Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
J Biol Chem. 2023 Feb;299(2):102864. doi: 10.1016/j.jbc.2022.102864. Epub 2022 Dec 31.
In response to environmental stresses, cells invoke translational control to conserve resources and rapidly reprogram gene expression for optimal adaptation. A central mechanism for translational control involves phosphorylation of the α subunit of eIF2 (p-eIF2α), which reduces delivery of initiator tRNA to ribosomes. Because p-eIF2α is invoked by multiple protein kinases, each responding to distinct stresses, this pathway is named the integrated stress response (ISR). While p-eIF2α lowers bulk translation initiation, many stress-related mRNAs are preferentially translated. The process by which ribosomes delineate gene transcripts for preferential translation is known to involve upstream open reading frames (uORFs) embedded in the targeted mRNAs. In this study, we used polysome analyses and reporter assays to address the mechanisms directing preferential translation of human IBTKα in the ISR. The IBTKα mRNA encodes four uORFs, with only 5'-proximal uORF1 and uORF2 being translated. Of importance, the 5'-leader of IBTKα mRNA also contains a phylogenetically conserved stem-loop of moderate stability that is situated 11 nucleotides downstream of uORF2. The uORF2 is well translated and functions in combination with the stem-loop to effectively lower translation reinitiation at the IBTKα coding sequence. Upon stress-induced p-eIF2α, the uORF2/stem loop element can be bypassed to enhance IBTKα translation by a mechanism that may involve the modestly translated uORF1. Our study demonstrates that uORFs in conjunction with RNA secondary structures can be critical elements that serve as the "bar code" by which scanning ribosomes can delineate which mRNAs are preferentially translated in the ISR.
在应对环境压力时，细胞会调用翻译控制来节约资源，并快速重新编程基因表达以实现最佳适应。翻译控制的一个核心机制涉及 eIF2 的 α 亚基（p-eIF2α）的磷酸化，这会减少起始 tRNA 向核糖体的传递。由于 p-eIF2α 是由多种蛋白激酶引发的，每种激酶对应不同的应激，因此该途径被命名为综合应激反应（ISR）。虽然 p-eIF2α 降低了总体翻译起始，但许多与应激相关的 mRNA 被优先翻译。核糖体区分优先翻译的基因转录本的过程，已知涉及靶向 mRNA 中嵌入的上游开放阅读框（uORFs）。在这项研究中，我们使用多核糖体分析和报告基因测定来解决在 ISR 中指导人类 IBTKα 优先翻译的机制。IBTKα mRNA 编码四个 uORFs，只有 5'-近端 uORF1 和 uORF2 被翻译。重要的是，IBTKα mRNA 的 5'-前导区还包含一个在 uORF2 下游 11 个核苷酸处具有中等稳定性的系统发育保守茎环结构。uORF2 被很好地翻译，并与茎环一起有效地降低 IBTKα 编码序列的翻译重新起始。在应激诱导的 p-eIF2α 后，uORF2/茎环元件可以被绕过，通过一种可能涉及适度翻译的 uORF1 的机制，增强 IBTKα 翻译。我们的研究表明，uORFs 与 RNA 二级结构可以是关键元素，充当扫描核糖体可以区分 ISR 中哪些 mRNA 被优先翻译的“条码”。
