Mechanistic investigation of long non-coding RNAs in Drosophila oogenesis

项目总结/摘要 长链非编码RNA（lncRNA），一种定义松散的长度超过200个核苷酸的RNA，组成了一个 真核生物转录组的重要部分。数以千计的lncRNA已经被编入 许多物种，但相对较少的已功能化，特别是在动物模型。 由于缺乏lncRNA功能的分子证据，lncRNA的信号一直很弱， 保护，其对生物体生物学和疾病的影响程度仍不清楚;更多的功能 动物模型的研究对于充分理解lncRNA生物学是必要的。这一长期目标 一项研究计划是研究lncRNA在经典动物卵子发生的关键发育过程中的功能 模型：果蝇。果蝇卵子发生的遗传学和发育学研究进展良好， 表征，促进lncRNA如何影响特定发育过程的研究， 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是共享的， 因此，对果蝇lncRNA生物学的深入了解将为人类生物学提供信息。这一建议 三个具体目标。首先，卵巢富集的lncRNA的定位将在卵子发生中表征。 对果蝇现有卵巢RNA-Seq数据集的分析已经确定了一组28个反义和基因间序列， lncRNA位点在卵巢中富集，并且可能在多种果蝇物种中保守，这表明 功能重要性。这些lncRNA在整个卵子发生过程中的细胞和细胞内定位将是 使用单分子荧光原位杂交（smFISH）测定。第二，卵子发生表型将 以lncRNA突变体为特征。标准的突变方法，如靶向基因缺失， 不足以研究lncRNA的功能，因为重叠的DNA调控元件是常见的， 混乱的解释相反，该提案旨在开发和实施突变方法， 特异性靶向lncRNA转录物。第三，lncRNA功能的机制将被表征。 lncRNA突变体和野生型果蝇之间的比较RNA-Seq实验将揭示特异性调节基因。 lncRNA的靶点以及lncRNA的功能是顺式还是反式。RNA/DNA双FISH将用于 证实lncRNA及其靶点的调节相互作用，以及用RNA聚合酶II抑制剂治疗 随后的smFISH将揭示lncRNA活性是由于RNA产物还是新生RNA 转录本身。该提案的成功完成将为lncRNA提供重要的见解 生物学由于许多lncRNA也与心血管疾病和癌症等疾病有关， 从果蝇获得的知识将被证明对理解lncRNA如何促进人类 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.