Translational regulation of cellular morphogenesis in early Drosophila embryos

描述（由申请人提供）：本研究的长期目标是阐明果蝇胚胎卵裂期细胞形态发生的mRNA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，并经历基因表达的母系到合子的转变（MZT）。尽管这一进化上保守的发育阶段具有根本的重要性，但在MZT期间特异性控制细胞形态发生的mRNA翻译调节机制尚不清楚。我们发现果蝇脆性X智力发育迟滞蛋白（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，低FMRP活性导致脆性X综合征（FXS），这是遗传性智力迟钝和自闭症最常见的形式。FMRP翻译调控的确切机制尚不清楚。我们已经证明，编码第二种翻译调节因子的尾链结（tral） mRNA的表达是dFMRP依赖性调控的直接靶标，并且dFMRP和tral在动态细胞质核糖核蛋白（RNP）体（MZT体）的MZT过程中起作用。阐明这些MZT小体的功能特性和鉴定它们所调节的mrna将大大提高我们对卵裂期胚胎细胞形态发生控制的分子机制的理解。以果蝇为模型，我们将对控制细胞形态发生的dfmrp依赖的翻译调控机制进行全面分析。我们将使用蛋白质组学筛选来鉴定dfmr1和试验突变胚胎中错误表达的蛋白质，并使用rna结合试验来确定哪些相应的mrna在体内是dFMRP的潜在直接靶标。遗传分析将确定哪些已鉴定的蛋白质对细胞形态发生至关重要。有了这些发现，我们将建立一个控制细胞形态发生的翻译调控途径。我们还将确定MZT机构的功能。MZT小体的蛋白质成分将通过已知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调节MZT体内特定mrna表达的机制，我们将使用体外翻译和多核糖体关联试验以及高分辨率荧光原位杂交相结合的方法来定位直接与dFMRP结合的mrna。这些机制研究将通过对新发现的dfmrp相关蛋白的生化和遗传特性进行补充，该蛋白与卵裂期胚胎的翻译起始和细胞分裂周期控制有关。我们的研究将为控制动物早期发育的分子机制和FXS的病因提供有价值的见解，从而为FXS提供新的治疗方法。公共卫生相关性：低FMR蛋白活性导致脆性X综合征（FXS），这是人类最常见的遗传性智力迟钝和自闭症形式。我们已经发现，果蝇（果蝇）中这种蛋白质的对应物在所有动物的进化保守的胚胎发育的早期阶段是必需的。我们的实验计划旨在阐明果蝇FMR蛋白功能的机制，为FXS的病因提供有意义的见解，可能导致儿童FXS的新治疗方法，并促进我们对控制动物早期发育的分子机制的基本认识。

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.