Department of Molecular and Cellular Biology, College of Biological Sciences, University of California, Davis, California, USA.
Department of Molecular and Cellular Biology, College of Biological Sciences, University of California, Davis, California, USA.
J Biol Chem. 2024 May;300(5):107242. doi: 10.1016/j.jbc.2024.107242. Epub 2024 Apr 1.
Messenger RNA (mRNA) recruitment to the 40S ribosomal subunit is mediated by eukaryotic initiation factor 4F (eIF4F). This complex includes three subunits: eIF4E (mG cap-binding protein), eIF4A (DEAD-box helicase), and eIF4G. Mammalian eIF4G is a scaffold that coordinates the activities of eIF4E and eIF4A and provides a bridge to connect the mRNA and 40S ribosomal subunit through its interaction with eIF3. While the roles of many eIF4G binding domains are relatively clear, the precise function of RNA binding by eIF4G remains to be elucidated. In this work, we used an eIF4G-dependent translation assay to reveal that the RNA binding domain (eIF4G-RBD; amino acids 682-720) stimulates translation. This stimulating activity is observed when eIF4G is independently tethered to an internal region of the mRNA, suggesting that the eIF4G-RBD promotes translation by a mechanism that is independent of the mG cap and mRNA tethering. Using a kinetic helicase assay, we show that the eIF4G-RBD has a minimal effect on eIF4A helicase activity, demonstrating that the eIF4G-RBD is not required to coordinate eIF4F-dependent duplex unwinding. Unexpectedly, native gel electrophoresis and fluorescence polarization assays reveal a previously unidentified direct interaction between eIF4G and the 40S subunit. Using binding assays, our data show that this 40S subunit interaction is separate from the previously characterized interaction between eIF4G and eIF3. Thus, our work reveals how eIF4F can bind to the 40S subunit using eIF3-dependent and eIF3-independent binding domains to promote translation initiation.
信使 RNA（mRNA）与 40S 核糖体亚基的募集由真核起始因子 4F（eIF4F）介导。该复合物包括三个亚基：eIF4E（mG 帽结合蛋白）、eIF4A（DEAD 盒解旋酶）和 eIF4G。哺乳动物 eIF4G 是一种支架，通过其与 eIF3 的相互作用协调 eIF4E 和 eIF4A 的活性，并提供连接 mRNA 和 40S 核糖体亚基的桥梁。虽然 eIF4G 结合域的许多作用相对清楚，但 eIF4G 对 RNA 的结合的确切功能仍有待阐明。在这项工作中，我们使用依赖 eIF4G 的翻译测定法揭示了 RNA 结合域（eIF4G-RBD；氨基酸 682-720）刺激翻译。当 eIF4G 独立地被连接到 mRNA 的内部区域时，观察到这种刺激活性，这表明 eIF4G-RBD 通过一种独立于 mG 帽和 mRNA 连接的机制促进翻译。使用动力学解旋酶测定法，我们表明 eIF4G-RBD 对 eIF4A 解旋酶活性的影响最小，这表明 eIF4G-RBD 不需要协调 eIF4F 依赖性双链体解旋。出乎意料的是，天然凝胶电泳和荧光偏振测定法揭示了 eIF4G 与 40S 亚基之间以前未识别的直接相互作用。通过结合测定，我们的数据表明，这种 40S 亚基相互作用与先前表征的 eIF4G 和 eIF3 之间的相互作用是分开的。因此，我们的工作揭示了 eIF4F 如何使用依赖 eIF3 和独立于 eIF3 的结合域结合到 40S 亚基，从而促进翻译起始。
