Functional Specialization of the Plant miR396 Regulatory Network through Distinct MicroRNA–Target Interactions

<div><p>MicroRNAs (miRNAs) are ∼21 nt small RNAs that regulate gene expression in animals and plants. They can be grouped into families comprising different genes encoding similar or identical mature miRNAs. Several miRNA families are deeply conserved in plant lineages and regulate key aspects of plant development, hormone signaling, and stress response. The ancient miRNA miR396 regulates conserved targets belonging to the <em>GROWTH-REGULATING FACTOR</em> (<em>GRF</em>) family of transcription factors, which are known to control cell proliferation in <em>Arabidopsis</em> leaves. In this work, we characterized the regulation of an additional target for miR396, the transcription factor <em>bHLH74</em>, that is necessary for <em>Arabidopsis</em> normal development. <em>bHLH74</em> homologs with a miR396 target site could only be detected in the sister families <em>Brassicaceae</em> and <em>Cleomaceae.</em> Still, <em>bHLH74</em> repression by miR396 is required for margin and vein pattern formation of <em>Arabidopsis</em> leaves. MiR396 contributes to the spatio-temporal regulation of <em>GRF</em> and <em>bHLH74</em> expression during leaf development. Furthermore, a survey of miR396 sequences in different species showed variations in the 5′ portion of the miRNA, a region known to be important for miRNA activity. Analysis of different miR396 variants in <em>Arabidopsis thaliana</em> revealed that they have an enhanced activity toward <em>GRF</em> transcription factors. The interaction between the <em>GRF</em> target site and miR396 has a bulge between positions 7 and 8 of the miRNA. Our data indicate that such bulge modulates the strength of the miR396-mediated repression and that this modulation is essential to shape the precise spatio-temporal pattern of <em>GRF2</em> expression. The results show that ancient miRNAs can regulate conserved targets with varied efficiency in different species, and we further propose that they could acquire new targets whose control might also be biologically relevant.</p>