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控制卵裂期果蝇胚胎细胞形态发生的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，并在基因表达中经历母体到合子的转变（MZT）。尽管这一进化上保守的发育阶段具有根本的重要性，但对在MZT期间发挥作用以特异性控制细胞形态发生的mRNA翻译调控机制知之甚少。我们已经发现果蝇脆性X智力低下蛋白（dFMRP），一个翻译调节因子，是卵裂期胚胎正常细胞形成所必需的。在人类中，低FMRP活性会导致脆性X综合征（FXS），这是遗传性精神发育迟滞和自闭症的最常见形式。FMRP翻译调控的确切机制尚不确定。我们已经表明，尾部结（TRAL）mRNA的表达，它编码的第二个翻译调节，是一个直接的目标dFMRP依赖性的调节和dFMRP和TRAL的功能，在动态胞质核糖核蛋白（RNP）机构（MZT机构）内的MZT。阐明这些MZT机构的功能特性，并确定它们调节的mRNA将显着提高我们的理解的分子机制控制细胞形态发生在卵裂期胚胎。使用果蝇作为模型，我们将进行一个全面的分析dFMRP依赖的翻译调控机制，控制细胞形态发生。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，并使用RNA结合试验来确定哪些相应的mRNA是体内dFMRP的潜在直接靶点。遗传分析将确定哪些已鉴定的蛋白质是细胞形态发生所必需的。有了这些发现，我们将建立一个翻译调控途径，控制细胞形态发生。我们还将确定MZT机构的功能。MZT体的蛋白质组分将通过已知RNP体（例如，应力颗粒）标记物。为了确定dFMRP调节MZT体内特异性mRNA表达的机制，我们将使用体外翻译和多核糖体缔合试验以及高分辨率荧光原位杂交的组合来定位dFMRP直接结合的mRNA。这些机制的研究将补充一个新发现的dFMRP相关蛋白的生物化学和遗传特性，涉及翻译起始和细胞分裂周期控制所需的卵裂期胚胎。我们的研究将为控制早期动物发育的分子机制和FXS的病因学提供有价值的见解，这可能指向FXS的新疗法。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精神发育迟滞和自闭症形式。我们已经发现，果蝇（果蝇）对应的这种蛋白质是必需的胚胎发育的早期阶段，是进化保守的所有动物。我们阐明果蝇FMR蛋白功能机制的实验计划应该为FXS的原因提供有意义的见解，这可能导致儿童FXS的新治疗方法，并促进我们对控制早期动物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.