microRNA-directed cleavage and translational repression of the copper chaperone for superoxide dismutase mRNA in Arabidopsis.
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.
Institut Jean Pierre Bourgin, UMR1318 INRA-AgroParisTech, INRA Centre de Versailles-Grignon, route de St Cyr, F-78026 Versailles, France.
Linda
L
Beauclair
Agnès
A
Yu
Nicolas
N
Bouché
D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000378:metabolism; D060565:Argonaute Proteins; D003300:Copper / Q000378:metabolism*; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism*; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics; D012323:RNA Processing, Post-Transcriptional; D018749:RNA, Plant / Q000235:genetics; D013482:Superoxide Dismutase / Q000378:metabolism*
D029681:Arabidopsis Proteins; D060565:Argonaute Proteins; D003300:Copper; D035683:MicroRNAs; D018832:Molecular Chaperones; D018749:RNA, Plant; D013482:Superoxide Dismutase; C529694:AT5G43810 protein, Arabidopsis; C543882:MIRN398 microRNA, Arabidopsis