Mechanistic investigation of long non-coding RNAs in Drosophila oogenesis

项目摘要/摘要 长非编码RNA(LncRNAs)是一类定义松散的长于200个核苷酸的RNAs，组成 真核转录组的重要部分。数以千计的lncRNA现在已经被编目在 许多物种，但相对较少的是功能特征，特别是在动物模型中。 由于缺乏关于lncRNA功能的分子证据，以及lncRNA信号一直很弱。 保护，它们对生物生物学和疾病的影响程度尚不清楚；更多的功能 在动物模型中的研究对于全面理解lncRNA生物学是必要的。这样做的长期目标是 建议研究lncRNA在经典动物卵子发生的关键发育过程中的功能。 模型：黑腹果蝇。果蝇卵子发生的遗传和发育状况良好 有助于研究LncRNA如何影响特定的发育过程和 与关键的卵子发生基因相互作用。此外，卵子发生的许多方面在所有动物中都是相同的，并且 因此，对果蝇lncRNA生物学的洞察将为人类生物学提供信息。这项建议具有 三个具体目标。首先，卵巢丰富的lncRNAs的定位将在卵子发生中得到表征。 对果蝇现有的卵巢RNA-Seq数据集的分析已经鉴定出28个反义和基因间的数据集 在卵巢中丰富并可能在多种果蝇物种中保守的lncRNA基因座，表明 功能重要性。这些lncRNAs在整个卵子发生过程中的细胞和细胞内定位将是 采用单分子荧光原位杂交技术(SmFISH)进行检测。其次，卵子表型将 被定性为lncRNA突变体。像靶向基因缺失这样的标准突变方法通常是 不足以研究lncRNA的功能，因为重叠的DNA调控元件很常见，并且可以 混乱的解释。相反，这项提议旨在开发和实施变异方法， 特别针对lncRNA转录本。第三，对lncRNA的作用机制进行了研究。 LncRNA突变体和野生型果蝇之间的比较RNA-Seq实验将揭示特定的调控 LncRNAs的靶点以及LncRNAs是在顺式还是在反式中起作用。RNA/DNA双重FISH将用于 确认lncRNAs与其靶标的调控相互作用，并用RNA聚合酶II抑制剂进行治疗 随后的smFISH将揭示lncRNA的活性是由于rna产物还是新生的rna。 抄本本身。这一提议的成功完成将为研究lncRNA提供重要的见解。 生物学。由于许多lncRNA还与心血管疾病和癌症等疾病有关， 从果蝇身上获得的知识将被证明对理解lncRNAs如何对人类做出贡献有价值 疾病。

Project Summary/Abstract Long non-coding RNAs (lncRNAs), a loosely-defined class of RNAs longer than 200 nucleotides, make up a significant fraction of the eukaryotic transcriptome. Thousands of lncRNAs have now been catalogued in numerous species, but relatively few have been functionally characterized, particularly in animal models. Because of the dearth of molecular evidence of lncRNA function and consistently weak signals of lncRNA conservation, the extent of their impact on organismal biology and disease remains unclear; more functional studies in animal models are necessary to fully understand lncRNA biology. The long term objective of this proposal is to investigate lncRNA functions in the key developmental process of oogenesis in a classic animal model: Drosophila melanogaster. The genetics and development of Drosophila oogenesis are well characterized, facilitating investigations of how lncRNAs may impact specific developmental processes and interact with key oogenesis genes. Further, many aspects of oogenesis are shared across all animals, and thus insights into lncRNA biology from Drosophila will be informative for human biology. This proposal has three specific aims. First, the localization of ovary-enriched lncRNAs will be characterized in oogenesis. Analyses of existing ovary RNA-Seq datasets in Drosophila have identified a set of 28 antisense and intergenic lncRNA loci that are enriched in the ovaries and likely conserved in multiple Drosophila species, suggesting functional importance. The cellular and intracellular localization of these lncRNAs throughout oogenesis will be determined using single-molecule fluorescent in situ hybridization (smFISH). Second, oogenic phenotypes will be characterized for lncRNA mutants. Standard mutational approaches like targeted gene deletion are often insufficient for investigating lncRNA function, as overlapping DNA regulatory elements are common and can confound interpretations. Instead, this proposal aims to develop and implement mutational approaches that specifically target the lncRNA transcript. Third, the mechanisms of lncRNA function will be characterized. Comparative RNA-Seq experiments between lncRNA mutants and wild-type flies will reveal specific regulatory targets of lncRNAs and whether the lncRNAs function in cis or in trans. RNA/DNA double-FISH will be used to confirm regulatory interactions of lncRNAs and their targets, and treatment with an RNA polymerase II inhibitor followed with smFISH will reveal whether lncRNA activity is due to the RNA product or nascent RNA transcription itself. The successful completion of this proposal will provide significant insights into lncRNA biology. As many lncRNAs are also associated with diseases like cardiovascular disease and cancer, knowledge gained from Drosophila will prove valuable to understanding how lncRNAs contribute to human disease.