High evidence targets of conserved plant microRNAs and the complexities of their ancient microRNA binding-sites

In plants, high complementarity between microRNAs (miRNAs) and their target genes is a prerequisite for a miRNA-target interaction (MTI). However, evidence suggests there are complexities beyond complementarity that impacts the strength of the MTI. To explore this, the bioinformatic pipeline TRUEE (Targets Ranked Using Experimental Evidence) was applied to strongly conserved miRNAs to identity their high evidence (HE) targets across species. For each miRNA family, HE targets predominantly consisted of homologues from one conserved target gene family (primary family). If an additional HE target family(s) was identified (secondary family), it was likely functionally related to the primary family. Many primary target families contained highly conserved nucleotide sequences flanking their miRNA binding-sites that were enriched in HE homologues across species, suggesting these sequences facilitate miRNA-mediated regulation. A subset of these flanking sequences are predicted to form conserved RNA secondary structures that preferentially base-pair with the miRNA binding-site, implying that these sites are highly structured. Functional testing of the conserved flanking sequences of the miR160 target, AUXIN RESPONSE FACTOR 10 (ARF10), found that mutations within these flanking sequences resulted in attenuated ARF10 silencing. Our findings support the notion that features beyond complementarity at highly conserved miRNA binding-sites underpin these ancient MTIs.