Institut Jean Pierre Bourgin, UMR1318 INRA-AgroParisTech, INRA Centre de Versailles-Grignon, route de St Cyr, F-78026 Versailles, France.
Plant J. 2010 May;62(3):454-62. doi: 10.1111/j.1365-313X.2010.04162.x. Epub 2010 Feb 1.
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.
miR398（miR398）是植物中的一种保守 miRNA，通过触发其 mRNA 的切割或抑制翻译，靶向拟南芥（CSD1 和 CSD2）中的三种铜/锌超氧化物歧化酶（SOD）中的两种。我们分析了 miR398 产生缺陷的突变体的转录组，发现编码超氧化物歧化酶铜伴侣（CCS1）的 mRNA 是 miR398 的未被发现的靶标，CCS1 将铜递送给不同细胞区室的 CSD1 和 CSD2 脱辅基蛋白。我们通过 5'-RACE PCR 鉴定了 CCS1 mRNA 中的切割位点。我们进一步表明，CCS1 蛋白和 mRNA 水平与 miR398 的数量紧密相关，而 miR398 的数量本身又依赖于培养基中的铜含量。我们生成了携带对 miR398 切割具有抗性的 CCS1 mRNA 版本的转基因植物，并证明当 miR398 丰富且铜有限时，CCS1 mRNA 和蛋白质在这些植物中积累。此外，我们表明拟南芥的十种 ARGONAUTE 蛋白之一（AGO10）参与 miR398 指导的 CCS1 mRNA 的翻译抑制，因为 AGO10（zll）突变体中积累了 CCS1 蛋白，但不是 CCS1 mRNA。因此，miR398 介导了 CCS1 编码 mRNA 的切割和翻译抑制，CCS1 是生成拟南芥成熟铜/锌 SOD 的必需伴侣蛋白。我们的结果还表明，即使对于像 miR398 这样经过广泛研究的 miRNA，计算分析尚未识别的新靶标仍有待发现。
