microRNA-directed cleavage and translational repression of the copper chaperone for superoxide dismutase mRNA in Arabidopsis
microRNA-directed cleavage and translational repression of the copper chaperone for superoxide dismutase mRNA in Arabidopsis
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DOI：
10.1111/j.1365-313x.2010.04162.x
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发表时间：
2010-05-01
期刊：
影响因子：
7.2
通讯作者：
Bouche, Nicolas
中科院分区：
文献类型：
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作者：
Beauclair, Linda;Yu, Agnes;Bouche, Nicolas
微小RNA398（miR398）是植物中一种保守的微小RNA，它通过触发裂解或抑制其信使RNA（mRNA）的翻译，靶向拟南芥三种铜/锌超氧化物歧化酶（SOD）中的两种（CSD1和CSD2）。我们分析了miR398产生受损的突变体的转录组，发现编码超氧化物歧化酶铜伴侣蛋白（CCS1）的mRNA是miR398未被发现的靶标，CCS1在不同的细胞区室中将铜传递给CSD1和CSD2脱辅基蛋白。我们通过5'-RACE PCR确定了CCS1 mRNA中的裂解位点。我们进一步表明，CCS1蛋白和mRNA水平都与miR398的量紧密相关，而miR398的量本身又取决于培养基中的铜含量。我们培育了携带一种对miR398裂解有抗性的CCS1 mRNA版本的转基因植物，并证明当miR398含量丰富且铜受限时，这些植物中CCS1的mRNA和蛋白都会积累。此外，我们表明拟南芥的十种AGO蛋白之一（AGO10）参与了miR398对CCS1 mRNA的翻译抑制，因为在ago10（zll）突变体中，CCS1蛋白积累而CCS1 mRNA不积累。因此，miR398介导了编码CCS1的mRNA的裂解和翻译抑制，CCS1是对产生拟南芥成熟的铜/锌SOD至关重要的伴侣蛋白。我们的结果还意味着，即使对于像miR398这样被广泛研究的微小RNA，仍有尚未通过计算分析鉴定出来的新靶标有待发现。
P>microRNA398 (miR398) is a conserved miRNA of plants that targets two of the three copper/zinc superoxide dismutases (SOD) of Arabidopsis (CSD1 and CSD2) by triggering cleavage or inhibiting translation of their mRNAs. We analysed the transcriptomes of mutants impaired in miR398 production, and found that the mRNAs encoding the copper chaperone for superoxide dismutase (CCS1), which delivers copper to CSD1 and CSD2 apoproteins in different cellular compartments, are undiscovered targets of miR398. We identified the cleavage site in CCS1 mRNAs by 5'-RACE PCR. We further show that both CCS1 protein and mRNA levels are tightly linked to the quantities of miR398, which are themselves dependent on the copper content in the medium. We generated transgenic plants carrying a CCS1 mRNA version resistant to cleavage by miR398, and demonstrated that both CCS1 mRNAs and proteins accumulate in these plants when miR398 is abundant and copper limiting. Moreover, we show that one of the ten ARGONAUTE proteins of Arabidopsis (AGO10) is involved in miR398-directed translational inhibition of CCS1 mRNAs, as CCS1 protein, but not CCS1 mRNAs accumulates in ago10 (zll) mutants. Thus, miR398 mediates the cleavage and translational inhibition of mRNAs encoding CCS1, the chaperone protein that is essential for generating the mature copper/zinc SODs of Arabidopsis. Our results also imply that new targets that have not been identified by computing analyses have yet to be discovered, even for an extensively studied miRNA such as miR398.