Identification of MicroRNA Processing Determinants by Random Mutagenesis of Arabidopsis MIR172a Precursor
Identification of MicroRNA Processing Determinants by Random Mutagenesis of Arabidopsis MIR172a Precursor
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DOI：
10.1016/j.cub.2009.10.072
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发表时间：
2010-01-12
期刊：
影响因子：
9.2
通讯作者：
Palatnik, Javier F.
中科院分区：
文献类型：
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作者：
Mateos, Julieta L.;Bologna, Nicolas G.;Palatnik, Javier F.
microRNA（miRNAs）是广泛存在的基因表达转录后调节因子。它们是从含有折返结构的较长初级转录本加工而来的（在[1，2]中进行了综述）。在动物中，Drosha和DGCR 8/Pasha形成的复合物识别单链RNA序列和茎环之间的过渡，以产生miRNA生物发生中的第一个切割步骤[3]。而动物的前体是统一的大小和形状，其植物对应物包括一组可变的茎环，并在其处理过程中识别的结构线索知之甚少。在此，我们设计了一种基于MIR 172 a前体随机诱变的无偏方法来研究植物中的miRNA加工。随机突变的前体在拟南芥中过表达，并在体内测定其活性。我们从这些转基因中收集序列数据，并使用它来构建MIR 172 a前体图谱，突出显示其处理的相关和中性位置。miRNA/miRNA（星星）双链体下方的15个核苷酸茎段对于MIR 172 a加工是必需的。相比之下，末端环区域的突变大多是中性的，但miR 172的生物发生需要一个环。结果可以扩展到其他前体，表明至少部分植物前体存在共同特征。
MicroRNAs (miRNAs) are widespread posttranscriptional regulators of gene expression. They are processed from longer primary transcripts that contain foldback structures (reviewed in [1, 2]). In animals, a complex formed by Drosha and DGCR8/Pasha recognizes the transition between the single-stranded RNA sequences and the stem loop to produce the first cleavage step in miRNA biogenesis [3]. Whereas animal precursors are of uniform size and shape, their plant counterparts comprise a collection of variable stem loops, and little is known about the structural clues recognized during their processing. Here, we designed an unbiased approach based on the random mutagenesis of the MIR172a precursor to study miRNA processing in plants. Randomly mutated precursors were overexpressed in Arabidopsis, and their activity was determined in vivo. We gathered sequence data from these transgenes and used it to build a MIR172a precursor map highlighting relevant and neutral positions for its processing. A 15 nucleotide stem segment below the miRNA/miRNA(star) duplex was essential for MIR172a processing. In contrast, mutations in the terminal-loop region were mostly neutral, yet a loop was required for miR172 biogenesis. The results could be extended to other precursors, suggesting the existence of common features in at least part of the plant precursors.