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

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，小鼠胚胎干细胞（Embryonic Stem Cells, ESCs）及其分化过程中的转录组图谱已受到广泛关注，但迄今为止的相关研究均局限于RNA的聚腺苷酸化组分，而非聚腺苷酸化（non-polyadenylated, NPA）组分在很大程度上仍未得到充分探索。尽管如此，NPA RNA超家族主要由RNA翻译装置、RNA剪接装置以及RNA-RNA或RNA-DNA相互作用复合物的核心组分构成，极有可能参与多能性（pluripotency）与干细胞命运的调控。本研究结合全基因组平铺芯片（whole genome tiling arrays）与下一代测序技术，对小鼠胚胎干细胞及胚胎干细胞来源的神经前体细胞（Neural Progenitor Cells, NPCs）中的NPA RNA进行了全面分析。该研究策略不仅可在这一独特的生物学模型中对已明确的NPA RNA亚类进行转录组学表征，还能鉴定出这些功能性非编码RNA（non-coding 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——小保守非聚腺苷酸化RNA（small non-polyadenylated conserved RNAs, snacRNAs），其中部分在胚胎干细胞与神经前体细胞中存在差异表达，首次为一类潜在参与多能性与干细胞命运调控的新型功能性非编码RNA家族提供了实验证据。本研究进一步发现，属于非聚腺苷酸化类型的次要剪接体小核RNA在胚胎干细胞中几乎完全缺失，而在分化过程中会上调表达。最后，本研究证实多梳家族基因（polycomb group gene）Eed的次要内含子存在差异剪接加工。本研究数据表明，无论是已知还是新发现的非聚腺苷酸化RNA，均在胚胎干细胞中发挥重要功能。实验设计概述：本研究从未分化的小鼠胚胎干细胞以及培养7天的神经前体细胞中提取总RNA。采用改良的Oligotex提取法（Qiagen，凯杰公司）将总RNA分为聚腺苷酸化富集组分（poly(A+)）与非聚腺苷酸化富集组分（poly(A-)）。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-乙醇沉淀法获取非聚腺苷酸化富集组分。本次提交的为该研究的RNA测序（RNA-Seq）部分数据。