Systematic Identification and Characterization of Long Non-coding RNAs in Mouse Mature Sperm

Increasingly, studies have shown that mature spermatozoa contain many transcripts including mRNAs and miRNAs. However, the expression profile of long noncoding RNAs (lncRNAs) in mammalian sperm has not been systematically investigated. Here, we used highly purified RNA to investigate lncRNA expression profiles in mouse mature sperm by stranded-specific RNA-seq. We identified 20907 known and 4088 novel lncRNAs transcripts, and the existence of intact lncRNAs was confirmed by RT-PCR and fluorescence in situ hybridization on two representative lncRNAs. Compared with testes, 2873 upregulated and 6629 downregulated lncRNAs and 1237 upregulated and 2513 downregulated mRNAs were identified in sperm. Based on the "Cis and Trans" RNA-RNA interaction principle, we found 1793 targeted coding genes of differently expressed lncRNAs. In terms of GO analysis, differentially expressed lncRNAs targeted genes mainly related to nucleic acid metabolic, protein modification, chromatin and histone modification, sperm function, and spermatogenesis. In contrast, differentially expressed transcripts of mRNAs were highly enriched for nucleic acid metabolic, spermatogenesis, sperm function, and chromatin organization. Furthermore, KEGG pathway analysis showed that the differentially expressed lncRNAs were involved in mRNA surveillance and AMPK signaling pathways. Besides these two pathways, RNA transport splicesome was another pathway incorporated with the differentially expressed mRNA. In summary, this study provides a preliminary database valuable for identifying lncRNAs critical in the late stage of spermatogenesis or important for sperm function regulation. LncRNA profiles of mice (3-5 months) were generated by stranded-specific RNA-seq, using Illumina HiSeqTM 4000 with PE100.越来越多的研究证实，成熟精子（mature spermatozoa）中存在包括信使RNA（mRNAs）和微RNA（miRNAs）在内的多种转录本。然而，哺乳动物精子中长链非编码RNA（long noncoding RNAs, lncRNAs）的表达谱尚未得到系统解析。本研究利用高纯度RNA，通过链特异性RNA测序（stranded-specific RNA-seq）探究了小鼠成熟精子中的lncRNA表达谱。我们共鉴定出20907个已知lncRNA转录本与4088个新型lncRNA转录本，并通过逆转录聚合酶链反应（reverse transcription-polymerase chain reaction, RT-PCR）以及针对两个代表性lncRNA的荧光原位杂交（fluorescence in situ hybridization）验证了完整lncRNA的存在。与睾丸组织相比，精子中分别鉴定出2873个上调、6629个下调的lncRNA，以及1237个上调、2513个下调的信使RNA。基于“顺式（Cis）与反式（Trans）”RNA-RNA互作原理，我们共获得1793个差异表达lncRNA的靶编码基因。基因本体（Gene Ontology, GO）分析结果显示，差异表达lncRNA的靶基因主要富集于核酸代谢、蛋白质修饰、染色质与组蛋白修饰、精子功能及精子发生相关过程。与之相对，差异表达的信使RNA转录本则显著富集于核酸代谢、精子发生、精子功能及染色质组织相关通路。进一步的京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路分析表明，差异表达lncRNA参与了mRNA监控通路与AMPK信号通路。除上述两条通路外，RNA转运及剪接体通路也与差异表达的信使RNA密切相关。综上，本研究构建了一个初步的数据库，可为鉴定精子发生后期关键lncRNA或调控精子功能的重要lncRNA提供有价值的参考。本研究采用Illumina HiSeq™ 4000平台、以双端100bp（PE100）测序模式，对3-5月龄小鼠的精子lncRNA表达谱进行了链特异性RNA测序。