Genome-wide non-polyadenylated transcription in differentiating mouse embryonic stem cells reveals novel non-coding RNA related to pluripotency and differentiation. Mus musculus

The transcriptional landscape in embryonic stem cells (ESCs) and during ESC differentiation has received considerable attention in recent years, but reports have so far been confined to the polyadenylated fraction of RNA, while the non-polyadenylated (NPA) fraction remained largely unexplored. Notwithstanding, the NPA RNA super-family, which is mainly comprised of fundamental components of the RNA translational machinery, the RNA splicing machinery, and RNA-RNA or RNA-DNA interacting complexes, has every potential to participate in the regulation of pluripotency and stem cell fate. We conducted a comprehensive analysis of NPA RNA in murine ESCs and ESC-derived Neural Progenitor Cells (NPCs) using a combination of whole genome tiling arrays and Next Generation Sequencing technologies. This strategy allowed for the transcriptional characterization of already well-defined NPA RNA subclasses in this unique biological context as well as the identification of many new members of these functional ncRNA classes. In addition, we describe a group of novel, conserved RNAs (snacRNAs: small, non-polyadenylated conserved), some of which are differentially expressed between ESC and NPCs, providing the first evidence of a novel group of potentially functional ncRNAs involved in the regulation of pluripotency and stem cell fate. We further show that minor spliceosomal snRNAs, which are non-polyadenylated, are almost completely absent in ESCs and are upregulated in differentiation. Finally, we show differential processing of the minor intron of the polycomb group gene Eed. Our data suggest that non-polyadenylated RNA, both known and novel, play important roles in embryonic stem cells. Overall design: We extracted total RNA from undifferentiated ESCs and 7 day NPCs. Total RNA was divided into poly(A+) and poly(A-) enriched fractions using a modified version of Oligotex extraction (Qiagen). Briefly, after the hybridization of the total RNA to oligo(dT) beads, supernatants were saved rather than discarded and then subjected to isopropanol-ethanol extraction to obtain the Poly(A-) fraction. This submission represents RNA-Seq component of study.

近年来，胚胎干细胞（ESCs）及其分化过程中的转录组图谱受到了广泛关注，但迄今为止相关研究均局限于RNA的聚腺苷酸化组分，而非聚腺苷酸化（NPA）组分基本未被探索。尽管如此，NPA RNA超家族主要由RNA翻译系统、RNA剪接系统以及RNA-RNA或RNA-DNA相互作用复合物的核心组分构成，其极有可能参与多能性调控与干细胞命运决定。本研究结合全基因组平铺阵列与下一代测序技术，对小鼠ESCs及ESCs衍生的神经前体细胞（NPCs）中的NPA RNA进行了全面分析。该策略不仅可在这一独特的生物学背景下对已明确的NPA RNA亚类进行转录组特征分析，还能鉴定出这些功能性非编码RNA（ncRNA）家族的诸多新成员。此外，本研究还报道了一类新型保守RNA（snacRNAs: small, non-polyadenylated conserved，即小型非聚腺苷酸化保守RNA），其中部分在ESCs与NPCs间存在差异表达，为一类参与多能性调控与干细胞命运决定的新型功能性非编码RNA的存在提供了首个实验证据。本研究进一步证实，非聚腺苷酸化的次要剪接体小核RNA（snRNAs）在ESCs中几乎完全缺失，且在分化过程中表达上调。最后，本研究揭示了多梳家族基因Eed的次要内含子存在差异加工过程。本研究数据表明，无论是已知还是新型的非聚腺苷酸化RNA，均在胚胎干细胞中发挥重要功能。实验设计概述：本研究从未分化ESCs及培养7天的NPCs中提取总RNA。通过改良的Oligotex提取试剂盒（Qiagen）法，将总RNA分为聚腺苷酸化富集（poly(A+)）组分与非聚腺苷酸化富集（poly(A-)）组分。简要而言，将总RNA与寡聚(dT)磁珠杂交后，保留上清液而非弃去，随后通过异丙醇-乙醇沉淀提取得到poly(A-)组分。本次提交的数据集为本研究的RNA测序（RNA-Seq）部分。