MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People's Republic of China.
PLoS Negl Trop Dis. 2012;6(7):e1745. doi: 10.1371/journal.pntd.0001745. Epub 2012 Jul 31.
The complex life cycle of the genus Schistosoma drives the parasites to employ subtle developmentally dependent gene regulatory machineries. Small non-coding RNAs (sncRNAs) are essential gene regulatory factors that, through their impact on mRNA and genome stability, control stage-specific gene expression. Abundant sncRNAs have been identified in this genus. However, their functionally associated partners, Argonaute family proteins, which are the key components of the RNA-induced silencing complex (RISC), have not yet been fully explored.
METHODOLOGY/PRINCIPAL FINDINGS: Two monoclonal antibodies (mAbs) specific to Schistosoma japonicum Argonaute protein Ago2 (SjAgo2), but not SjAgo1 and SjAgo3, were generated. Soluble adult worm antigen preparation (SWAP) was subjected to immunoprecipitation with the mAbs and the captured SjAgo2 protein was subsequently confirmed by Western blot and mass spectrometry (MS) analysis. The small RNA population associated with native SjAgo2 in adult parasites was extracted from the immunoprecipitated complex and subjected to library construction. High-through-put sequencing of these libraries yielded a total of ≈50 million high-quality reads. Classification of these small RNAs showed that endogenous siRNAs (endo-siRNAs) generated from transposable elements (TEs), especially from the subclasses of LINE and LTR, were prominent. Further bioinformatics analysis revealed that siRNAs derived from ten types of well-defined retrotransposons were dramatically enriched in the SjAgo2-specific libraries compared to small RNA libraries constructed with total small RNAs from separated adult worms. These results suggest that a key function of SjAgo2 is to maintain genome stability through suppressing the activities of retrotransposons.
CONCLUSIONS/SIGNIFICANCE: In this study, we identified and characterized one of the three S. japonicum Argonautes, SjAgo2, and its associated small RNAs were found to be predominantly derived from particular classes of retrotransposons. Thus, a major function of SjAgo2 appears to associate with the maintenance of genome stability via suppression of retroelements. The data advance our understanding of the gene regulatory mechanisms in the blood fluke.
曼森血吸虫属的复杂生命周期促使寄生虫采用微妙的、依赖发育的基因调控机制。小非编码 RNA（sncRNA）是必不可少的基因调控因子，通过影响 mRNA 和基因组稳定性，控制特定阶段的基因表达。在这个属中已经鉴定出大量的 sncRNA。然而，它们功能相关的伴侣，Argonaute 家族蛋白，是 RNA 诱导沉默复合物（RISC）的关键组成部分，尚未得到充分探索。
方法/主要发现：生成了两种针对日本血吸虫 Argonaute 蛋白 Ago2（SjAgo2）的单克隆抗体（mAb），但不针对 SjAgo1 和 SjAgo3。可溶性成虫抗原制剂（SWAP）用 mAb 进行免疫沉淀，随后通过 Western blot 和质谱（MS）分析确认捕获的 SjAgo2 蛋白。从免疫沉淀复合物中提取与天然 SjAgo2 相关的成虫寄生虫小 RNA 群体，并进行文库构建。对这些文库进行高通量测序共获得约 5000 万个高质量读数。对这些小 RNA 的分类表明，来源于转座元件（TEs）的内源性 siRNA（endo-siRNA），特别是来源于 LINE 和 LTR 的亚类，是突出的。进一步的生物信息学分析表明，与从分离的成虫总小 RNA 构建的小 RNA 文库相比，来源于 10 种明确的逆转录转座子的 siRNA 在 SjAgo2 特异性文库中显著富集。这些结果表明，SjAgo2 的一个主要功能是通过抑制逆转录转座子的活性来维持基因组稳定性。
结论/意义：在这项研究中，我们鉴定并表征了日本血吸虫属的三个 Argonautes 之一，SjAgo2，并且发现其相关的小 RNA 主要来源于特定类别的逆转录转座子。因此，SjAgo2 的主要功能似乎与通过抑制逆转录元件来维持基因组稳定性有关。该数据增进了我们对血吸虫基因调控机制的理解。
