Functional characterization of the mRNA-binding protein ZC3HAV1 in stem cell renewal and differentiation

RNA结合蛋白结合转录本上的顺式调控元件，协调mRNA的生物发生、翻译和周转。RBPs在胚胎干细胞中的详细功能及其在分化过程中结合活性的动态变化尚未得到解决。为此，我们应用多能小鼠胚胎干细胞的mRNA相互作用组捕获和它们向神经元的分化来鉴定差异结合的限制性商业惯例。MRNA结合蛋白ZC3HAV1在分化过程中出现了一过性的mRNA结合变化。ZC3HAV主要作为一种抗病毒因子被研究，参与病毒转录本的腐烂和翻译。我们将提供三个相互独立的主要里程碑，但累积到对ZC3HAV1在干细胞自我更新和分化中的特异性和调节的系统范围的描述中，提供第一个关于ZC3HAV1在细胞mRNA靶标转录中的功能的描述。我们将使用蛋白质-RNA交联、免疫沉淀和下一代测序来绘制ZC3HAV1的mRNA交互作用组图，以确定ZC3HAV1与mRNAs结合的位置、mRNAs内的区域以及可能的靶点如何变化。我们将通过邻近生物素化和质谱学表征蛋白质相互作用伙伴和ZC3HAV1-蛋白质复合体的谱系。为了表征ZC3HAV1功能的全球分子影响，我们将耗尽和过度表达RNA结合蛋白，并测定mRNA半衰期和核糖体占有率的变化。这三种检测方法提供了假定的目标转录本在其mRNA稳定性和翻译方面可能经历的变化的概述。此外，我们还将研究ZC3HAV1在细胞分化过程中的作用。

RNA-binding proteins bind cis-regulatory elements on transcripts to orchestrate mRNA biogenesis, translation and turnover. The detailed functions of RBPs in embryonic stem cells and their dynamic changes in binding activity during differentiation have yet to be addressed. To this end, we applied mRNA interactome capture of pluripotent mouse embryonic stem cells and their differentiation to neurons to identify differentially binding RBPs. The mRNA binding protein ZC3HAV1 showed transient changes in mRNA-binding during the course of differentiation. ZC3HAV has been mostly studied as an antiviral factor involved in decay and translation of viral transcripts. We will deliver three major milestones that are independent from each other, but cumulate into a system-wide description of ZC3HAV1 specificity and regulation in stem cell self-renewal and differentiation, providing the first description of ZC3HAV1 function on cellular mRNA target transcripts. We will map the mRNA interactome of ZC3HAV1 using protein-RNA crosslinking, immunoprecipitation, and next-generation sequencing to identify mRNAs where ZC3HAV1 binds, the region within the mRNA, and how the repertoire of putative targets changes. We will characterize the repertoire of protein interaction partners and ZC3HAV1-protein complexes through proximity biotinylation followed by mass spectrometry. To characterize the global molecular impact of ZC3HAV1 function, we will deplete and overexpress RNA-binding protein 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. In addition, we will examine the role of ZC3HAV1 during cell differentiation.