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Increased levels of eIF2A inhibit translation by sequestering 40S ribosomal subunits
bioRxiv - Biochemistry Pub Date : 2023-08-03 , DOI: 10.1101/2022.11.18.517125
Daisy J. Grove , Daniel J. Levine , Michael G. Kearse

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:

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。
更新日期:2023-08-04
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