Unravelling structural and functional communications between the Dhh1/DDX6 helicase and the Ccr4-Not deadenylase complex

转录后基因表达调控的一个关键方面是信使核糖核酸（mRNA）的协调破坏。真核细胞中存在两种主要的mRNA降解途径，具有3 ′-5 ′或5 ′-3 ′方向性。两者都是由专门的酶（deadenylases）缩短3 ′聚腺苷酸尾（poly（A）tail）开始的。细胞中主要的去腺苷化复合物是多组分Ccr 4-Not复合物。Ccr 4-Not包含两个去腺苷化酶，Caf 1和Ccr 4，它们彼此相互作用并形成不同的去腺苷化酶模块。Dhh 1/DDX 6 RNA解旋酶是一种已知的mRNA降解因子，是Ccr 4-Not的相互作用因子，Ccr 4-Not与去腺苷酸酶模块紧密结合。然而，没有研究Dhh 1/DDX 6对Ccr 4-Not的去腺苷化的影响。此外，Dhh 1/DDX 6与去腺苷酶模块的相互作用的结构信息是不存在的。引人注目的是，mRNA降解和翻译延长是密切相关的事件。然而，关于翻译延伸动力学如何与5 '-3' mRNA衰变机制传递的分子细节目前还很少。有趣的是，Dhh 1/DDX 6直接与核糖体结合，Dhh 1/DDX 6和Caf 1都被证明可以区分次优转录物和最优转录物，直接将它们与翻译动力学联系起来。在该项目的一部分中，我将研究Dhh 1/DDX 6对Ccr 4-Not去腺苷化的影响。为此，我将首先映射Dhh 1/DDX 6和Ccr 4-Not的相互作用网络，特别是与去腺苷酶模块，重组表达的Ccr 4-Not组件和Dhh 1/DDX 6变体。基于这些结果，我将阐明Dhh 1/DDX 6与Ccr 4-Not deadenylase模块复合的结构。此外，我将描述Dhh 1/DDX 6和Ccr 4-Not的功能相互作用，并测试对去腺苷化的影响。这项工作的功能方面，包括在体外脱腺苷酸测定重组纯化的蛋白质和在酵母中的体内研究。在我的提案的第二部分，我将从结构上研究5 ′-3 ′ mRNA衰变途径通过与Dhh 1/DDX 6的直接相互作用与核糖体的通讯。结构研究将包括冷冻电子显微镜、大分子晶体学和各种质谱法的组合。从这项研究的结果将推进主要去腺苷酶复合物，Ccr 4-不和Dhh 1/DDX 6解旋酶的转录后基因表达调控的基本理解。此外，这项工作将揭示翻译和5 ′-3 ′ mRNA衰变的复杂耦合机制。

A key aspect of post-transcriptional gene expression regulation is the coordinated destruction of messenger ribonucleic acids (mRNAs). Two major mRNA decay pathways exist in the eukaryotic cell, with either a 3´-5´, or a 5´-3´ directionality. Both start with the shortening of the 3´ polyadenylate tail (poly(A) tail) by specialized enzymes (deadenylases). The major deadenylation complex in the cell is the multi-component Ccr4-Not complex. Ccr4-Not contains two deadenylases, Caf1 and Ccr4, which interact with each other and form a distinct deadenylase module. The Dhh1/DDX6 RNA helicase, which is a known mRNA decay factor, is an interactor of Ccr4-Not, which binds in close proximity to the deadenylase module. The effect of Dhh1/DDX6 on deadenylation by Ccr4-Not is, however, not investigated. In addition, structural information on the interaction of Dhh1/DDX6 with the deadenylase module is absent. Strikingly, mRNA decay and translation elongation are intimately coupled events. Molecular details, of how translation elongation dynamics are communicated to the 5´-3´ mRNA decay machinery, are, however, presently sparse. Interestingly, Dhh1/DDX6 binds directly to the ribosome and both, Dhh1/DDX6 and Caf1, have been shown to distinguish suboptimal transcripts from optimal ones, directly linking them to translational dynamics. In one part of the proposed project, I will investigate the effect of Dhh1/DDX6 on deadenylation by Ccr4-Not. To this end, I will initially map the interaction network of Dhh1/DDX6 and Ccr4-Not, in particular with the deadenylase module, with recombinantly expressed Ccr4-Not components and Dhh1/DDX6 variants. Based on these results, I will elucidate the structure of Dhh1/DDX6 in complex with the Ccr4-Not deadenylase module. Furthermore, I will delineate the functional interplay of Dhh1/DDX6 and Ccr4-Not and test the effect on deadenylation. Functional aspects of this work include in vitro deadenylation assays with recombinantly purified proteins and in vivo studies in yeast. In the second part of my proposal, I will investigate structurally the communication of the 5´-3´ mRNA decay pathway with the ribosome through direct interactions with Dhh1/DDX6. Structural studies will comprise a combination of cryo-electron microscopy, macromolecular crystallography and various mass-spectrometry methods. Results from this study will advance the fundamental understanding of post-transcriptional gene expression regulation by the major deadenylase complex, Ccr4-Not and the Dhh1/DDX6 helicase. Furthermore, this work will shed light on the intricate coupling mechanisms of translation and 5´-3´ mRNA decay.