系统在体外对被封顶的mrna进行了启动。eIF4E-cap相互作用刺激了5′端起始复合物的形成，并遵循“第一个AUG”规则，表明反向扫描没有发生。起始复合物甚至在mRNA的5 '端形成，这意味着Met-tRNAiMet从第一个核苷酸开始检查mRNA，并且起始没有“盲点”。在组装的起始复合物中，帽不再与eIF4E相关。eIF4A的缺失或eIF4E - eif4g - eif3相互作用的破坏可将eIF4E转化为一种特异性抑制剂，抑制被封顶mrna的起始。综上所述，这些结果与eIF4E - eif4g - eif3 - 40S相互作用将eIF4E置于40S亚基的前沿的模型一致，并且mRNA被插入mRNA结合通道中，因此Met-tRNAiMet可以从第一个核苷酸开始检查它。在进入之前，eIF4E可能从帽上解离以克服空间位挡。我们还发现m7G帽特异性地与eIF3l相互作用。
Here, Kumar et al. investigated the mechanism by which mRNA enters the mRNA-binding channel of the 40S ribosomal subunit. Using an in vitro reconstituted translation system that recapitulates initiation on capped mRNAs, their results support a model in which eIF4E–eIF4G–eIF3–40S interactions place eIF4E at the leading edge of the 40S subunit, and mRNA is threaded into the mRNA-binding channel such that Met-tRNAiMet can inspect it from the first nucleotide. Ribosomal attachment to mammalian capped mRNAs is achieved through the cap–eukaryotic initiation factor 4E (eIF4E)–eIF4G–eIF3–40S chain of interactions, but the mechanism by which mRNA enters the mRNA-binding channel of the 40S subunit remains unknown. To investigate this process, we recapitulated initiation on capped mRNAs in vitro using a reconstituted translation system. Formation of initiation complexes at 5′-terminal AUGs was stimulated by the eIF4E–cap interaction and followed “the first AUG” rule, indicating that it did not occur by backward scanning. Initiation complexes formed even at the very 5′ end of mRNA, implying that Met-tRNAiMet inspects mRNA from the first nucleotide and that initiation does not have a “blind spot.” In assembled initiation complexes, the cap was no longer associated with eIF4E. Omission of eIF4A or disruption of eIF4E–eIF4G–eIF3 interactions converted eIF4E into a specific inhibitor of initiation on capped mRNAs. Taken together, these results are consistent with the model in which eIF4E–eIF4G–eIF3–40S interactions place eIF4E at the leading edge of the 40S subunit, and mRNA is threaded into the mRNA-binding channel such that Met-tRNAiMet can inspect it from the first nucleotide. Before entering, eIF4E likely dissociates from the cap to overcome steric hindrance. We also found that the m7G cap specifically interacts with eIF3l.