Detailed characterization of the mouse embryonic stem cell transcriptome reveals novel genes and intergenic splicing associated with pluripotency
Detailed characterization of the mouse embryonic stem cell transcriptome reveals novel genes and intergenic splicing associated with pluripotency
复制标题
DOI：
10.1186/1471-2164-9-155
复制
发表时间：
2008-04-09
期刊：
影响因子：
4.4
通讯作者：
Lipovich, Leonard
中科院分区：
文献类型：
--
作者：
Kunarso, Galih;Wong, Kee-Yew;Lipovich, Leonard
背景：胚胎干（ES）细胞多能性的转录控制一直是深入研究的主题。 Oct4（Oct3/4 指数）、Sox2 和 Nanog 等转录调控因子对于维持未分化状态至关重要。然而，ES 细胞转录组并不限于其靶标，并且在使用微阵列、MPSS、cDNA/EST 测序和 SAGE 技术进行分析时表现出相当的复杂性。为了鉴定与多能性相关的新基因，我们在全球范围内搜索与已知基因不对应的 ES 转录本，验证其序列，确定其表达谱，并采用 RNAi 测试其功能。结果：基因识别特征 (GIS) 分析（一种通过配对 5' 和 3' 转录本末端标签区分的 SAGE 衍生物）在小鼠 E14 ES mRNA 文库中鉴定出 153 个与已知基因不同的候选新转录单位 (TU)。我们重点研究了 16 个没有人工制品和图谱差异的 TU，其中 5 个通过 RTPCR 产品测序进行了验证。通过注释揭示了其中两个 TU 代表新的蛋白质编码基因：PRY 结构域簇成员和 KRAB 结构域锌指。其他三个 TU 代表基因间剪接事件，涉及以相同方向转录的相邻、功能上不相关的蛋白质编码基因，其中一个事件可能编码包含来自两个组成基因（Clk2 和 Scamp3）的结构域的融合蛋白。使用胚胎样本和成体组织组的表达谱证实，其中三个 TU 是 ES 细胞所独有的或在 ES 细胞中表达最高。在对培养的 ES 细胞进行三种不同的化学诱导分化处理期间，所有 5 个 TU 的表达水平均急剧下降。然而，TU 的 siRNA 敲低并没有改变多能性或分化标记物的 mRNA 水平，也没有影响细胞形态。结论：尽管最近进行了大量的 cDNA 和 EST 测序工作，但转录组文库在发现新基因方面仍然具有相当大的潜力；这些 ES 细胞 TU 的 cDNA 和 EST 证据有限或不存在。 RTPCR 和全长测序对于解决从高通量测序推断出的大量候选新转录本与可验证的小部分转录本之间的瓶颈仍然至关重要。 RNAi 结果表明，尽管这五种转录组学新颖性与多能性密切相关，但它们可能不需要维持多能性。
Background: Transcriptional control of embryonic stem (ES) cell pluripotency has been a subject of intense study. Transcriptional regulators including Oct4 (Oct3/4 index), Sox2 and Nanog are fundamental for maintaining the undifferentiated state. However, the ES cell transcriptome is not limited to their targets, and exhibits considerable complexity when assayed with microarray, MPSS, cDNA/EST sequencing, and SAGE technologies. To identify novel genes associated with pluripotency, we globally searched for ES transcripts not corresponding to known genes, validated their sequences, determined their expression profiles, and employed RNAi to test their function.Results: Gene Identification Signature (GIS) analysis, a SAGE derivative distinguished by paired 5' and 3' transcript end tags, identified 153 candidate novel transcriptional units (TUs) distinct from known genes in a mouse E14 ES mRNA library. We focused on 16 TUs free of artefacts and mapping discrepancies, five of which were validated by RTPCR product sequencing. Two of the TUs were revealed by annotation to represent novel protein-coding genes: a PRY-domain cluster member and a KRAB-domain zinc finger. The other three TUs represented intergenic splicing events involving adjacent, functionally unrelated protein-coding genes transcribed in the same orientation, with one event potentially encoding a fusion protein containing domains from both component genes (Clk2 and Scamp3). Expression profiling using embryonic samples and adult tissue panels confirmed that three of the TUs were unique to or most highly expressed in ES cells. Expression levels of all five TUs dropped dramatically during three distinct chemically induced differentiation treatments of ES cells in culture. However, siRNA knockdowns of the TUs did not alter mRNA levels of pluripotency or differentiation markers, and did not affect cell morphology.Conclusion: Transcriptome libraries retain considerable potential for novel gene discovery despite massive recent cDNA and EST sequencing efforts; cDNA and EST evidence for these ES cell TUs had been limited or absent. RTPCR and full-length sequencing remain essential in resolving the bottleneck between numerous candidate novel transcripts inferred from high-throughput sequencing and the small fraction that can be validated. RNAi results indicate that, despite their strong association with pluripotency, these five transcriptomic novelties may not be required for maintaining it.