Molecular mechanisms and functional implications of Ded1p phaseseparation.

Ded1p （DDX3）是一种RNA DEAD盒解旋酶，可去除mRNA 5 ' UTR（非翻译区）中的二级结构元素。这有助于扫描40S核糖体亚基，从而可以识别翻译起始密码子。在细胞应激时，dep1p被招募到含有mrna（信使核糖核蛋白）的细胞质应激颗粒中，这些mrna（信使核糖核蛋白）被翻译停滞。在功能上，应激颗粒的形成保护嵌入的mrna免受降解，并诱导基因表达的变化，从而上调应激反应。在这里，我们讨论了相互作用背后的分子细节，这些相互作用是导致dep1p凝结成应力颗粒的原因。此外，我们还讨论了这种缩合过程如何调节Ded1p活性。实验上，我们将使用生化方法来确定哪些Ded1p氨基酸有助于酶的LLPS（液-液相分离）倾向。基于生物物理研究，特别是核磁共振波谱，我们将揭示在凝结时发生的瞬时分子间接触以及这些相互作用如何影响蛋白质的动力学。这些数据将与计算方法相结合，以预测和可视化Ded1p的LLPS行为。为了解决LLPS如何调节Ded1p解旋酶活性，我们将确定显示冷凝倾向改变的Ded1p变体的周转率。基于大量的这些突变蛋白，我们的目标是建立活性和相分离之间的直接关联。这些结构和催化研究将辅以（带帽）RNA和mRNA帽结合复合物组分存在的实验。总之，我们的数据将有助于理解压力颗粒形成如何调节Ded1p活性。我们对dep1p缩合、相互作用和活性的体外研究将与我们在体内的研究结果相辅相成。总之，我们的工作将提供对导致Ded1p LLPS的原子细节的机械见解，并揭示LLPS如何影响催化活性和细胞功能。此外，我们的结果将增强对LLPS分子基础的一般理解。

Ded1p (DDX3) is an RNA DEAD box helicase that removes secondary structure elements in the 5’ UTR (untranslated region) of an mRNA. This facilitates the scanning of the 40S ribosomal subunit such that the translation initiation codon can be identified. Upon cellular stress, Ded1p is recruited to cytoplasmic stress granules that contain mRNPs (messenger ribonucleoproteins) that are stalled in translation. Functionally, stress granule formation protects the embedded mRNAs against degradation and induces changes in gene expression that up-regulate stress response. Here, we address the molecular details behind the interactions that are responsible for Ded1p condensation into stress granules. In addition, we address how Ded1p activity is modulated by this condensation process. Experimentally, we will make use of biochemical methods to determine which Ded1p amino-acids contribute to the LLPS (liquid-liquid phase separation) propensity of the enzyme. Based on biophysical studies, in particular NMR spectroscopy, we will reveal which transient intermolecular contacts take place upon condensation and how the dynamics of the protein is affected by these interactions. These data will be combined with computational methods to predict and visualize the LLPS behavior of Ded1p. To address how Ded1p helicase activity is modulated by LLPS we will determine the turnover rates of Ded1p varients that show altered condensation propensities. Based on a large number of these mutated proteins we aim to establish a direct correlation between activity and phase separation. These structural and catalytic studies will be complemented with experiment in the presence of (capped) RNA and components of the mRNA cap binding complex. Together, our data will thereby contribute to the understanding of how Ded1p activity is regulated through stress granule formation. Our in vitro insights into Ded1p condensation, interactions and activity will be complemented with experiments that address the relevance of our findings in vivo.In summary, our work will provide mechanistical insights into the atomic details that result in Ded1p LLPS and reveal how LLPS effects catalytic activity and cellular function. Furthermore, our results will enhance the general understanding of the molecular basis of LLPS.