New insights into miR398 functions in Arabidopsis

Abstract

We recently identified a new target of microRNA398 (miR398), a conserved miRNA in plants. In Arabidopsis, miR398 targets the mRNAs of two copper/zinc superoxide dismutases (Cu/Zn SODs) by triggering their cleavage or repressing their translation. We analysed the transcriptomes of mutants impaired in miR398 production, revealing that the mRNAs encoding the chaperone (CCS1), essential for copper delivering to the Cu/Zn SODs of Arabidopsis and to generate the mature proteins, were undiscovered targets of miR398. It is likely that CCS1 was not identified by previous bioinformatic predictions because of the number of mismatches between the mRNA and its target. Since CCS1 has four mismatches and one GU wobble, it would have been excluded by the majority of prediction algorithms. miR398 directs the post-transcriptional regulation of CCS1 mRNAs by cleavage and ARGONAUTE10 (AGO10)-mediated translational repression. Indeed, CCS1 protein accumulate in zwille (ago10) mutants while both miR398 and CCS1 mRNAs levels remain identical to the Landsberg erecta WT plants. 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.

Figure 1

miR398 targets in Arabidopsis. The sequence (Col-0) of the miR398 complementary sites in the target mRNA s is aligned with the sequences of miR398a and miR398b/c. In Arabidopsis, miR398 is encoded by three genes, MIR398a, MIR398b and MIR398c.– The miRNAs produced from MIR398b and c are identical and differ by one nucleotide from the one produced by MIR398a. The arrows indicate the cleavage sites experimentally validated by a modified version of the 5′-RACE PCR and localized between the nucleotides 10 and 11 of the miRNA , like for many other targets of miRNAs in Arabidopsis. The region corresponding to the seed is in bold.

Figure 2

CCS1 protein levels in ago1, ago10 and ago1 ago10 Col mutants. Immunodetection of CCS1 and CSD2 proteins from Col, ago1-27 hypomorphic mutant, ago10-3, (SALK_519738) and the double ago1-27 ago10-3, mutant. Plants were grown for 12 days in vitro, with (0.5 µM of CuSO₄) or without copper added to the medium. In limiting copper conditions, miR398 levels are kept high, and as a consequence, CSD1, CSD2, and CCS1, transcripts are cleaved resulting in low amounts of proteins. Thus, the changes of Cu contents in the medium are directly modulating the levels of miR398. Total proteins (10 µg) were extracted in bulks (10 to 15 plants) and CCS1 or CSD2 proteins were detected with anti-CCS1 or anti-CSD1/2 polyclonal antibodies (Agrisera AB, Sweden) after SDS-PAGE. Staining of Rubisco small subunit (RbcS) by Coomassie blue served as control of equal loading. mRNA and miRNA levels are described in reference .