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 (premiRNA) by . 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 A9-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 baseflipping process (from the closed to the fully flipped state) of the 2-nt 39 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 39 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.

A Baculovirus-Encoded MicroRNA (miRNA) Suppresses Its Host miRNA Biogenesis by Regulating the Exportin-5 Cofactor Ran

“…Ran has been shown to be an important component of nucleocytoplasmic transport of small RNAs, as well as proteins (5,6,33,50,54), and its complete depletion may be lethal, but we did not observe such…”

“…Ran is a 25-kDa protein that plays an important role in nucleocytoplasmic transport of various noncoding RNAs and proteins (5). During nuclear export of pre-miRNAs, exportin-5 binds to the pre-miRNAs in a RanGTP-dependent manner, and depletion of Ran results in a severe reduction of pre-miRNA export (6,33,50,54). In humans, Ran also interacts with other transporters, like exportin-1 and exportin-t, which are involved in the transport of small nuclear RNAs (snRNAs) and tRNAs, respectively (44).…”

Nucleocytoplasmic Transport of RNAs and RNA–Protein Complexes

“…to the negatively charged phosphate backbone of the pre-miRNA [61,62]. This promotes recruitment of the pre-miRNA substrate and enables formation of hydrogen bonds and saltbridges between the amino acids and the ribose moieties.…”

Augmentation of Ras-induced cell transformation : a new role for miR-200a in malignancy.

“…Exportin5 is a nuclear transporter that exports pre-miRNAs in complex with RanGTP. The entire complex is shuttled across the nuclear membrane to the cytoplasm where RanGAP stimulates hydrolysis of GTP to GDP, and the pre-miRNA is released into the cytoplasm [98]. Once in the cytoplasm, they are cleaved by the class I Ribonuclease III, Dicer, to form imperfect duplexes.…”