RNA Localization in Xenopus Ooctytes

描述（由申请人提供）：卵中极性的建立可以被视为胚胎模式化的最早步骤。因此，早期卵裂细胞中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mRNA形式的局部母体决定子提供。虽然有证据表明在许多系统中存在定域决定子，但定域过程直到现在才被揭开。在脊椎动物中，Vg1 mRNA是被认为在图案形成中起作用的局部mRNA的突出例子。Vg1 mRNA编码一种生长因子样分子，在卵发生过程中定位于非洲爪蟾卵母细胞的植物细胞质中。Vgl蛋白在卵的植物半球中的限制性表达似乎对于胚胎的正确图案化是至关重要的，使得Vgl mRNA的定位成为理解母体分子如何定位以影响图案和极性的重要模型。该研究项目的目标是研究RNA分子如何靶向细胞质的特定区域，以产生空间限制的基因表达。这项调查的基础已经奠定了我们最近的工作研究之间的相互作用的反式作用的定位因子和Vg1 mRNA内的重要靶向序列。我们已经确定并表征了定位机制的关键组成部分，并定义了RNA靶向信号内的共有序列元件。此外，我们已经发现，定位途径在细胞核中启动，我们已经发现了不同的核和细胞质的步骤中的定位途径。我们现在试图扩展这些发现，以阐明负责Vg1 RNA的局部表达的分子机制，具体目标如下：I）分析导致细胞质RNA定位的核事件。II）表征细胞质RNP复合物的组装和转运。III）探测mRNA定位与其他转录后控制点的协调。拟议的研究旨在提供发育信号在脊椎动物胚胎中空间分布的机制。

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.