Construction of Specific Parallel Amplification of RNA Ends (SPARE) libraries for the systematic identification of plant microRNA processing intermediates
Construction of Specific Parallel Amplification of RNA Ends (SPARE) libraries for the systematic identification of plant microRNA processing intermediates
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DOI：
10.1016/j.ymeth.2013.08.032
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发表时间：
2013-12-15
期刊：
影响因子：
4.8
通讯作者：
Palatnik, Javier F.
中科院分区：
文献类型：
--
作者：
Schapire, Arnaldo L.;Bologna, Nicolas G.;Palatnik, Javier F.
MicroRNAs(MiRNAs)是一种小RNA，来源于具有折叠结构的内源性前体。植物miRNA前体在大小和形状上有很大的变化。尽管如此，由Disher-like 1(DCL1)和辅助蛋白组成的miRNA处理机制识别前体上的结构特征，在特定位置将其切割，释放成熟的miRNAs。植物中miRNA加工中间体的鉴定主要依赖于一种改进的5‘RACE方法，该方法旨在检测未封顶RNA的5’端。然而，这种方法很耗时，因此只适用于少数miRNAs的分析。在这里，我们提出了一种改进的方法，以执行全基因组分析的miRNA加工中间体。简而言之，用针对所有已知miRNA前体设计的特定引物的混合物进行逆转录。然后，对miRNA加工中间体进行特异性扩增，生成文库，并进行深度测序。这种方法被称为SPARE(5‘RNA末端的特异性并行扩增)，它可以识别大多数拟南芥miRNAs的加工中间产物。该结果有助于确定DCL1的加工方向和miRNA加工机制在前体中引入的切割位置。Sare方法可以很容易地用于检测其他系统中的miRNA加工中间体。(C)2013 Elsevier Inc.保留所有权利。
MicroRNAs (miRNAs) are small RNAs that derive from endogenous precursors harboring foldback structures. Plant miRNA precursors are quite variable in their size and shape. Still, the miRNA processing machinery, consisting of DICER-LIKE1 (DCL1) and accessory proteins recognize structural features on the precursors to cleave them at specific places releasing the mature miRNAs. The identification of miRNA processing intermediates in plants has mostly relied on a modified 5' RACE method, designed to detect the 5' end of uncapped RNAs. However, this method is time consuming and is, therefore, only practical for the analysis of a handful miRNAs. Here, we present a modification of this approach in order to perform genome-wide analysis of miRNA processing intermediates. Briefly, a reverse transcription is performed with a mixture of specific primers designed against all known miRNA precursors. miRNA processing intermediates are then specifically amplified to generate a library and subjected to deep sequencing. This method, called SPARE (Specific Parallel Amplification of 5' RNA Ends) allows the identification of processing intermediates for most of the Arabidopsis miRNAs. The results enable the determination of the DCL1 processing direction and the cleavage sites introduced by miRNA processing machinery in the precursors. The SPARE method can be easily adapted to detect miRNA-processing intermediates in other systems. (c) 2013 Elsevier Inc. All rights reserved.