Base-pair opening dynamics of primary miR156a using NMR elucidates structural determinants important for its processing level and leaf number phenotype in Arabidopsis

MicroRNAs originate from primary transcripts containing hairpin structures. The levels of mature miR156 influence the leaf number prior to flowering in the life cycle of plants. To understand the molecular mechanism of biogenesis of primary miR156a (pri-miR156a) to mature miR156, a base-pair opening dynamics study was performed using model RNAs mimicking the cleavage site of wild type and B5 bulge-stabilizing mutant pri-miR156a constructs. We also determined the mature miR156 levels and measured leaf numbers at flowering of plants overexpressing the wild type and mutant constructs. Our results suggest that the stabilities and/or opening dynamics of the C15·G98 and U16·A97 base-pairs at the cleavage site are essential for formation of the active conformation and for efficient processing of pri-miR156a, and that mutations of the B5 bulge can modulate mature miR156 levels as well as miR156-driven leaf number phenotypes via changes in the base-pair stability of the cleavage site.