Table_6_Role of Fasciola hepatica Small RNAs in the Interaction With the Mammalian Host.xlsx

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression being involved in many different biological processes and play a key role in developmental timing. Additionally, recent studies have shown that miRNAs released from parasites are capable of regulating the expression of host genes. In the present work, we studied the expression patterns of ncRNAs of various intra-mammalian life-cycle stages of the liver fluke, Fasciola hepatica, as well as those packaged into extracellular vesicles and shed by the adult fluke. The miRNA expression profile of the intra-mammalian stages shows important variations, despite a set of predominant miRNAs that are highly expressed across all stages. No substantial variations in miRNA expression between dormant and activated metacercariae were detected, suggesting that they might not be central players in regulating fluke gene expression during this crucial step in the invasion of the definitive host. We generated a curated pipeline for the prediction of putative target genes that reports only sites conserved between three different prediction approaches. This pipeline was tested against an iso-seq curated database of the 3’ UTR regions of F. hepatica genes to detect miRNA regulation networks within liver fluke. Several functions related to the host immune response or modulation were enriched among the targets of the most highly expressed parasite miRNAs, stressing that they might be key players during the establishment and maintenance of infection. Additionally, we detected fragments derived from the processing of tRNAs, in all developmental stages analyzed, and documented the presence of novel long tRNA fragments enriched in vesicles. We confirmed the presence of at least 5 putative vault RNAs (vtRNAs), that are expressed across different stages and enriched in vesicles. The presence of tRNA fragments and vtRNAs in vesicles raise the possibility that they could be involved in the host-parasite interaction.

微小RNA（miRNAs）是一类重要的基因表达转录后调控因子，参与诸多生物学过程，并在发育时序调控中发挥关键作用。此外，近期研究表明，寄生虫分泌释放的miRNA能够调控宿主基因的表达。本研究中，我们分析了肝片吸虫（Fasciola hepatica）在哺乳类体内的不同生命周期阶段的非编码RNA（non-coding RNAs, ncRNAs）表达模式，以及成虫分泌并包裹于细胞外囊泡（extracellular vesicles）中的非编码RNA。尽管在所有发育阶段均存在高表达的优势miRNA，但该寄生虫哺乳类体内阶段的miRNA表达谱仍存在显著差异。未检测到休眠态与激活态后囊蚴间的miRNA表达存在显著差异，这提示二者在终宿主入侵这一关键步骤中，可能并非调控肝片吸虫基因表达的核心因子。我们构建了一套人工注释的分析流程，仅保留三种不同预测算法共同保守的结合位点以预测推定靶基因。我们利用基于iso-seq全长转录组测序的肝片吸虫基因3’非翻译区（3’ UTR）人工注释数据库对该流程进行了验证，以解析肝片吸虫体内的miRNA调控网络。在高表达的寄生虫miRNA靶基因中，富集了诸多与宿主免疫应答或免疫调控相关的功能通路，这进一步凸显了它们在感染建立与维持过程中可能扮演的关键角色。此外，我们在所有分析的发育阶段中均检测到了tRNA加工产生的片段，并记录了一类富集于囊泡中的新型长tRNA片段。我们证实了至少5种推定的穹隆RNA（vault RNAs, vtRNAs）的存在，它们在不同发育阶段均有表达且在囊泡中富集。囊泡中存在tRNA片段与vtRNA这一现象，提示它们可能参与宿主-寄生虫的互作过程。