RNA Localization in Xenopus Ooctytes

描述（由申请人提供）：在卵子中建立极性可以被视为胚胎模式的最早步骤。因此，卵裂早期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母体mRNA形式的局部决定因素提供。虽然有证据表明在许多系统中存在局部决定因素，但局部化过程直到现在才被解开。在脊椎动物中，Vg1 mRNA是定位mRNA的一个突出例子，被认为在模式中发挥作用。Vg1 mRNA编码一种生长因子样分子，并在卵发生过程中定位于爪蟾卵母细胞的植物细胞质中。卵细胞植物半球中Vg1蛋白的受限表达似乎对胚胎的正确模式至关重要，这使得Vg1 mRNA的本地化成为理解母体分子如何本地化以影响模式和极性的重要模型。本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性基因表达。这项研究的基础是我们最近研究Vg1 mRNA中反式作用定位因子与基本靶向序列之间的相互作用。我们已经确定并表征了定位机制的关键组成部分，并在RNA靶向信号中定义了共识序列元素。此外，我们已经发现定位途径始于细胞核，并且我们已经发现了定位途径中不同的核和细胞质步骤。我们现在寻求扩展这些发现，以阐明负责Vg1 RNA定位表达的分子机制，具体目的如下：1)分析导致细胞质RNA定位的核事件。II)表征胞质RNP复合物的组装和运输。III)探讨mRNA定位与其他转录后控制点的协调性。提出的研究旨在提供机制的见解如何发育信号是空间分布在脊椎动物胚胎。

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.