The mRNA export protein DBP5 binds RNA and the cytoplasmic nucleoporin NUP214 in a mutually exclusive manner

Max-Planck-Institute of Biochemistry, Department of Structural Cell Biology, Am Klopferspitz 18, D-82152 Martinsried, Germany.
Nature Structural & Molecular Biology (Impact Factor: 13.31). 03/2009; 16(3):247-54. DOI: 10.1038/nsmb.1561
Source: PubMed

ABSTRACT The DEAD-box protein DBP5 is essential for mRNA export in both yeast and humans. It binds RNA and is concentrated and locally activated at the cytoplasmic side of the nuclear pore complex. We have determined the crystal structures of human DBP5 bound to RNA and AMPPNP, and bound to the cytoplasmic nucleoporin NUP214. The structures reveal that binding of DBP5 to nucleic acid and to NUP214 is mutually exclusive. Using in vitro assays, we demonstrate that NUP214 decreases both the RNA binding and ATPase activities of DBP5. The interactions are mediated by conserved residues, implying a conserved recognition mechanism. These results suggest a framework for the consecutive steps leading to the release of mRNA at the final stages of nuclear export. More generally, they provide a paradigm for how binding of regulators can specifically inhibit DEAD-box proteins.

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    • "The experiments were carried out essentially as previously described (52). Single-stranded 5′ biotinylated U20 RNA (Dharmacon) was mixed with 3 μg of a given protein and/or nucleotide in binding buffer (20 mM Hepes, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT, 10% glycerol, 0.1% Nonidet 40) to a final volume of 60 µl and was kept at 4°C overnight. "
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    Nucleic Acids Research 07/2013; DOI:10.1093/nar/gkt600 · 9.11 Impact Factor
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    • "However, because the kinetic cycle in these examples is dominated by a single rate-limiting step, it is not clear whether many of these effects have physiological repercussions. Additional effects reported to arise from cofactors include an increase (Ballut et al., 2005; Weirich et al., 2006) or decrease (von Moeller et al., 2009) in RNA affinity and a decrease (Maeder et al., 2009) in ATPase activity (Table 10.1). By modulating activity and potentially increasing specificity of RNA helicases, cofactors provide an additional level of regulation. "
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    Methods in enzymology 06/2012; 511:213-37. DOI:10.1016/B978-0-12-396546-2.00010-3 · 2.09 Impact Factor
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    • "RNA and Nup159 binding are mutually exclusive due to their similar binding site on Dbp5 (Fig. 1C; Napetschnig et al. 2009; von Moeller et al. 2009; Montpetit et al. 2011). Also, the human homolog of Nup159 has tight affinity for ADP-bound and nucleotide-free Dbp5, but has weak affinity for ATP-bound Dbp5 (von Moeller et al. 2009). Therefore, Nup159 likely binds only to RNAfree , ADP-bound Dbp5. "
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    ABSTRACT: It is commonly assumed that all DEAD-box ATPases function via a shared mechanism, since this is the case for the few proteins characterized thus far. Hodge and colleagues (pp. 1052-1064) and Noble and colleagues (pp. 1065-1077) now describe a novel model for Dbp5's ATPase cycle in mRNA (messenger RNA)/protein complex (mRNP) remodeling during nuclear export. Notably, unlike other DEAD-box proteins, Dbp5 uses a conformational change distinct from ATP hydrolysis for its activity and requires an ADP release factor to reset its ATPase cycle.
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