Intergenic long non-coding RNA vs mRNA pairs.

Long non-coding (lnc)RNAs are a class of eukaryotic RNA that do not code for protein and are linked with transcriptional regulation, amongst a myriad of other functions. Using a custom in silico pipeline we have identified 6,436 putative lncRNA transcripts in the liver fluke parasite, Fasciola hepatica, none of which are conserved with those previously described from Schistosoma mansoni. F. hepatica lncRNAs were distinct from F. hepatica mRNAs in transcript length, coding probability, exon/intron composition, expression patterns, and genome distribution. RNA-Seq and digital droplet PCR measurements demonstrated developmentally regulated expression of lncRNAs between intra-mammalian life stages; a similar proportion of lncRNAs (14.2%) and mRNAs (12.8%) were differentially expressed (p<0.001), supporting a functional role for lncRNAs in F. hepatica life stages. While most lncRNAs (81%) were intergenic, we identified some that overlapped protein coding loci in antisense (13%) or intronic (6%) configurations. We found no unequivocal evidence for correlated developmental expression within positionally correlated lncRNA:mRNA pairs, but global co-expression analysis identified five lncRNA that were inversely co-regulated with 89 mRNAs, including a large number of functionally essential proteases. The presence of micro (mi)RNA binding sites in 3135 lncRNAs indicates the potential for miRNA-based post-transcriptional regulation of lncRNA, and/or their function as competing endogenous (ce)RNAs. The same annotation pipeline identified 24,141 putative lncRNAs in F. gigantica. This first description of lncRNAs in F. hepatica provides an avenue to future functional and comparative genomics studies that will provide a new perspective on a poorly understood aspect of parasite biology.

长链非编码RNA（long non-coding RNA，简称lncRNA）是一类不编码蛋白质的真核RNA，除参与众多其他生物学功能外，还与转录调控过程密切相关。本研究通过自定义的计算机模拟分析流程，在肝片形吸虫（*Fasciola hepatica*）中鉴定出6436条推定的lncRNA转录本，且这些转录本均未与此前在曼氏血吸虫（*Schistosoma mansoni*）中报道的lncRNA序列具有保守性。肝片形吸虫的lncRNA在转录本长度、编码概率、外显子-内含子结构、表达模式以及基因组分布特征上，均与该物种的mRNA存在显著差异。通过RNA测序（RNA-Seq）与数字液滴PCR（digital droplet PCR）检测发现，lncRNA在肝片形吸虫寄生哺乳动物体内的生活史阶段呈现发育调控性表达；差异表达的lncRNA占比为14.2%，mRNA占比为12.8%，二者比例相近（p<0.001），这一结果支持lncRNA在肝片形吸虫生活史中发挥功能性作用。尽管81%的lncRNA位于基因间区域，但本研究也鉴定出部分lncRNA以反义链（占比13%）或内含子区（占比6%）的构型与蛋白质编码基因位点重叠。本研究未在位置相关的lncRNA与mRNA配对中发现明确的发育表达相关性，但全局共表达分析鉴定出5条lncRNA与89条mRNA存在反向共调控关系，其中包含大量功能必需的蛋白酶编码基因。在3135条lncRNA中发现了微小RNA（microRNA，简称miRNA）结合位点，这表明lncRNA可能通过miRNA介导的转录后调控发挥功能，或作为内源竞争RNA（competing endogenous RNA，简称ceRNA）行使功能。通过同一套注释流程，本研究还在巨片形吸虫（*Fasciola gigantica*）中鉴定出24141条推定的lncRNA。本次对肝片形吸虫lncRNA的首次系统性报道，为后续功能基因组学与比较基因组学研究提供了方向，有望为寄生虫生物学中这一尚未被充分阐明的领域提供全新的研究视角。