lincRNA Expression

There is growing recognition that mammalian cells produce many thousands of large intergenic transcripts. However, the functional significance of these transcripts has been particularly controversial. While there are some well-characterized examples, the vast majority (>95%) show little evidence of evolutionary conservation and have been suggested to represent transcriptional noise. Here, we report a new approach to identifying large non-coding RNAs (ncRNAs) by using chromatin-state maps to discover discrete transcriptional units intervening known protein-coding loci. Our approach identified ~1600 large multi-exonic RNAs across four mouse cell types. In sharp contrast to previous collections, these large intervening ncRNAs (lincRNAs) exhibit strong purifying selection in their genomic loci, exonic sequences, and promoter regions - with greater than 95% showing clear evolutionary conservation. We also developed a novel functional genomics approach that assigns putative functions to each lincRNA, revealing a diverse range of roles for lincRNAs in processes from ES pluripotency to cell proliferation. We obtained independent functional validation for the predictions for over 100 lincRNAs, using cell-based assays. In particular, we demonstrate that specific lincRNAs are transcriptionally regulated by key transcription factors in these processes such as p53, NFKB, Sox2, Oc4, and Nanog. Together, these results define a unique collection of functional lincRNAs that are highly conserved and implicated in diverse biological processes. lincRNA expression patterns were generated across a compendium of mouse tissues, cell types, and conditions. lincRNA expression profiles were generated on custom NimbleGen arrays (Samples GSM346609..GSM346650) and correlated with known protein coding genes on Affymetrix gene expression arrays (Samples GSM347910..GSM347943).越来越多的研究表明，哺乳动物细胞可产生数千种大型基因间转录本。然而，此类转录本的功能意义一直存在较大争议。尽管已有部分得到充分表征的实例，但绝大多数（>95%）未显示出进化保守性的相关证据，因此被认为属于转录噪声。本研究报道了一种识别大型非编码RNA（large non-coding RNAs，ncRNAs）的新方法：通过染色质状态图谱，发掘已知蛋白质编码基因座之间的离散转录单元。我们的方法在四种小鼠细胞类型中鉴定出约1600个大型多外显子RNA。与既往收集的转录本集形成鲜明对比的是，这些大型基因间非编码RNA（large intervening ncRNAs，lincRNAs）在其基因组基因座、外显子序列及启动子区域均表现出强烈的纯化选择特性——其中超过95%具有明确的进化保守性。我们还开发了一种全新的功能基因组学方法，可为每个lincRNA赋予推定功能，由此揭示了lincRNA在从胚胎干细胞（ES）多能性到细胞增殖等多种生物学过程中的多样作用。我们通过细胞实验对100余种lincRNA的预测结果进行了独立功能验证。具体而言，我们证实了在这些过程中，特定lincRNA的转录受到p53、NFKB、Sox2、Oc4及Nanog等关键转录因子的调控。综上，本研究构建了一套独特的功能性lincRNA集，此类lincRNA具有高度保守性，并参与多种生物学过程。本研究获取了涵盖小鼠组织、细胞类型及实验条件的全数据集内的lincRNA表达模式。通过定制化NimbleGen基因芯片（样本编号：GSM346609至GSM346650）生成lincRNA表达谱，并将其与Affymetrix基因表达芯片（样本编号：GSM347910至GSM347943）上的已知蛋白质编码基因表达数据进行关联分析。