Complex and dynamic landscape of RNA polyadenylation revealed by PAS-Seq
Complex and dynamic landscape of RNA polyadenylation revealed by PAS-Seq
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DOI：
10.1261/rna.2581711
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发表时间：
2011-04-01
期刊：
影响因子：
4.5
通讯作者：
Shi, Yongsheng
中科院分区：
文献类型：
--
作者：
Shepard, Peter J.;Choi, Eun-A;Shi, Yongsheng
在高等真核生物中，mRNAs的选择性多聚腺苷酸化(APA)已成为转录后基因调控的重要机制。尽管微阵列最近被用来在全球范围内表征APA，但它们有许多严重的限制，阻碍了全面和高度定量的分析。为了更好地表征APA及其调控，我们开发了一种基于深度测序的方法，称为Poly(A)Site Sequence(PAS-Seq)，用于在转录组水平上定量分析RNA的聚腺苷酸化。PAS-Seq不仅准确和全面地识别mRNAs和非编码RNAs中的PolyA连接，而且还提供了关于多腺苷化RNAs相对丰度的定量信息。对人和小鼠转录本的PAS-Seq分析表明，所有表达的基因中有40%-50%产生交替的多腺化mRNAs。此外，我们的研究检测到了进化上保守的组蛋白mRNAs的聚腺苷酸化，并揭示了线粒体RNA聚腺苷酸化的新特征。最后，对小鼠胚胎干细胞(ES)、神经干细胞/祖细胞(NSP)和神经元的PAS-SEQ分析不仅确定了比整个小鼠EST数据库中发现的更多的聚(A)位点，而且还检测到全球APA谱的显著变化，导致在干细胞分化过程中许多mRNAs中的3‘非翻译区(UTR)变长。总之，我们的PAS-Seq分析揭示了哺乳动物细胞中RNA多腺化的复杂图景和干细胞分化过程中APA的动态调节。
Alternative polyadenylation (APA) of mRNAs has emerged as an important mechanism for post-transcriptional gene regulation in higher eukaryotes. Although microarrays have recently been used to characterize APA globally, they have a number of serious limitations that prevents comprehensive and highly quantitative analysis. To better characterize APA and its regulation, we have developed a deep sequencing-based method called Poly(A) Site Sequencing (PAS-Seq) for quantitatively profiling RNA polyadenylation at the transcriptome level. PAS-Seq not only accurately and comprehensively identifies poly(A) junctions in mRNAs and noncoding RNAs, but also provides quantitative information on the relative abundance of polyadenylated RNAs. PAS-Seq analyses of human and mouse transcriptomes showed that 40%-50% of all expressed genes produce alternatively polyadenylated mRNAs. Furthermore, our study detected evolutionarily conserved polyadenylation of histone mRNAs and revealed novel features of mitochondrial RNA polyadenylation. Finally, PAS-Seq analyses of mouse embryonic stem (ES) cells, neural stem/progenitor (NSP) cells, and neurons not only identified more poly(A) sites than what was found in the entire mouse EST database, but also detected significant changes in the global APA profile that lead to lengthening of 3' untranslated regions (UTR) in many mRNAs during stem cell differentiation. Together, our PAS-Seq analyses revealed a complex landscape of RNA polyadenylation in mammalian cells and the dynamic regulation of APA during stem cell differentiation.