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. 2023 Jul;299(7):104936. doi: 10.1016/j.jbc.2023.104936. Epub 2023 Jun 17.
The canonical DEAD-box helicase, eukaryotic initiation factor (eIF) 4A, unwinds 5' UTR secondary structures to promote mRNA translation initiation. Growing evidence has indicated that other helicases, such as DHX29 and DDX3/ded1p, also function to promote the scanning of the 40S subunit on highly structured mRNAs. It is unknown how the relative contributions of eIF4A and other helicases regulate duplex unwinding on an mRNA to promote initiation. Here, we have adapted a real-time fluorescent duplex unwinding assay to monitor helicase activity precisely in the 5' UTR of a reporter mRNA that can be translated in a cell-free extract in parallel. We monitored the rate of 5' UTR-dependent duplex unwinding in the absence or presence of an eIF4A inhibitor (hippuristanol), a dominant negative eIF4A (eIF4A-R362Q), or a mutant eIF4E (eIF4E-W73L) that can bind the mG cap but not eIF4G. Our experiments reveal that the duplex unwinding activity in the cell-free extract is roughly evenly split between eIF4A-dependent and eIF4A-independent mechanisms. Importantly, we show that the robust eIF4A-independent duplex unwinding is not sufficient for translation. We also show that the mG cap structure, and not the poly(A) tail, is the primary mRNA modification responsible for promoting duplex unwinding in our cell-free extract system. Overall, the fluorescent duplex unwinding assay provides a precise method to investigate how eIF4A-dependent and eIF4A-independent helicase activity regulates translation initiation in cell-free extracts. We anticipate that potential small molecule inhibitors could be tested for helicase inhibition using this duplex unwinding assay.
真核起始因子（eIF）4A 是一种典型的 DEAD -box 解旋酶，它能解开 5'UTR 二级结构，促进 mRNA 翻译起始。越来越多的证据表明，其他解旋酶，如 DHX29 和 DDX3/ded1p，也能促进 40S 亚基在高度结构化的 mRNA 上的扫描。目前尚不清楚 eIF4A 和其他解旋酶如何调节 mRNA 上双链的解旋，以促进起始。在这里，我们通过实时荧光双链解旋测定法来监测报告 mRNA 5'UTR 中的解旋酶活性，该报告 mRNA 可以在无细胞提取物中平行翻译。我们在不存在或存在 eIF4A 抑制剂（hippuristanol）、显性负性 eIF4A（eIF4A-R362Q）或不能结合 mG 帽但能结合 eIF4G 的突变型 eIF4E（eIF4E-W73L）的情况下，监测 5'UTR 依赖性双链解旋的速率。我们的实验表明，无细胞提取物中的双链解旋活性大致均匀地分为 eIF4A 依赖性和 eIF4A 非依赖性机制。重要的是，我们表明，在我们的无细胞提取物系统中，强大的 eIF4A 非依赖性双链解旋不足以促进翻译。我们还表明，mG 帽结构，而不是 poly(A) 尾，是促进无细胞提取物中双链解旋的主要 mRNA 修饰。总的来说，荧光双链解旋测定法为研究 eIF4A 依赖性和 eIF4A 非依赖性解旋酶活性如何调节无细胞提取物中的翻译起始提供了一种精确的方法。我们预计可以使用这种双链解旋测定法来测试潜在的小分子抑制剂对解旋酶的抑制作用。
