Evolution of MIR168 paralogs in Brassicaceae.

Background: In plants, expression of ARGONAUTE1 (AGO1), the catalytic subunit of the RNA-Induced Silencing Complexresponsible for post-transcriptional gene silencing, is controlled through a feedback loop involving the miR168 microRNA. Thiscomplex auto-regulatory loop, composed of miR168-guided AGO1-catalyzed cleavage of AGO1 mRNA and AGO1-mediatedstabilization of miR168, was shown to ensure the maintenance of AGO1 homeostasis that is pivotal for the correct functioningof the miRNA pathway.Results: We applied different approaches to studying the genomic organization and the structural and functional evolution ofMIR168 homologs in Brassicaeae. A whole genome comparison of Arabidopsis and poplar, phylogenetic footprinting andphylogenetic reconstruction were used to date the duplication events originating MIR168 homologs in these genomes. Whileorthology was lacking between Arabidopsis and poplar MIR168 genes, we successfully isolated orthologs of both loci present inArabidopsis (MIR168a and MIR168b) from all the Brassicaceae species analyzed, including the basal species Aethionemagrandiflora, thus indicating that (1) independent duplication events took place in Arabidopsis and poplar lineages and (2) the originof MIR168 paralogs predates both the Brassicaceae radiation and the Arabidopsis alpha polyploidization. Different phylogeneticfootprints, corresponding to known functionally relevant regions (transcription starting site and double-stranded structuresresponsible for microRNA biogenesis and function) or for which functions could be proposed, were found to be highlyconserved among MIR168 homologs. Comparative predictions of the identified microRNAs also indicate extreme conservationof secondary structure and thermodynamic stability.Conclusion: We used a comparative phylogenetic footprinting approach to identify the structural and functional constraintsthat shaped MIR168 evolution in Brassicaceae. Although their duplication happened at least 40 million years ago, we foundevidence that both MIR168 paralogs have been maintained throughout the evolution of Brassicaceae, most likely functionally asindicated by the extremely high conservation of functionally relevant regions, predicted secondary structure and thermodynamicprofile. Interestingly, the expression patterns observed in Arabidopsis indicate that MIR168b underwent partialsubfunctionalization as determined by the experimental characterization of its expression pattern provided in this study. Wefound further evolutionary evidence that pre-miR168 lower stem (the RNA-duplex structure adjacent to the miR-miR* stem)is significantly longer than animal lower stems and probably plays a relevant role in multi-step miR168 biogenesis.