Small RNA Discovery and Analysis in Ascaris

描述(由申请人提供)：寄生线虫在世界许多地区仍然是一个严重的公共卫生问题。蛔虫感染了10多亿人，降低了儿童的身体和认知发育，并阻碍了流行地区的社会经济发展。为了帮助抗击这些寄生虫感染，需要更多关于寄生虫生物学的知识。寄生线虫研究的一个新途径是小RNA，它在基因沉默中起到“向导”的作用。小RNA(19-32nt)通过异染色质的形成调节mRNA的翻译和稳定性，导致基因沉默，并参与DNA的消除和重排。关于寄生线虫中存在的小RNA的光谱，目前还一无所知。蛔虫代表了一种独特的线虫系统，用于分析小RNA，因为1)可以获得大量同步发育的胚胎，2)可以分离出具有离散发育区域的单个组织，如男性和女性生殖系统，以及3)在体细胞谱系的早期发育过程中会发生一种不寻常的DNA消除形式(染色质减少，其中25%的基因组被消除)。我们将从男性和女性生殖系统的几个早期发育阶段和区域制备和“深测序”蛔虫小RNA文库。来自小RNA文库的序列将使我们能够检验以下假设：1)一组离散的小RNA的表达在时间上与蛔虫早期胚胎中染色质的减少有关，2)小RNA在合子转录开始之前和早期发育期间与mRNA翻译和稳定性的调节在时间上存在差异，以及3)小RNA与生殖系、配子发生和器官发生唯一相关。此外，我们还将开展研究，利用我们的无细胞和活体系统，深入分析小RNA在蛔虫mRNA翻译和稳定性中的作用。我们的研究将识别和表征寄生线虫在特定阶段和组织中的小RNA，以及迄今为止在线虫中未曾做过的深度。总体而言，我们对蛔虫中的小RNA的分析可能会识别与寄生线虫相关的新的小RNA。蛔虫是一种具有巨大繁殖能力的寄生线虫。这些研究可能发现寄生线虫基因表达和调控的新角色，可能成为开发针对这些寄生虫的新疗法的新药物靶点。公共卫生相关性：在世界许多地区，寄生线虫仍然是一个严重的公共卫生问题。仅蛔虫一项就感染了超过10亿人，并阻碍了流行地区的社会经济发展。这些研究可能在寄生线虫基因表达和调控方面发现新的参与者，从而可能导致开发针对寄生线虫的新疗法。

DESCRIPTION (provided by applicant): Parasitic nematodes remain a significant public health problem in many parts of the world. Ascaris infects over 1 billion people, decreases physical and cognitive development in children, and hinders socioeconomic development in endemic areas. In order to help combat these parasitic infections, greater knowledge of parasite biology is needed. One new avenue of research yet to be explored in parasitic nematodes is small RNAs that act as "guides" in gene silencing. Small RNAs (19-32 nt) regulate mRNA translation and stability, lead to gene silencing through the formation of heterochromatin, and are involved in DNA elimination and rearrangements. Nothing is known regarding the spectrum of small RNAs present in parasitic nematodes. Ascaris represents a unique nematode system for analysis of small RNAs because 1) large numbers of synchronously developing embryos can be obtained, 2) individual tissues with discrete developmental regions such as the male and female reproductive systems can be isolated, and 3) an unusual form of DNA elimination (chromatin diminution in which 25% of the genome is eliminated) in the somatic lineages occurs during early development. We will prepare and "deep sequence" Ascaris small RNA libraries from several early developmental stages and regions of the male and female reproductive systems. Sequences from the small RNA libraries will enable us to test the following hypotheses: 1) Expression of a discrete set of small RNAs is temporally associated with chromatin diminution in early Ascaris embryos, 2) Small RNAs are differentially expressed and are temporally associated with regulation of mRNA translation and stability prior to the onset of zygotic transcription and in early development, and 3) Small RNAs are uniquely associated with the germline, gametogenesis, and organogenesis. Furthermore, we will also initiate studies to develop methods for in depth mechanistic analyses of the role of small RNAs in mRNA translation and stability in Ascaris using our cell-free and in vivo systems. Our studies will identify and characterize parasitic nematode small RNAs in specific stages and tissues and at a depth heretofore not previously done in a nematode. Overall, our analysis of small RNAs in Ascaris, a parasitic nematode with tremendous reproductive capacity, may identify novel small RNAs associated with parasitic nematodes. These studies are likely to identify new players in parasitic nematode gene expression and regulation that could be new drug targets for the development of novel therapeutics against these parasites. PUBLIC HEALTH RELEVANCE: Parasitic nematodes remain a significant public health problem in many parts of the world. Ascaris alone infects upwards of 1 billion people and hinders socioeconomic development in endemic areas. These studies are likely to identify new players in parasitic nematode gene expression and regulation that could lead to the development of novel therapeutics against parasitic nematodes.