Alternative splicing regulates mouse embryonic stem cell pluripotency and differentiation
Alternative splicing regulates mouse embryonic stem cell pluripotency and differentiation
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DOI：
10.1073/pnas.0912260107
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发表时间：
2010-06-08
影响因子：
11.1
通讯作者：
Conklin, Bruce R.
中科院分区：
文献类型：
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作者：
Salomonis, Nathan;Schlieve, Christopher R.;Conklin, Bruce R.
再生医学的两个主要目标是重复地将成年体细胞转化为多能状态，并控制它们分化为特定的细胞命运。通过选择性剪接(AS)和选择性启动子选择(APS)获得这些细胞产生的RNA异构体的完整图像，将极大地有助于实现这些目标的进展。为了研究AS和APS的作用，我们在全基因组水平上利用Affymetrix基因芯片原型研究了小鼠胚胎干细胞分化过程中的相互外显子-外显子连接。利用最近发布的开源软件包AltAnalyze，我们鉴定了170个可能的异构体变体的144个基因，其中大部分(67%)被预测会改变蛋白质序列和结构域组成。已证实的可选外显子在很大程度上与Wnt信号和细胞周期控制的途径有关，并且大多数在老鼠和人类之间保守。为了研究AS的功能影响，我们研究了两个基因的异构体。正如AltAnalyze预测的那样，我们发现SERCA2基因的不同亚型被不同的microRNAs(miRNA-200b，miRNA-214)靶向，这表明AS在心脏发育中起着关键作用。对Wnt转录因子Tcf3的分析表明，在ES细胞中存在几个不同的转录抑制靶点(Stmn2、CCND2、ATF3、Klf4、Nodal和Jun)以及不同的分化结果。这里的发现说明了AS在ES细胞分化规范中的关键作用，并强调了AS的全球功能分析的实用性。
Two major goals of regenerative medicine are to reproducibly transform adult somatic cells into a pluripotent state and to control their differentiation into specific cell fates. Progress toward these goals would be greatly helped by obtaining a complete picture of the RNA isoforms produced by these cells due to alternative splicing (AS) and alternative promoter selection (APS). To investigate the roles of AS and APS, reciprocal exon-exon junctions were interrogated on a genome-wide scale in differentiating mouse embryonic stem (ES) cells with a prototype Affymetrix microarray. Using a recently released open-source software package named AltAnalyze, we identified 144 genes for 170 putative isoform variants, the majority (67%) of which were predicted to alter protein sequence and domain composition. Verified alternative exons were largely associated with pathways of Wnt signaling and cell-cycle control, and most were conserved between mouse and human. To examine the functional impact of AS, we characterized isoforms for two genes. As predicted by AltAnalyze, we found that alternative isoforms of the gene Serca2 were targeted by distinct microRNAs (miRNA-200b, miRNA-214), suggesting a critical role for AS in cardiac development. Analysis of the Wnt transcription factor Tcf3, using selective knockdown of an ES cell-enriched and characterized isoform, revealed several distinct targets for transcriptional repression (Stmn2, Ccnd2, Atf3, Klf4, Nodal, and Jun) as well as distinct differentiation outcomes in ES cells. The findings herein illustrate a critical role for AS in the specification of ES cells with differentiation, and highlight the utility of global functional analyses of AS.