MicroRNA MIMIC binding sites: Minor flanking nucleotide alterations can strongly impact MIMIC silencing efficacy in Arabidopsis

In plants, microRNA (miRNA) target MIMICs (MIMs) have been widely used to inhibit miRNA function. They are based on the Arabidopsis INSENSITIVE TO PHOSPATE STARVATION 1 (IPS1) gene that corresponds to a non‐coding RNA containing a miR399 binding site that can be modified to sequester and inhibit any miRNA of interest. However, the efficacy of miRNA inhibition of these different MIMs can vary greatly. Using MIMs that have strong efficacy (MIM159) and poor efficacy (MIM165), we investigate the underlying cause of this variation. Firstly, sequence alignments of IPS1 homologs from the Brassicaceae identified a highly conserved sequence immediately downstream of the miRNA binding site. Mutating this sequence in the context of the MIM159 attenuates its strong efficacy. This conserved flanking region contains a predicted stem‐loop structure that is also predicted to be present in most modified MIMs that appear to have a strong efficacy, but not in MIM165 that has a poor efficacy. Restoring this predicted stem‐loop in MIM165 via mutation of only three or five nucleotides within the conserved flanking region resulted in MIM165 variants that have very strong efficacies of miRNA inhibition. However, specifically mutating this predicted stem‐loop in the MIM159 context failed to significantly reduce efficacy, and additional mutations to restore this predicted stem‐loop weakened efficacy further. Although this shows there is no simple correlation between this predicted stem‐loop and efficacy, these results add to the growing evidence that the sequence context of miRNA binding sites is important, and that minor nucleotide substitutions to flanking sequences of miRNA binding sites can strongly enhance or attenuate the miRNA‐target interaction.