Global discovery of erythroid long non-coding RNAs reveals novel regulators of red cell maturation

Erythropoiesis is regulated at multiple levels to ensure the proper generation of mature red cells under multiple physiological conditions. To probe the contribution of long non-coding RNAs (lncRNAs) to this process, we examined >1 billion RNA-Seq reads of polyadenylated and nonpolyadenylated RNA from differentiating mouse fetal liver red blood cells, and identified 655 lncRNA genes including not only intergenic, antisense and intronic but also pseudogene and enhancer loci. Over 100 of these genes are previously unrecognized and highly erythroidspecific. By integrating genome-wide surveys of chromatin states, transcription factor occupancy, and tissue expression patterns, we identify multiple lncRNAs that are dynamically expressed during erythropoiesis, show epigenetic regulation and are targeted by key erythroid transcription factors GATA1, TAL1 or KLF1. We focus on 12 such candidates and find that they are nuclear-localized and exhibit complex developmental expression patterns. Depleting them severely impaired erythrocyte maturation, inhibiting cell size reduction and subsequent enucleation. One of them, alncRNA-EC7, is transcribed from an enhancer and is specifically needed for activation of the neighboring gene encoding BAND3. Our study provides an annotated catalog of erythroid lncRNAs, readily available through an online resource, and shows that diverse types of lncRNAs participate in the regulatory circuitry underlying erythropoiesis. one Ter119- total RNA, one Ter119+ total RNA, one Ter119+ poly(A)+ RNA, one Ter119+ poly(A)- RNA, all are Hi-seq Ilumina data

红细胞生成过程会在多个层面受到调控，以确保在多种生理条件下能够正常生成成熟红细胞。为探究长链非编码RNA（long non-coding RNAs, lncRNAs）在该过程中的作用，我们对来自分化中小鼠胎肝红细胞的多聚腺苷酸化与非多聚腺苷酸化RNA进行了超过10亿条RNA-Seq读段分析，共鉴定出655个lncRNA基因，其来源不仅涵盖基因间区、反义链及内含子区，还包括假基因与增强子位点。其中超过100个基因为此前未被发现的高红细胞特异性基因。通过整合全基因组范围的染色质状态、转录因子结合占有率及组织表达模式分析，我们鉴定出多个在红细胞生成过程中动态表达、受表观遗传调控且被关键红细胞系转录因子GATA1、TAL1或KLF1靶向的lncRNAs。我们针对其中12个候选lncRNA展开深入研究，发现它们定位于细胞核内，并呈现复杂的发育阶段特异性表达模式。敲低这些lncRNA会严重损伤红细胞成熟过程，抑制细胞体积缩小及后续的去核步骤。其中一个名为alncRNA-EC7的lncRNA由增强子区域转录而来，其对于邻近编码带3蛋白（BAND3）的基因的激活具有特异性必需作用。本研究构建了一个带有注释的红细胞系lncRNA目录，可通过在线资源便捷获取，并揭示了多种类型的lncRNAs参与红细胞生成的调控网络。本次测序共包含4组样本：1份Ter119阴性总RNA、1份Ter119阳性总RNA、1份Ter119阳性多聚腺苷酸化阳性RNA及1份Ter119阳性多聚腺苷酸化阴性RNA，所有数据均为Illumina HiSeq测序所得。