Structures of microRNA-precursor apical junctions and loops reveal non-canonical base pairs important for processing

Metazoan pri-miRNAs and pre-miRNAs fold into characteristic hairpins that are recognized by the processing machinery. Essential to the recognition of these miR-precursors are their apical junctions where double-stranded stems meet single-stranded hairpin loops. Little is known about how apical junctions and loops fold in three-dimensional space. Here we developed a scaffold-directed crystallography method and determined the structures of eight human miR-precursor apical junctions and loops. Six structures contain non-canonical base pairs stacking on top of the hairpin stem. U-U pair contributes to thermodynamic stability in solution and is highly enriched at human miR-precursor apical junctions. Our systematic mutagenesis shows that U-U is among the most efficiently processed variants. The RNA-binding heme domain of pri-miRNA-processing protein DGCR8 binds longer loops more tightly and non-canonical pairs at the junction appear to modulate loop length. Our study provides structural and biochemical bases for understanding miR-precursors and molecular mechanisms of microRNA maturation.