Correlating eIF4A conformational dynamics with eIF4A-, eIF4B-, and eIF4G-dependence and translation efficiencies of yeast mRNAs

将 eIF4A 构象动力学与酵母 mRNA 的 eIF4A、eIF4B 和 eIF4G 依赖性以及翻译效率相关联

• 批准号: 537881349
• 负责人: Professorin Dr. Dagmar Klostermeier
• 金额: --
• 依托单位: Institut für Physikalische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/537881349?language=en
• 关键词: Correlating; eIF4A; conformational; dynamics; eIF4B

Translation of eukaryotic mRNAs starts with the recognition of the 5’-cap by eIF4F, a complex of the cap-binding protein eIF4E, the scaffold protein eIF4G, and the DEAD-box helicase eIF4A. eIF4A unwinds secondary structures in the 5’-untranslated region (5’-UTR) during recruitment of the pre-initiation complex (PIC) and scanning of the PIC towards the start codon. Its activity is coupled to a conformational cycle, which is stimulated by eIF4B and 4G, as well as the 5’-UTR itself, making eIF4A a regulatory hub in translation initiation. Transcriptome-wide studies identified sets of yeast mRNAs with hyper-dependence on eIF4A, 4B, or 4G, but the molecular basis for the different factor dependence is unclear. We have generated a series of yeast strains that enable us to prepare cell-free extracts lacking translation initiation factors, either individually or in combination. By supplementing these extracts with recombinant factors, we can now perform single-molecule FRET experiments to interrogate eIF4A conformational dynamics and in vitro translation assays to determine translation efficiencies under identical conditions, and over a wide range of concentrations of these factors. Building on this resource, we will test the following hypotheses: In single-molecule experiments and in vitro translation assays using luciferase reporters with 5’-UTRs of mRNAs showing different levels of eIF4A-dependence in vivo, we will test whether the capacity of 5’-UTRs to stimulate eIF4A conformational changes correlates with eIF4A-dependence. Using reporters with 5’-UTRs of mRNAs with different levels of eIF4B- and 4G-dependence, we will map the effects of these factors on eIF4A conformational dynamics and on translation efficiencies. We will also dissect the contributions of individual domains of eIF4B and 4G to eIF4A-dependent and -independent effects. These experiments may provide insights into reprogramming mechanisms of translation in cancer, and may define a minimal system of factors for synthetic biology approaches. Finally, we will test the hypothesis that factor dependence is encoded in the 5’-UTR. Using luciferase reporters fused to 5’-UTRs of selected eIF4A-, 4B- and 4G-dependent mRNAs, we will (1) delete individual elements in these 5’-UTRs and test for loss of factor dependence, and (2) insert these elements into the 5’-UTR of a factor-independent mRNA and test for gain of factor dependence. These studies will identify the structural features that make an mRNA eIF4A-, 4B-, or 4G-dependent, and will demonstrate whether these elements are sufficient to confer factor dependence. Collectively, these studies will define the link between eIF4A conformational dynamics and translation efficiencies, will delineate eIF4A-dependent and -independent effects of eIF4B and 4G, and will uncover the 5’-UTR elements that make an mRNA 4A-, 4B-, or 4G-dependent. The results will provide important insight into the regulation of translation and its dysregulation in disease.

真核生物mRNA的翻译始于eIF 4F对5 '-帽的识别，eIF 4F是帽结合蛋白eIF 4 E、支架蛋白eIF 4G和DEAD盒解旋酶eIF 4A的复合物。eIF 4A在前起始复合物（PIC）的募集和PIC向起始密码子的扫描期间解旋5 '-非翻译区（5'-UTR）中的二级结构。其活性与构象循环偶联，该构象循环受到eIF 4 B和4G以及5 '-UTR本身的刺激，使得eIF 4A成为翻译起始中的调控中心。转录组范围的研究确定了对eIF 4A、4 B或4G具有高度依赖性的酵母mRNA组，但不同因子依赖性的分子基础尚不清楚。我们已经产生了一系列的酵母菌株，使我们能够制备无细胞提取物缺乏翻译起始因子，无论是单独或组合。通过补充这些提取物与重组因子，我们现在可以进行单分子FRET实验来询问eIF 4A构象动力学和体外翻译测定，以确定在相同条件下的翻译效率，并在这些因素的浓度范围很广。在此基础上，我们将测试以下假设：在单分子实验和体外翻译试验中，使用荧光素酶报告基因与体内显示不同水平eIF 4A依赖性的mRNA的5 '-UTR，我们将测试5'-UTR刺激eIF 4A构象变化的能力是否与eIF 4A依赖性相关。使用具有不同水平eIF 4 B和4G依赖性的mRNA的5 '-UTR的报告基因，我们将绘制这些因素对eIF 4A构象动力学和翻译效率的影响。我们还将剖析eIF 4 B和4G的各个结构域对eIF 4A依赖性和非依赖性效应的贡献。这些实验可能为癌症翻译的重编程机制提供见解，并可能为合成生物学方法定义最小的因子系统。最后，我们将检验因子依赖性在5 '-UTR中编码的假设。使用与选定的eIF 4A-、4 B-和4G-依赖性mRNA的5 '-UTR融合的荧光素酶报告基因，我们将（1）删除这些5'-UTR中的单个元件并测试因子依赖性的丧失，以及（2）将这些元件插入因子非依赖性mRNA的5 '-UTR中并测试因子依赖性的获得。这些研究将确定使mRNA eIF 4A-，4 B-或4G依赖的结构特征，并将证明这些元素是否足以赋予因子依赖性。总的来说，这些研究将确定eIF 4A构象动力学和翻译效率之间的联系，将描绘eIF 4 B和4G的eIF 4A依赖性和非依赖性作用，并将揭示使mRNA 4A-，4 B-或4G依赖性的5 '-UTR元件。这些结果将为理解翻译的调节及其在疾病中的失调提供重要的见解。