Long noncoding RNA function in the Drosophila germ line

长链非编码RNA（lncRNA）最初是由几个突出的例子定义的。的应用 高通量基因组方法已经揭示了候选lncRNA的显著扩大的库存， 在动物基因组的普遍转录中产生。一些lncRNA与癌症有关， 许多其他的基因定位在暗示与癌症有关的基因组区域。了解lncRNA的功能 因此符合NIH的总体使命。大量预测的lncRNA（人类中超过9000种） 为充分认识它们的功能设置了一系列挑战。一个杰出的分析模型， lncRNA功能由果蝇oskar（osk）基因提供。尽管osk RNA编码蛋白质， 也有非编码功能。编码功能和非编码功能明显不同，作用于不同的 卵子发生和胚胎发生阶段，并有助于不同的过程。的lncRNA功能 osk在卵子发生过程中是必不可少的，osk RNA的缺失会导致某些 来自nuage的蛋白质，nuage是在所有动物的生殖细胞中发现的核周结构。Nuage是一个网站， 产生piRNA。piRNA的作用是限制转座子的活性，从而保护生殖系 DNA损伤值得注意的是，osk lncRNA活性的丧失导致piRNA水平急剧下降，并且 转座子mRNA水平的相应增加。本提案的目标是了解osk如何 RNA在nuage组织和piRNA产生中起作用，这些特征在所有动物中都是保守的 包括人类目的之一是完成osk突变体和基因的遗传特征 编码与osk RNA中的关键元件结合的蛋白质，该关键元件在其lncRNA功能中起作用。一个目的是 定义nuage如何受到osk RNA丢失的影响，检查单个组件以确定是否特异性 nuage蛋白的子集主要受到影响，并通过以下方法评估nuage的整体组织： 超微结构研究这一目标的一部分是探索和测试可能的解释的要求， osk RNA包含从保育细胞（具有nuage的细胞）运输到卵母细胞的信号。 另一个目标集中在与osk lncRNA功能元件结合的蛋白质上。这是建立在我们的工作， 定义这些要素，并确定与这些要素相结合的因素。目标是获得 更完整地了解所有与功能元件结合的蛋白质，了解复合物 从这些蛋白质和osk RNA组装，从而了解潜在的机制。的 最后的目的是寻找哺乳动物中与介导osk lncRNA的关键序列元件等价的基因 活动候选人将在小鼠中进行测试，以确定该特征是否在所有小鼠中发挥保守作用。 考虑到nuage、piRNA和许多nuage组分的保守性， 参与piRNA的生产。总的来说，这些研究很有可能揭示 与人类生殖系细胞直接相关的nuage功能的细节。

Long noncoding RNAs (lncRNAs) were initially defined by a few prominent examples. Application of high-throughput genomic methods has revealed a dramatically expanded inventory of candidate lncRNAs, produced in the pervasive transcription of animal genomes. Some lncRNAs are implicated in cancer, and many others map to regions of the genome suggestive of a link to cancer. Learning how lncRNAs function thus fits with the overall mission of the NIH. The large number of predicted lncRNAs (over 9000 in humans) sets up a series of challenges for fully appreciating their functions. An outstanding model for analysis of lncRNA function is provided by the Drosophila oskar (osk) gene. Although osk RNA encodes a protein, it also has a noncoding function. The coding and noncoding functions are clearly distinct, acting at different stages of oogenesis and embryogenesis, and contributing to different processes. The lncRNA function of osk is essential for progression through oogenesis, and absence of osk RNA results in loss of certain proteins from nuage, a perinuclear structure found in germ line cells of all animals. Nuage is the site where piRNAs are produced. piRNAs act in restricting the activity of transposons, thus protecting the germ line DNA from damage. Notably, loss of osk lncRNA activity leads to a dramatic reduction in piRNA levels, and a corresponding increase in levels of transposon mRNAs. The goal of this proposal is to understand how osk RNA acts in nuage organization and piRNA production, features that are conserved among all animals including humans. One Aim is to complete the genetic characterization of osk mutants and of genes encoding proteins that bind to the key elements in osk RNA acting in its lncRNA function. One Aim is to define how nuage is affected by loss of osk RNA, examining individual components to determine if specific subsets of nuage proteins are primarily affected, and evaluating the overall organization of nuage by ultrastructural studies. A part of this Aim is to explore and test possible explanations for the requirement that osk RNA contains signals for transport out of the nurse cells (the cells with nuage) and into the oocyte. Another Aim centers on the proteins that bind to osk lncRNA functional elements. This builds on our work in defining these elements, and on identifying the factors that bind to these elements. The goal is to obtain a more complete understanding of all proteins that bind to the functional elements, to learn about the complex assembled from these proteins and the osk RNA, and thus to understand the underlying mechanisms. The final Aim is to search for mammalian equivalents of the critical sequence element that mediates osk lncRNA activity. Candidates will be tested in mice to determine if this feature plays a conserved role across all animals, as might be expected given the conservation of nuage, piRNAs, and many nuage components involved in piRNA production. Collectively, these studies have a high probability of revealing mechanistic details of nuage function that have direct relevance to germ line cells in humans.