RNA Localization in Xenopus Ooctytes

描述(申请人提供)：在卵子中建立极性可以被视为胚胎模式的最早步骤。因此，早期卵裂细胞之间细胞命运的差异是受精前卵细胞质中信息分子不对称分布的结果，这种极性的基础可以由以mRNA形式存在的定位的母系决定因素提供。虽然有证据表明在许多系统中存在局部化决定因素，但局部化过程直到现在才被揭开。在脊椎动物中，Vg1信使核糖核酸是一个被认为在模式形成中起作用的局部信使核糖核酸的突出例子。Vg1基因编码一种类似生长因子的分子，并在卵子发生过程中定位于非洲爪哇卵母细胞的营养细胞质。Vg1蛋白在卵子植物半球的限制表达似乎对胚胎的正确模式至关重要，使Vg1 mRNA的定位成为理解母体分子如何定位以影响模式和极性的重要模型。这个研究项目的目标是研究RNA分子如何定位于细胞质的特定区域，以产生空间受限的基因表达。我们最近对反式作用的定位因子和Vg1mRNA中的基本靶向序列之间的相互作用的研究为这一研究奠定了基础。我们已经确定并表征了定位机制的关键组件，并定义了RNA靶向信号中的共识序列元素。此外，我们还发现定位途径起始于细胞核，并在定位途径中发现了明显的核步骤和细胞质步骤。我们现在试图扩展这些发现，以阐明Vg1 RNA定位表达的分子机制，具体目的如下：i)分析导致细胞质RNA定位的核事件。Ii)表征细胞质RNP复合体的组装和运输。3)探讨mRNA定位与其他转录后控制点的协调。这项拟议的研究旨在提供对脊椎动物胚胎中发育信号如何在空间上分布的机械性见解。

DESCRIPTION (provided by applicant): Establishment of polarity in the egg can be viewed as the earliest step in embryonic patterning. Thus, differences in cell fate among the early cleavage cells are a consequence of asymmetric distributions of informational molecules in the egg cytoplasm before fertilization, the basis for such polarity can be provided by localized maternal determinants in the form of mRNA. While there is evidence for the existence of localized determinants in many systems, the localization process is only now being unraveled. Among vertebrates, Vg1 mRNA is a prominent example of a localized mRNA that is thought to play a role in patterning. Vg1 mRNA encodes a growth factor-like molecule, and is localized during oogenesis to the vegetal cytoplasm of Xenopus oocytes. Restricted expression of Vgl protein in the vegetal hemisphere of the egg appears to be critical for correct patterning of the embryo, making Iocalization of Vg1 mRNA an important model for understanding how maternal molecules are localized to influence pattern and polarity. The goal of this research project is to investigate how RNA molecules can be targeted to specific regions of the cell cytoplasm to generate spatially restricted gene expression. The foundation for this investigation has been laid by our recent work studying the interactions between trans-acting localization factors and essential targeting sequences within Vg1 mRNA. We have identified and characterized key components of the localization machinery and defined consensus sequence elements within an RNA targeting signal. Moreover, we have discovered that the localization pathway initiates in the nucleus and we have uncovered distinct nuclear and cytoplasmic steps in the localization pathway. We now seek to extend these findings to elucidate the molecular mechanisms responsible for localized expression of Vg1 RNA with the following specific aims: I) To analyze nuclear events leading to cytoplasmic RNA localization. II) To characterize assembly and transport of the cytoplasmic RNP complex. III) To probe coordination of mRNA localization with other post-transcriptional control points. The proposed research is designed to provide mechanistic insight into how developmental signals are spatially distributed in the vertebrate embryo.