Transcriptome-wide identification of A > I RNA editing sites by inosine specific cleavage
Transcriptome-wide identification of A > I RNA editing sites by inosine specific cleavage
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DOI：
10.1261/rna.036202.112
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发表时间：
2013-02-01
期刊：
影响因子：
4.5
通讯作者：
Mattick, John S.
中科院分区：
文献类型：
--
作者：
Cattenoz, Pierre B.;Taft, Ryan J.;Mattick, John S.
由蛋白质的阿达尔家族催化的腺苷至肌苷（A > I）RNA编辑是产生转录组多样性的基本机制之一。事实上，许多全基因组分析表明，A > I编辑并不像最初认为的那样仅限于少数mRNA，而是广泛发生在转录组中，特别是在大脑中。重要的是，越来越多的证据表明A > I编辑对动物发育和神经系统功能至关重要。为了更有效地表征哺乳动物转录组中阿达尔事件的完整目录，我们开发了一种高通量方案来鉴定A > I编辑位点，其利用乙二醛保护鸟苷而不是肌苷免受RNA酶T1处理的能力，从而促进在其末端具有肌苷碱基的RNA片段的提取以用于高通量测序。使用这种方法，我们确定了小鼠大脑RNA中的665个编辑位点，包括大多数已知的位点和一套新的位点，包括蛋白质编码基因的非同义变化，已知调节p53的基因的超编辑，以及非蛋白质编码RNA的改变。该方法适用于任何生物系统，用于从头发现A > I编辑位点，并且避免了与使用传统RNA测序数据的编辑位点鉴定相关的复杂的信息学和实际问题。这种方法有可能大大增加我们对RNA编辑的程度和功能的理解，从而阐明转录可塑性在进化，发育和认知中的作用。
Adenosine to inosine( A > I) RNA editing, which is catalyzed by the ADAR family of proteins, is one of the fundamental mechanisms by which transcriptomic diversity is generated. Indeed, a number of genome-wide analyses have shown that A > I editing is not limited to a few mRNAs, as originally thought, but occurs widely across the transcriptome, especially in the brain. Importantly, there is increasing evidence that A > I editing is essential for animal development and nervous system function. To more efficiently characterize the complete catalog of ADAR events in the mammalian transcriptome we developed a high-throughput protocol to identify A > I editing sites, which exploits the capacity of glyoxal to protect guanosine, but not inosine, from RNAse T1 treatment, thus facilitating extraction of RNA fragments with inosine bases at their termini for high-throughput sequencing. Using this method we identified 665 editing sites in mouse brain RNA, including most known sites and suite of novel sites that include nonsynonymous changes to protein-coding genes, hyperediting of genes known to regulate p53, and alterations to non-protein-coding RNAs. This method is applicable to any biological system for the de novo discovery of A > I editing sites, and avoids the complicated informatic and practical issues associated with editing site identification using traditional RNA sequencing data. This approach has the potential to substantially increase our understanding of the extent and function of RNA editing, and thereby to shed light on the role of transcriptional plasticity in evolution, development, and cognition.