Bioinformatic prediction and experimental validation of a microRNA-directed tandem trans-acting siRNA cascade in Arabidopsis
Bioinformatic prediction and experimental validation of a microRNA-directed tandem trans-acting siRNA cascade in Arabidopsis
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DOI：
10.1073/pnas.0611119104
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发表时间：
2007-02-27
影响因子：
11.1
通讯作者：
Wu, Shu-Hsing
中科院分区：
文献类型：
--
作者：
Chen, Ho-Ming;Li, Yi-Hang;Wu, Shu-Hsing
小分子RNA在高等真核生物基因表达调控中起着重要作用。其中，反式作用siRNA（ta-siRNA）是一类调控植物发育的小分子RNA。ta-siRNA的生物发生取决于microRNA靶向切割，然后是DCL 4介导的小RNA的产生，相对于两条链上的切割位点以21-nt增量定相。为了找到TAS基因，我们利用这些特征开发了第一个计算算法，该算法允许从任何给定的小RNA数据库中对推定的TAS基因进行全面搜索和统计评估。用该算法在拟南芥小RNA大规模并行签名测序（MPSS）数据库中的搜索揭示了已知和先前未知的ta-siRNA产生位点。我们通过实验验证了来自两个PPR基因的ta-siRNA的生物发生和其中一个ta-siRNA的反式作用活性。从鉴定的PPR基因产生ta-siRNA是通过切割TAS2衍生的ta-siRNA来指导的，而不是如先前报道的TASia、-B、-c、TAS2和TAS3基因的MicroRNA。我们的研究结果表明存在一个小RNA调控级联反应，该级联反应由miR173定向切割启动，随后由两个TAS基因连续产生ta-siRNA。
Small RNAs play pivotal roles in regulating gene expression in higher eukaryotes. Among them, trans-acting siRNAs (ta-siRNAs) are a class of small RNAs that regulate plant development. The biogenesis of ta-siRNA depends on microRNA-targeted cleavage followed by the DCL4-mediated production of small RNAs phased in 21-nt increments relative to the cleavage site on both strands. To find TAS genes, we have used these characteristics to develop the first computational algorithm that allows for a comprehensive search and statistical evaluation of putative TAS genes from any given small RNA database. A search in Arabidopsis small RNA massively parallel signature sequencing (MPSS) databases with this algorithm revealed both known and previously unknown ta-siRNA-producing loci. We experimentally validated the biogenesis of ta-siRNAs from two PPR genes and the trans-acting activity of one of the ta-siRNAs. The production of ta-siRNAs from the identified PPR genes was directed by the cleavage of a TAS2derived ta-siRNA instead of by MicroRNAs as was reported previously for TASia, -b, -c, TAS2, and TAS3 genes. Our results indicate the existence of a small RNA regulatory cascade initiated by miR173-directed cleavage and followed by the consecutive production of ta-siRNAs from two TAS genes.