Genetic and molecular analysis of germ granule components in C. elegans

描述(由申请人提供)：生殖细胞对于物种的维持具有明显的重要性，并且在几个基本方面与分化的体细胞不同。尽管它们作为细胞谱系具有重要意义，但它们到底是如何获得并保持其独特特征的尚不清楚。生殖粒是脊椎动物和无脊椎动物生殖细胞中一种保守而独特的成分。生育需要几种胚芽颗粒成分；然而，它们的生化功能尚不清楚。这一建议解决了这样的假设，即生殖粒蛋白与mRNA相互作用，调节其翻译和/或稳定性。分子、遗传和细胞生物学技术将用于线虫，线虫，以解决两个特定的目标。首先，将使用pos-1mRNA来确定RNA定位到胚芽颗粒的意义，pos-1是以前发现的一种胚芽颗粒RNA。在其他物种中，已知mRNAs的特定3‘UTR区域对于将RNA靶向生殖细胞颗粒是必要的和充分的。在线虫中，已经确定了pos-1 3‘非编码区中的两个保守元件；将使用定点突变来产生这些区域的突变。转基因株系将使用GFP报告程序与不同形式的pos-1 3‘非编码区融合。3‘非编码区突变对RNA定位到胚芽颗粒、RNA稳定性和翻译调控的影响将被检测，以确定3’非编码区元件和RNA定位如何影响RNA稳定性和翻译调控。第二个具体目标是解决卵母细胞中胚泡颗粒在卵子发生受阻时的功能。卵子发生受阻发生在年老的野生型蠕虫和几个缺乏功能精子的突变体中。在受阻的卵母细胞中，RNA和生殖粒蛋白可逆地形成巨大的聚集体。受精后，聚集体迅速解离。重要的是，如果受精，带有聚集体的受阻卵母细胞是可以存活的，因此，假设聚集体对调节RNA稳定性或翻译调节具有积极作用。实验将确定调节聚集体形成和解离的线索，并识别和表征聚集体形成缺陷的突变体。更好地了解线虫生殖细胞的功能可能适用于其他生物体的生殖细胞以及干细胞，后者与生殖细胞有几个共同的特征。这些研究还可能揭示卵母细胞用来随着时间的推移保持其完整性的保守机制，这对生育至关重要。

DESCRIPTION (provided by applicant): Germ cells are of clear importance for the maintenance of species and differ from differentiated somatic cells in several fundamental ways. Despite their significance as a cell lineage, precisely how they acquire and maintain their unique characteristics is unknown. Germ granules are a conserved and unique component of germ cells in vertebrates and invertebrates. Several germ granule components are required for fertility; however, their biochemical functions are unknown. This proposal addresses the hypothesis that germ granule proteins interact with mRNA to regulate its translation and/or stability. Molecular, genetic, and cell biology techniques will be used in the nematode, C. elegans, to address two specific aims. First, the significance of RNA localization to germ granules will be determined, using pos-1 mRNA, a previously identified germ granule RNA. In other species, specific 3' UTR regions of mRNAs are known to be necessary and sufficient to target RNA to germ granules. In C. elegans two conserved elements in the pos-1 3'UTR have been identified; site-directed mutagenesis will be used to generate mutations in these regions. Transgenic lines will be made using a GFP reporter fused to various forms of the pos-1 3' UTR. Effects of mutations in the 3' UTR on RNA localization to germ granules, RNA stability, and translational regulation will be assayed to determine how 3' UTR elements and RNA localization affect RNA stability and translational regulation. The second specific aim addresses the function of germ granules in oocytes when oogenesis is arrested. Arrested oogenesis occurs in old-aged wild-type worms and in several mutants that lack functional sperm. Giant aggregates of RNA and germ granule proteins reversibly form in arrested oocytes. Upon fertilization, the aggregates rapidly dissociate. Importantly, the arrested oocytes with aggregates are viable if fertilized and thus, the hypothesis is that aggregates function positively to modulate RNA stability or translational regulation. Experiments will determine the cues that regulate the formation and dissociation of aggregates and identify and characterize mutants defective in aggregate formation. A better understanding of how C. elegans germ cells function may be applicable to germ cells in other organisms as well as to stem cells, which share several characteristics with germ cells. These studies may also uncover conserved mechanisms that oocytes use to preserve their integrity over time and are critical for fertility.