German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany; CellNetworks - Cluster of Excellence, 69120 Heidelberg University, Germany; Heidelberg University, 69120 Heidelberg, Germany; Heidelberg Biosciences International Graduate School (HBIGS), 69120 Heidelberg, Germany; National Center for Tumor Diseases (NCT) partner site, 69120 Heidelberg, Germany.
Center for Molecular Biology of Heidelberg University (ZMBH) and German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.
Mol Cell. 2020 Aug 20;79(4):561-574.e5. doi: 10.1016/j.molcel.2020.06.005. Epub 2020 Jun 25.
Translation regulation occurs largely during the initiation phase. Here, we develop selective 40S footprinting to visualize initiating 40S ribosomes on endogenous mRNAs in vivo. This reveals the positions on mRNAs where initiation factors join the ribosome to act and where they leave. We discover that in most human cells, most scanning ribosomes remain attached to the 5' cap. Consequently, only one ribosome scans a 5' UTR at a time, and 5' UTR length affects translation efficiency. We discover that eukaryotic initiation factor 3B (eIF3B,) eIF4G1, and eIF4E remain bound to 80S ribosomes as they begin translating, with a decay half-length of ∼12 codons. Hence, ribosomes retain these initiation factors while translating short upstream open reading frames (uORFs), providing an explanation for how ribosomes can reinitiate translation after uORFs in humans. This method will be of use for studying translation initiation mechanisms in vivo.
翻译调控主要发生在起始阶段。在这里，我们开发了选择性的 40S 足迹法，以可视化体内内源性 mRNA 上起始的 40S 核糖体。这揭示了起始因子与核糖体结合并离开的 mRNA 位置。我们发现，在大多数人类细胞中，大多数扫描核糖体仍然附着在 5'帽上。因此，一次只有一个核糖体扫描 5'UTR，并且 5'UTR 长度会影响翻译效率。我们发现，真核起始因子 3B（eIF3B）、eIF4G1 和 eIF4E 在开始翻译时仍然与 80S 核糖体结合，半衰期约为 12 个密码子。因此，核糖体在翻译短的上游开放阅读框（uORF）时保留这些起始因子，为核糖体如何在人类中翻译 uORF 后重新起始翻译提供了解释。这种方法将有助于研究体内翻译起始机制。
