eIF2A was the first eukaryotic initiator tRNA carrier discovered but its exact function has remained enigmatic. Uncharacteristic of translation initiation factors, eIF2A is reported to be non-cytosolic in multiple human cancer cell lines. Attempts to study eIF2A mechanistically have been limited by the inability to achieve high yield of soluble recombinant protein. Here, we developed a purification paradigm that yields ~360-fold and ~6,000-fold more recombinant human eIF2A from E. coli and insect cells, respectively, than previous reports. Using a mammalian in vitro translation system, we found that increased levels of recombinant human eIF2A inhibit translation of multiple reporter mRNAs, including those that are translated by cognate and near-cognate start codons, and does so prior to start codon recognition. eIF2A also inhibited translation directed by all four types of cap-independent viral IRESs, including the CrPV IGR IRES that does not require initiation factors or initiator tRNA, suggesting excess eIF2A sequesters 40S subunits. Supplementation with additional 40S subunits prevented eIF2A-mediated inhibition and pull-down assays demonstrated direct binding between recombinant eIF2A and purified 40S subunits. These data support a model that eIF2A must be kept away from the translation machinery to avoid sequestering 40S ribosomal subunits.

中文翻译：

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eIF2A 水平升高通过隔离 40S 核糖体亚基抑制翻译

eIF2A 是第一个被发现的真核起始 tRNA 载体，但其确切功能仍然是个谜。据报道，eIF2A 在多种人类癌细胞系中是非胞质的，这与翻译起始因子的特征不同。由于无法获得高产量的可溶性重组蛋白，对 eIF2A 机制进行研究的尝试受到限制。在这里，我们开发了一种纯化范例，从大肠杆菌和昆虫细胞中获得的重组人 eIF2A 产量分别比之前的报道多约 360 倍和约 6,000 倍。使用哺乳动物体外翻译系统，我们发现重组人eIF2A水平的增加会抑制多个报告基因mRNA的翻译，包括那些由同源和近同源起始密码子翻译的mRNA，并且在起始密码子识别之前就发生这种情况。eIF2A 还抑制所有四种类型的帽依赖性病毒 IRES 指导的翻译，包括不需要起始因子或起始子 tRNA 的 CrPV IGR IRES，表明过量的 eIF2A 隔离了 40S 亚基。补充额外的 40S 亚基可防止 eIF2A 介导的抑制，下拉实验证明重组 eIF2A 与纯化的 40S 亚基之间存在直接结合。这些数据支持这样一个模型：eIF2A 必须远离翻译机器，以避免隔离 40S 核糖体亚基。补充额外的 40S 亚基可防止 eIF2A 介导的抑制，下拉实验证明重组 eIF2A 与纯化的 40S 亚基之间存在直接结合。这些数据支持这样一个模型：eIF2A 必须远离翻译机器，以避免隔离 40S 核糖体亚基。补充额外的 40S 亚基可防止 eIF2A 介导的抑制，下拉实验证明重组 eIF2A 与纯化的 40S 亚基之间存在直接结合。这些数据支持这样一个模型：eIF2A 必须远离翻译机器，以避免隔离 40S 核糖体亚基。