Single Base-Pair Conformational Switch Modulates miR-34a Targeting of Sirt1 mRNA

Abstract

MicroRNAs (miRNAs) regulate numerous messenger RNAs levels and translation, via binding to the protein Argonaute (Ago). The base pairing between the miRNA and mRNA in the seed region governs target selection and repression efficiency. Biochemical studies and X-ray crystallography structures elucidated the role of Ago and miRNA in this complex, however, structural information on the miRNA-mRNA duplex and its dynamics are still missing. We employ Nuclear Magnetic Resonance R1ρ Relaxation Dispersion and molecular simulations to study the structure and dynamics of miR-34a binding to mRNA of Sirt1, regulating p53 activity. We reveal a switching mode based on a single base pair rearrangement in the miRNA-mRNA duplex, elongating the 6/7mer seed to an 8mer seed. The base pair switch correlates with changing in overall structure of these conformers, which, in hAgo2, indicates two distinct binding modes and the stabilization of the minor conformer exhibit enhanced target repression in cells, shown by luciferase reporter assay. This change in activity indicates that the conformational switch in the miRNA-mRNA duplex can push the complex from an initial “screening” state to an “active” state. Our observations tie together current understanding of the step-wise miRNA target recognition process and shed light on the role of miRNA binding to mRNA beyond the seed.