microRNA-directed cleavage and translational repression of the copper chaperone for superoxide dismutase mRNA in Arabidopsis

Abstract

SUMMARY 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.

Short tandem target mimic rice lines uncover functions of miRNAs in regulating important agronomic traits

“…We further investigated miR398, which has been extensively studied and is thought to regulate the response to abiotic and biotic stresses by promoting cleavage of its target genes, Cu/Znsuperoxide dismutases (CSD1 and CSD2) (20)(21)(22)(23). Although no developmental or architectural phenotypes have been associated with this miRNA in Arabidopsis, we found that silencing of miR398 resulted in rice lines (STTM398) that were significantly shorter than wild-type (WT) plants (62.7 ± 4.1 cm vs. 86.7 ± 2.4 cm, respectively) ( Fig.…”

Literature review of baseline information to support the risk assessment of RNAi‐based GM plants

“…Similarly to mammalian miRNAs, plant miRNAs can also mediate translational repression and sequence-specific cleavage of cognate mRNAs (e.g. Rhoades et al, 2002;Brodersen et al, 2008;Mallory and Bouche, 2008;Beauclair et al, 2010;. At the same time, activities of plant mRNAs differ from their animal counterparts in several aspects.…”

New insights into miR398 functions in Arabidopsis

“…In the Landsberg erecta (Ler) accession, the CSD2 protein accumulate in zll (ago10) mutants whereas both the levels of the corresponding mRNA and the level of miR398 remain almost unchanged. 5,15 This is suggesting that AGO10 is probably involved in miR398-mediated translational inhibition of CSD2 mRNA. We have shown that CCS1 protein accumulate in zll mutants in a similar way, suggesting that AGO10 is also involved in miR398-directed posttranscriptional regulation of CCS1.…”

“…We have shown that CCS1 protein accumulate in zll mutants in a similar way, suggesting that AGO10 is also involved in miR398-directed posttranscriptional regulation of CCS1. 5 Thus, miR398 acts through both translational finding that CCS1 protein accumulate in these plants even when the miRNA is abundant. Altogether, the number of targets revealed by our transcriptome analyses (designed to identify cleaved targets) is very limited.…”

“…Moreover, since AGO10 is a negative regulator of AGO1, the CCS1 protein is more abundant in a double ago1-27 ago10-3 Col mutant compared to the single hypomorphic ago1-27 mutant, as previously shown for CSD2. 5 that delivers the copper cofactor to the three Cu/Zn SODs (CSD1, 2 and 3). miR398 seems to be involved in responses to biotic and abotic stresses.…”