Division of Animal and Nutritional Sciences, West Virginia University, Morgantown, WV, 26506, USA.
Reprod Biol Endocrinol. 2020 Feb 21;18(1):13. doi: 10.1186/s12958-020-00573-4.
Long non-coding RNAs (lncRNAs) are key regulators of diverse cellular processes. Although a number of studies have reported the identification of bovine lncRNAs across many tissues, very little is known about the identity and characteristics of lncRNAs in bovine oocytes.
A bovine oocyte cDNA library was constructed and sequenced using the Illumina HiSeq 2000 sequencing system. The oocyte transcriptome was constructed using the ab initio assembly software Scripture and Cufflinks. The assembled transcripts were categorized to identify the novel intergenic transcripts, and the coding potential of these novel transcripts was assessed using CPAT and PhyloCSF. The resulting candidate long intergenic non-coding RNAs (lincRNAs) transcripts were further evaluated to determine if any of them contain any known protein coding domains in the Pfam database. RT-PCR was used to analyze the expression of oocyte-expressed lincRNAs in various bovine tissues.
A total of 85 million raw reads were generated from sequencing of the bovine oocyte library. Transcriptome reconstruction resulted in the assembly of a total of 42,396 transcripts from 37,678 genomic loci. Analysis of the assembled transcripts using the step-wide pipeline resulted in the identification of 1535 oocyte lincRNAs corresponding to 1183 putative non-coding genes. A comparison of the oocyte lincRNAs with the lncRNAs reported in other bovine tissues indicated that 970 of the 1535 oocyte lincRNAs appear to be unique to bovine oocytes. RT-PCR analysis of 5 selected lincRNAs showed either specific or predominant expression of 4 lincRNAs in the fetal ovary. Functional prediction of the oocyte-expressed lincRNAs suggested their involvement in oogenesis through regulating their neighboring protein-coding genes.
This study provides a starting point for future research aimed at understanding the roles of lncRNAs in controlling oocyte development and early embryogenesis in cattle.
长非编码 RNA（lncRNA）是多种细胞过程的关键调节因子。尽管许多研究已经报道了在许多组织中鉴定牛 lncRNA，但对牛卵母细胞中 lncRNA 的身份和特征知之甚少。
构建了牛卵母细胞 cDNA 文库，并使用 Illumina HiSeq 2000 测序系统进行测序。使用从头组装软件 Scripture 和 Cufflinks 构建卵母细胞转录组。将组装的转录物进行分类，以鉴定新的基因间转录物，并使用 CPAT 和 PhyloCSF 评估这些新转录物的编码潜力。进一步评估所得候选长基因间非编码 RNA（lincRNA）转录本，以确定它们是否在 Pfam 数据库中包含任何已知的蛋白质编码结构域。使用 RT-PCR 分析了各种牛组织中卵母细胞表达的 lincRNA 的表达。
从牛卵母细胞文库测序中产生了总共 8500 万条原始读数。转录组重建导致从 37678 个基因组座总共组装了 42396 个转录物。使用分步宽管道对组装的转录物进行分析，确定了 1535 个卵母细胞 lincRNA，对应于 1183 个推定的非编码基因。将卵母细胞 lincRNA 与其他牛组织中报道的 lncRNA 进行比较表明，1535 个卵母细胞 lincRNA 中有 970 个似乎是牛卵母细胞所特有的。对 5 个选定 lincRNA 的 RT-PCR 分析表明，在胎儿卵巢中，4 个 lincRNA 表现出特异性或主要表达。对卵母细胞表达的 lincRNA 的功能预测表明，它们通过调节其邻近的蛋白质编码基因参与卵母细胞发生。
本研究为进一步研究 lncRNA 在控制牛卵母细胞发育和早期胚胎发生中的作用提供了起点。
