Understanding the molecular interactions and mechanisms of the RNA helicase DDX6 in posttranscriptional regulation

RNA命运的决定是基因表达调控的核心，影响着mrna的稳定性、定位和翻译效率。在这里，我们提出表征RNA解旋酶DDX6及其在转录后调控中的作用。已知DDX6通过miRNA途径参与mRNA在不同蛋白-mRNA颗粒中的定位和储存，mRNA降解酶的募集以及翻译抑制。尽管DDX6具有重要意义，但其rna靶标特异性和详细的分子功能仍然难以捉摸。我们将提供三个相互独立的主要里程碑，但将以人类HEK293细胞为例，提供DXX6s特异性和调控的全系统描述。我们将使用蛋白质- rna交联、免疫沉淀和下一代测序来绘制DDX6和突变变体的mRNA相互作用组，以确定DDX6结合的mRNA， mRNA内的区域，以及假设靶点的库如何变化。我们将通过接近生物素化和质谱分析来描述蛋白质相互作用伙伴和ddx6蛋白复合物的特征。为了描述DDX6功能的整体分子影响，我们将耗尽解旋酶并确定mRNA半衰期和核糖体占用的变化。这三种分析提供了假设的目标转录本可能经历的mRNA稳定性和翻译变化的概述。这些拟议的研究之前都没有以全面的、全系统的方式进行过，它们的结合将提供关于DDX6功能和分子靶点变化的协同信息，这取决于它与其他假定的调节剂的相互作用。由于rna结合蛋白的系统研究仍在进行中，我们将为基因表达转录后调控中的关键角色提供重要的见解。

RNA fate determination is at the center of gene expression regulation, influencing the stability, localization, and translation efficiency of mRNAs. Here, we propose to characterize the RNA helicase DDX6 and its role in posttranscriptional regulation. DDX6 is known to be involved in mRNA localization and storage in different protein-mRNA granules, recruitment of mRNA degradation enzymes, and translation repression through the miRNA pathway. Despite its importance the RNA-target specificity and the detailed molecular functions of DDX6 remain elusive. We will deliver three major milestones that are independent from each other, but feed into a system-wide description of DXX6s specificity and regulation at the example of human HEK293 cells. We will map the mRNA interactome of DDX6 and mutant variants using protein-RNA crosslinking, immunoprecipitation, and next-generation sequencing to identify mRNAs where DDX6 binds, the region within the mRNA, and how the repertoire of putative targets changes. We will characterize the repertoire of protein interaction partners and DDX6-protein complexes through proximity biotinylation followed by mass spectrometry. To characterize the global molecular impact of DDX6 function, we will deplete the helicase and determine changes in mRNA half-lives and ribosome occupancy. These three assays provide an overview of the changes that putative target transcripts may undergo with respect to their mRNA stability and translation. None of these proposed studies has been performed before in a comprehensive, system-wide manner, and their combination will provide synergistic information on changes in the functions and molecular targets of DDX6, depending on its interactions with other putative regulators. As RNA-binding proteins are still systematically understudied, we will provide important insights of the roles of a key player in post-transcriptional regulation of gene expression.