RNA Localization in Xenopus Oocytes

描述(由申请人提供)：细胞质RNA定位是产生胚胎和体细胞极性的一种广泛的机制。在脊椎动物中，Vg1 mRNA是一个在胚胎模式中发挥作用的局部mRNA的突出例子。Vg1基因编码一种多肽生长因子，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分子如何通过空间受限的蛋白质表达来靶向细胞质的特定区域来产生细胞极性。我们对支配这一过程的分子相互作用的研究进展为这一研究奠定了基础。我们的实验定义了关键的RNA-蛋白质和蛋白质-蛋白质相互作用，导致组装一个具有运输能力的RNP复合体，并发现了定位途径中的不同步骤。我们目前的建议试图通过研究本地化途径中这些步骤之间的动态转换来扩展这些发现。我们将使用体内和体外相结合的方法来确定Vg1 mRNA局部表达的分子机制。在目标I中，我们将表征细胞质Vg1 RNP复合体的动力学和运输，在目标II中，我们将分析RNP运输颗粒的组装和活性，在目标III中，我们将研究翻译抑制和RNA定位的耦合机制。拟议的研究旨在揭示mRNA分子在细胞内运输以产生空间受限的蛋白质表达的机制，并将为了解发育信号在脊椎动物胚胎中的空间分布提供洞察力。这项工作将影响与人类健康相关的问题，如出生缺陷，以及与RNA运输缺陷和局部蛋白质合成有关的神经疾病。公共卫生相关性本研究项目的目标是阐明负责RNA定位和蛋白质合成定位的分子机制。这些过程对胚胎发育和神经功能都是至关重要的。特别是，某些人类疾病，包括脆性X智力低下综合征和脊髓性肌萎缩症，被认为与RNA定位和定位蛋白质合成缺陷有关。

DESCRIPTION (provided by applicant): Cytoplasmic RNA localization is a widespread mechanism for generating both embryonic and somatic cell polarity. Among vertebrates, Vg1 mRNA is a prominent example of a localized mRNA that plays a role in embryonic patterning. Vg1 mRNA encodes a peptide growth factor, and is localized during oogenesis to the vegetal cytoplasm of Xenopus oocytes. Restricted expression of Vg1 protein in the vegetal hemisphere of the egg is critical for correct patterning of the embryo, making localization 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 cell polarity through spatially restricted protein expression. The foundation for this investigation has been laid by our progress studying the molecular interactions that govern this process. Our experiments have defined critical RNA-protein and protein-protein interactions leading to assembly of a transport-competent RNP complex and have uncovered distinct steps in the localization pathway. Our current proposal seeks to extend these findings by studying the dynamic transitions between these steps in the localization pathway. We will use a combination of in vivo and in vitro approaches to determine the molecular mechanisms responsible for localized expression of Vg1 mRNA. Three specific aims are proposed: In Aim I, we will characterize the dynamics and transport of the cytoplasmic Vg1 RNP complex, in Aim II, we will analyze the assembly and activity of RNP transport granules and in Aim III we will investigate the mechanisms that couple translational repression and RNA localization The proposed research is designed to reveal the mechanisms by which mRNA molecules are transported within cells to generate spatially restricted protein expression, and will provide insight into how developmental signals are spatially distributed in the vertebrate embryo. This work will impact issues related to human health such as birth defects, as well as neurological diseases that have been linked to defective RNA transport and localized protein synthesis. PUBLIC HEALTH RELEVANCE The goal of this research project is to elucidate the molecular mechanisms responsible for RNA localization and localized protein synthesis. These processes are critical for both embryonic development and neurological function. In particular, certain human diseases, including fragile X mental retardation syndrome and spinal muscular atrophy, have been linked to defects in RNA localization and localized protein synthesis.