Translational regulation of cellular morphogenesis in early Drosophila embryos

描述(申请人提供)：本研究的长期目标是阐明控制卵裂期果蝇胚胎细胞形态发生的信使核糖核酸翻译调控机制。所有动物的胚胎发生都始于卵裂阶段，此时细胞通过分裂增殖而不生长，并在基因表达上经历母体向合子的转变(MZT)。尽管这一进化保守的发育阶段具有基本的重要性，但在MZT过程中发挥作用的特定控制细胞形态发生的mRNA翻译调控机制却知之甚少。我们发现，果蝇脆性X智力低下蛋白(DFMRP)是卵裂期胚胎正常细胞形成所必需的翻译调节因子。在人类中，FMRP活性低会导致脆性X综合征(FXS)，这是最常见的遗传性智力低下和自闭症形式。FMRP翻译调控的确切机制尚不确定。我们已经证明，编码第二个翻译调节因子的拖尾连接(Tral)mRNA的表达是dFMRP依赖的调控的直接靶点，并且dFMRP和tral在动态细胞质核糖核蛋白(RNP)小体(MZT小体)的MZT过程中发挥作用。阐明这些MZT小体的功能特性并确定它们所调控的mRNAs将大大增强我们对卵裂期胚胎细胞形态发生的分子机制的理解。以果蝇为模型，我们将对控制细胞形态发生的依赖dFMRP的翻译调控机制进行全面的分析。我们将使用蛋白质组学筛选来鉴定dfmr1和tral突变胚胎中错误表达的蛋白质，并进行RNA结合分析，以确定哪些相应的mRNAs是dFMRP在体内的潜在直接靶点。遗传分析将确定哪些已识别的蛋白质对细胞形态发生至关重要。有了这些发现，我们将建立一条控制细胞形态发生的翻译调控途径。我们还将确定MZT小体的功能。MZT小体的蛋白质成分将通过已知的RNP小体(如应激颗粒)标记的免疫荧光定位来鉴定。为了确定dFMRP调控MZT小体内特定mRNAs表达的机制，我们将使用体外翻译和多聚核糖体结合分析相结合的方法，以及高分辨率荧光原位杂交来定位与dFMRP直接结合的mRNAs。这些机制研究将得到新发现的dFMRP相关蛋白的生化和遗传特征的补充，该蛋白参与翻译启动，并在卵裂阶段的胚胎中控制细胞分裂周期。我们的研究将为控制动物早期发育的分子机制和FXS的病因学提供有价值的见解，可能为FXS的新疗法指明方向。与公共卫生相关：FMR蛋白活性低会导致脆性X综合征(FXS)，这是人类最常见的遗传性智力低下和自闭症形式。我们已经发现，这种蛋白质的果蝇(果蝇)对应于胚胎发育的早期阶段是必需的，这在所有动物中都是进化保守的。我们阐明果蝇FMR蛋白功能机制的实验计划将为FXS的原因提供有意义的见解，从而为儿童FXS提供新的治疗方法，并促进我们对控制动物早期发育的分子机制的基本理解。

DESCRIPTION (provided by applicant): The long-term objective of this research is to elucidate the mRNA translational regulatory mechanisms that control cellular morphogenesis in cleavage stage Drosophila embryos. Embryogenesis in all animals begins with a cleavage stage when cells proliferate through division without growing and undergo a maternal to zygotic transition (MZT) in gene expression. Despite the fundamental importance of this evolutionarily conserved phase of development, the mRNA translational regulatory mechanisms that function during the MZT to specifically control cellular morphogenesis are poorly understood. We have found that Drosophila Fragile X mental retardation protein (dFMRP), a translational regulator, is required for normal cell formation in cleavage stage embryos. In humans, low FMRP activity causes Fragile X syndrome (FXS), the most common form of heritable mental retardation and autism. The precise mechanism of FMRP translational regulation is uncertain. We have shown that the expression of trailer hitch (tral) mRNA, which encodes a second translational regulator, is a direct target of dFMRP-dependent regulation and that dFMRP and TRAL function during the MZT within dynamic cytoplasmic ribonucleoprotein (RNP) bodies (MZT bodies). Elucidating the functional properties of these MZT bodies and identifying the mRNAs that they regulate will significantly enhance our understanding of the molecular mechanisms controlling cellular morphogenesis in cleavage stage embryos. Using Drosophila as a model, we will conduct a comprehensive analysis of the dFMRP-dependent translational regulatory mechanisms that control cellular morphogenesis. We will use proteomic screens to identify proteins misexpressed in dfmr1 and tral mutant embryos and RNA-binding assays to determine which of the corresponding mRNAs are potentially direct targets of dFMRP in vivo. Genetic analysis will determine which identified proteins are essential for cellular morphogenesis. With these findings we will establish a translational regulatory pathway that controls cellular morphogenesis. We will also determine the function of the MZT bodies. The protein components of MZT bodies will be identified through immunofluorescence localization of known RNP body (e.g., stress granule) markers. To determine the mechanism by which dFMRP regulates expression of specific mRNAs within MZT bodies we will use a combination of in vitro translation and polyribosome-association assays, and high resolution fluorescence in situ hybridization to localize mRNAs directly bound by dFMRP. These mechanistic studies will be complemented by the biochemical and genetic characterization of a newly identified dFMRP-associated protein that is implicated in translational initiation and required for cell division cycle control in cleavage stage embryos. Our study will provide valuable insights into the molecular mechanisms that control early animal development and the etiology of FXS that could point to new therapies for FXS. PUBLIC HEALTH RELEVANCE: Low FMR protein activity causes Fragile X syndrome (FXS), the most common form of heritable mental retardation and autism in humans. We have found that the Drosophila (fruit fly) counterpart of this protein is required for an early stage of embryonic development that is evolutionarily conserved in all animals. Our experimental plan to elucidate the mechanism of Drosophila FMR protein function should provide meaningful insights into the causes of FXS that could lead to new treatments for FXS in children, and advance our basic understanding of the molecular mechanisms that control early animal development.