Zhao Jing, Luo Rong, Xu Xindong, Zou Ying, Zhang Qingfeng, Pan Weiqing
Institute for Infectious Diseases and Vaccine Development, Tongji University School of Medicine, Shanghai, China.
Department of Tropical Infectious Diseases, Second Military Medical University, Shanghai, China.
Parasit Vectors. 2015 Nov 14;8:589. doi: 10.1186/s13071-015-1203-9.
Schistosomiasis, caused by schistosomes, is one of the most prevalent and serious parasitic diseases in tropical and subtropical countries. This pathogen has a complex life cycle and harbors a unique repertoire of genes expressed at different life-stages. Understanding the gene regulation of schistosomes will contribute to identification of novel drug targets and vaccine candidates. Some conserved and novel microRNAs (miRNAs) have been identified in schistosomes as key transcriptional and post-transcriptional regulators in the past few years; however, little is known about their specific targets.
High-throughput sequencing of RNAs isolated by cross-linking immunoprecipitation (HITS-CLIP) was used to covalently crosslink native Argonaute protein-RNA complexes in Schistosoma japonicum. An antibody against S.japonicum Argonaute proteins, was generated and used for immunoprecipitation of the crosslinked SjAgo-RNA complex from soluble adult worm extract. Small RNAs, including miRNAs and their target mRNAs associated with the native SjAgo in adult parasites, were enriched and extracted for library construction.
High-throughput sequencing produced a total of ~7.4 million high-quality reads, of which approximately 45.07 % were composed of 769 miRNAs and 35.54 % were composed of 11,854 mRNAs target sites. Further bioinformatics analysis identified 43 conserved known miRNAs and 256 novel miRNAs in the SjAgo-associated small RNA population. An average of approximately 15 target sites were predicted for each miRNA. Moreover, a positive rate of 50 % has been achieved in a small-scale verification test of the putative target sites of miRNA1.
In this study, we isolated and identified small RNAs including miRNAs and their targets associated with the S. japonicum Argonaute proteins, by the HITS-CLIP method combined with bioinformatics and biologic experimental analysis. These data reveal a genome-wide miRNA-mRNA interaction map in S. japonicum in vivo, which will help us understand the complex gene regulatory network in this pathogen and thereby facilitate the development of novel drug approaches against schistosomiasis.
血吸虫病由血吸虫引起，是热带和亚热带国家最普遍且严重的寄生虫病之一。这种病原体具有复杂的生命周期，并拥有在不同生命阶段表达的独特基因库。了解血吸虫的基因调控将有助于鉴定新的药物靶点和候选疫苗。在过去几年中，已在血吸虫中鉴定出一些保守和新的微小RNA（miRNA），它们是关键的转录和转录后调节因子；然而，对其特定靶点知之甚少。
采用交联免疫沉淀法（HITS-CLIP）对RNA进行高通量测序，以共价交联日本血吸虫中的天然AGO蛋白-RNA复合物。制备了针对日本血吸虫AGO蛋白的抗体，并用于从可溶性成虫提取物中免疫沉淀交联的SjAgo-RNA复合物。富集并提取与成年寄生虫中天然SjAgo相关的小RNA，包括miRNA及其靶mRNA，用于文库构建。
高通量测序共产生约740万个高质量读段，其中约45.07%由769个miRNA组成，35.54%由11854个mRNA靶位点组成。进一步的生物信息学分析在与SjAgo相关的小RNA群体中鉴定出43个保守的已知miRNA和256个新的miRNA。每个miRNA平均预测约15个靶位点。此外，在miRNA1假定靶位点的小规模验证试验中，阳性率达到了50%。
在本研究中，我们通过HITS-CLIP方法结合生物信息学和生物学实验分析，分离并鉴定了与日本血吸虫AGO蛋白相关的小RNA，包括miRNA及其靶标。这些数据揭示了日本血吸虫体内全基因组的miRNA-mRNA相互作用图谱，这将有助于我们了解该病原体中复杂的基因调控网络，从而促进针对血吸虫病的新药研发。
