Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome
Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome
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DOI：
10.1016/j.cub.2008.04.042
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发表时间：
2008-05-20
期刊：
影响因子：
9.2
通讯作者：
Axtell, Michael J.
中科院分区：
文献类型：
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作者：
Addo-Quaye, Charles;Eshoo, Tifani W.;Axtell, Michael J.
MicroRNAs(MiRNAs)调节植物和动物中目标mRNAs的表达[1]。植物miRNA靶标的预测是基于它们与miRNAs的广泛且往往保守的互补性[2-4]，以及miRNA过表达实验[5]；许多这些靶标预测已经通过分离miRNA指导的切割产物而得到证实。在这里，我们提出了一种转录组范围的实验方法，称为“降解dome测序”，可以直接检测被切割的miRNA靶标，而不依赖于预测或过度表达。来自拟南芥的多腺苷化的、未封顶的mRNAs的5‘端被直接采样，得到了降解穹顶的经验快照。MiRNA介导的切割产物很容易从降解的mRNAs的广泛背景中辨别出来，这些mRNAs共同覆盖了注释转录组的大部分。许多已知的拟南芥miRNA靶点得到了确认，也发现了几个新的靶点。对切割片段的定量分析表明，那些来自TAS转录本的片段积累到了非常高的水平，这些转录本产生了大量的次级小干扰RNA(SiRNAs)。已知二级siRNAs的一个子集直接切割反式[6]中的靶标；降解基因组测序显示这些反式作用的siRNAs(ta-siRNAs)有许多切割的靶标。这种经验方法广泛适用于没有先验预测的小RNA裂解靶的发现和量化。
MicroRNAs (miRNAs) regulate the expression of target mRNAs in plants and animals [1]. Plant miRNA targets have been predicted on the basis of their extensive and often conserved complementarity to the miRNAs [2-4], as well as on miRNA overexpression experiments [5]; many of these target predictions have been confirmed by isolation of the products of miRNA-directed cleavage. Here, we present a transcriptome-wide experimental method, called "degradome sequencing," to directly detect cleaved miRNA targets without relying on predictions or overexpression. The 5' ends of polyadenylated, uncapped mRNAs from Arabidopsis were directly sampled, resulting in an empirical snapshot of the degradome. miRNA-mediated-cleavage products were easily discerned from an extensive background of degraded mRNAs, which collectively covered the majority of the annotated transcriptome. Many previously known Arabidopsis miRNA targets were confirmed, and several novel targets were also discovered. Quantification of cleavage fragments revealed that those derived from TAS transcripts, which are unusual in their production of abundant secondary small interfering RNAs (siRNAs), accumulated to very high levels. A subset of secondary siRNAs are also known to direct cleavage of targets in trans [6]; degradome sequencing revealed many cleaved targets of these trans-acting siRNAs (ta-siRNAs). This empirical method is broadly applicable to the discovery and quantification of cleaved targets of small RNAs without a priori predictions.