Laboratory of Regulation of Gene Expression, Institute of Microbiology of the Czech Academy of Sciences, Prague, Videnska 1083, 142 20, the Czech Republic.
Nucleic Acids Res. 2020 Feb 28;48(4):1969-1984. doi: 10.1093/nar/gkz1185.
One of the key roles of the 12-subunit eukaryotic translation initiation factor 3 (eIF3) is to promote the formation of the 43S and 48S pre-initiation complexes (PICs). However, particular contributions of its individual subunits to these two critical initiation reactions remained obscure. Here, we adapted formaldehyde gradient cross-linking protocol to translation studies and investigated the efficiency of the 43S and 48S PIC assembly in knockdowns of individual subunits of human eIF3 known to produce various partial subcomplexes. We revealed that eIF3d constitutes an important intermolecular bridge between eIF3 and the 40S subunit as its elimination from the eIF3 holocomplex severely compromised the 43S PIC assembly. Similarly, subunits eIF3a, c and e were found to represent an important binding force driving eIF3 binding to the 40S subunit. In addition, we demonstrated that eIF3c, and eIF3k and l subunits alter the efficiency of mRNA recruitment to 43S PICs in an opposite manner. Whereas the eIF3c knockdown reduces it, downregulation of eIF3k or eIF3l increases mRNA recruitment, suggesting that the latter subunits possess a regulatory potential. Altogether this study provides new insights into the role of human eIF3 in the initial assembly steps of the translational machinery.
真核翻译起始因子 3（eIF3）的十二亚基之一的主要作用之一是促进 43S 和 48S 起始前复合物（PIC）的形成。然而，其各个亚基对这两个关键起始反应的特定贡献仍然不清楚。在这里，我们采用甲醛梯度交联方案进行翻译研究，并在已知产生各种部分亚复合物的人类 eIF3 单个亚基的敲低中研究了 43S 和 48S PIC 组装的效率。我们发现 eIF3d 作为 eIF3 和 40S 亚基之间的重要分子间桥，其从 eIF3 全复合物中的消除严重损害了 43S PIC 的组装。同样，亚基 eIF3a、c 和 e 被发现代表一种重要的结合力，驱动 eIF3 与 40S 亚基结合。此外，我们证明 eIF3c 和 eIF3k 和 l 亚基以相反的方式改变 mRNA 向 43S PIC 募集的效率。eIF3c 的敲低降低了它，而 eIF3k 或 eIF3l 的下调增加了 mRNA 的募集，表明后两个亚基具有调节潜力。总的来说，这项研究为人类 eIF3 在翻译机器初始组装步骤中的作用提供了新的见解。
