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方仍然是一个重大的公共卫生问题。蛔虫感染超过10亿人，降低儿童的身体和认知发育，并阻碍流行地区的社会经济发展。为了帮助对抗这些寄生虫感染，需要更多的寄生虫生物学知识。在寄生线虫中尚待探索的一个新的研究途径是在基因沉默中充当“向导”的小RNA。小RNA（19-32 nt）调节mRNA的翻译和稳定性，通过形成异染色质导致基因沉默，并参与DNA消除和重排。关于寄生线虫中存在的小RNA的谱尚不清楚。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）可以分离具有离散发育区域的个体组织，如雄性和雌性生殖系统，3）体细胞谱系中的DNA消除（染色质减少，其中25%的基因组被消除）发生在早期发育期间。我们将从几个早期发育阶段和男性和女性生殖系统的区域制备和“深度测序”蛔虫小RNA文库。来自小RNA文库的序列将使我们能够测试以下假设：1）一组离散的小RNA的表达与早期蛔虫胚胎中的染色质减少在时间上相关，2）小RNA是差异表达的，并且在合子转录开始之前和早期发育中与mRNA翻译和稳定性的调节在时间上相关，3）小RNA与生殖系、配子发生和器官发生独特相关。此外，我们还将启动研究，以开发使用我们的无细胞和体内系统深入分析蛔虫中小RNA在mRNA翻译和稳定性中的作用的方法。我们的研究将确定和表征寄生线虫的小RNA在特定的阶段和组织，并在一个深度，迄今为止没有在线虫。总之，我们对蛔虫（一种具有巨大生殖能力的寄生线虫）中小RNA的分析，可能会发现与寄生线虫相关的新型小RNA。这些研究有可能确定寄生线虫基因表达和调控的新参与者，这些新参与者可能成为开发针对这些寄生虫的新型疗法的新药物靶点。公共卫生相关性：寄生线虫在世界许多地方仍然是一个重大的公共卫生问题。仅蛔虫就感染了10亿多人，阻碍了流行地区的社会经济发展。这些研究有可能确定寄生线虫基因表达和调控的新参与者，这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.