Plant microRNAs (miRNAs) typically form near-perfect duplexes with their targets and mediate mRNA cleavage. Here, we describe an unconventional miRNA target of miR398 in Arabidopsis, an mRNA encoding the blue copper-binding protein (BCBP). BCBP mRNA carries an miR398 complementary site in its 5′-untranslated region (UTR) with a bulge of six nucleotides opposite to the 5′ region of the miRNA. Despite the disruption of a target site region thought to be especially critical for function, BCBP mRNAs are cleaved by ARGONAUTE1 between nucleotides 10th and 11th, opposite to the miRNA, like conventional plant target sites. Levels of BCBP mRNAs are inversely correlated to levels of miR398 in mutants lacking the miRNA, or transgenic plants overexpressing it. Introducing two mutations that disrupt the miRNA complementarity around the cleavage site renders the target cleavage-resistant. The BCBP site functions outside of the context of the BCBP mRNA and does not depend on 5′-UTR location. Reducing the bulge does not interfere with miR398-mediated regulation and completely removing it increases the efficiency of the slicing. Analysis of degradome data and target predictions revealed that the miR398-BCBP interaction seems to be rather unique. Nevertheless, our results imply that functional target sites with non-perfect pairings in the 5′ region of an ancient conserved miRNA exist in plants.
The extent of epigenetic variation is currently well documented, but the number of natural epialleles described so far remains very limited. Determining the relevance of epigenetic changes for natural variation is an important question of research that we investigate by isolating natural epialleles segregating in Arabidopsis recombinant populations. We previously described a genetic incompatibility among Arabidopsis strains based on the silencing of a gene involved in fitness. Here, we isolated a new epiallele resulting from the silencing of a transfer-RNA editing gene in an Arabidopsis accession from the Netherlands (Nok-1). Crosses with the reference accession Col-0 show a complete incompatibility between this epiallele and another locus localized on a different chromosome. We demonstrate that conversion of an unmethylated version of this allele occurs in hybrids, associated with modifications of small RNA populations. These epialleles can also spontaneously revert within the population. Furthermore, we bring evidence that neither METHYLTRANSFERASE 1, maintaining methylation at CGs, nor components of RNA-directed DNA methylation, are key factors for the transmission of the epiallele over generations. This depends only on the self-reinforcing loop between CHROMOMETHYLASE 3 and KRYPTONITE, involving DNA methylated in the CHG context and histone H3 lysine 9 methylation. Our findings reveal a predominant role of this loop in maintaining a natural epiallele.
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