title: High-throughput sequencing of RNAs isolated by cross-linking immunoprecipitation (HITS-CLIP) reveals Argonaute-associated microRNAs and targets in Schistosoma japonicum.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute for Infectious Diseases and Vaccine Development, Tongji University School of Medicine, Shanghai, China. zhj88717@live.cn.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Institute for Infectious Diseases and Vaccine Development, Tongji University School of Medicine, Shanghai, China. luorong.1008@163.com.
authors.forename: Rong
authors.initials: R
authors.lastname: Luo
authors.affiliation: Institute for Infectious Diseases and Vaccine Development, Tongji University School of Medicine, Shanghai, China. xuxd1981@139.cm.
authors.forename: Xindong
authors.initials: X
authors.lastname: Xu
authors.affiliation: Institute for Infectious Diseases and Vaccine Development, Tongji University School of Medicine, Shanghai, China. zouying620@hotmail.com.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zou
authors.affiliation: Institute for Infectious Diseases and Vaccine Development, Tongji University School of Medicine, Shanghai, China. qfzhangsh@aliyun.com.
authors.forename: Qingfeng
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Institute for Infectious Diseases and Vaccine Development, Tongji University School of Medicine, Shanghai, China. wqpan0912@aliyun.com.|Department of Tropical Infectious Diseases, Second Military Medical University, Shanghai, China. wqpan0912@aliyun.com.
authors.forename: Weiqing
authors.initials: W
authors.lastname: Pan
mesh_terms: D000818:Animals; D060565:Argonaute Proteins / Q000276:immunology; D005786:Gene Expression Regulation; D015801:Helminth Proteins / Q000276:immunology; D059014:High-Throughput Nucleotide Sequencing; D047468:Immunoprecipitation; D035683:MicroRNAs / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D018510:RNA, Helminth / Q000235:genetics* / Q000302:isolation & purification; D012549:Schistosoma japonicum / Q000235:genetics*; D017422:Sequence Analysis, DNA
chemical_list: D060565:Argonaute Proteins; D015801:Helminth Proteins; D035683:MicroRNAs; D018510:RNA, Helminth