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（Erythropoiesis）在多个层面受到精准调控，以确保在多种生理条件下可正常生成成熟红细胞。为探究长链非编码RNA（long non-coding RNAs, lncRNAs）在该过程中的贡献，我们对分化中的小鼠胎肝红细胞的聚腺苷酸化与非聚腺苷酸化RNA开展了超过10亿条RNA测序（RNA-Seq）读段分析，最终鉴定出655个lncRNA基因，这些基因不仅涵盖基因间区、反义链及内含子区域，还包含假基因与增强子位点。其中超100个基因为此前未被报道的、且具有高度红细胞特异性的基因。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的lncRNAs：这些lncRNAs受表观遗传调控，且可被关键红细胞转录因子GATA1、TAL1或KLF1靶向调控。我们聚焦于其中12个候选基因，发现它们定位于细胞核内，并呈现复杂的发育表达模式。敲低这些基因会严重损害红细胞成熟过程，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由增强子转录而来，其对于邻近编码BAND3的基因的激活具有特异性必需作用。本研究提供了一份经过注释的红细胞lncRNA目录，可通过在线资源直接获取，并证实多种类型的lncRNAs参与了红细胞生成背后的调控环路。本次实验包含1份Ter119阴性总RNA样本、1份Ter119阳性总RNA样本、1份Ter119阳性聚腺苷酸化（poly(A)+）RNA样本及1份Ter119阳性非聚腺苷酸化（poly(A)-）RNA样本，所有上述样本的测序数据均为Illumina HiSeq测序数据。