Dynamic mechanisms for pre-miRNA binding and export by Exportin-5

Abstract

The biogenesis and function of mature microRNAs (miRNAs) is dependent on the nuclear export of miRNA precursors (pre-miRNA) by Exportin-5 (Exp5). To characterize the molecular mechanisms of how pre-miRNA is recognized and transported by Exp5, we have performed 21 molecular dynamic (MD) simulations of RNA-bound Exp5 (Exp5–RanGTP–premiRNA, Exp5–RanGDP–premiRNA, Exp5–premiRNA), RNA-unbound Exp5 (Exp5–RanGTP, Exp5–RanGDP, apo–Exp5), and pre-miRNA. Our simulations with standard MD, steered molecular dynamics (SMD), and energy analysis have shown that (1) Free Exp5 undergoes extensive opening motion, and in this way facilitates the RanGTP binding. (2) RanGTP efficiently regulates the association/dissociation of pre-miRNA to its complex by inducing conformational changes in the HEAT-repeat helix stacking of Exp5. (3) The GTP hydrolysis prevents Ran from rebinding to Exp5 by regulating the hydrophobic interfaces and salt bridges between Ran and Exp5. (4) The transition from the A′-form to the A-form of the pre-miRNA modulates the structural complementarities between the protein and the pre-miRNA, thus promoting efficient assembly of the complex. (5) The base-flipping process (from the closed to the fully flipped state) of the 2-nt 3′ overhang is a prerequisite for the pre-miRNA recognition by Exp5, which occurs in a sequence-independent manner as evidenced by the fact that different 2-nt 3′ overhangs bind to Exp5 in essentially the same way. And finally, a plausible mechanism of the pre-miRNA export cycle has been proposed explaining how the protein–protein and protein–RNA interactions are coordinated in physiological conditions.