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


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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    ABSTRACT: Translational repression and deadenylation of eukaryotic mRNAs result either in the sequestration of the transcripts in a nontranslatable pool or in their degradation. Removal of the 5′ cap structure is a crucial step that commits deadenylated mRNAs to 5′-to-3′ degradation. Pat1, Edc3 and the DEAD-box protein Dhh1 are evolutionary conserved factors known to participate in both translational repression and decapping, but their interplay is currently unclear. We report the 2.8 Å resolution structure of yeast Dhh1 bound to the N-terminal domain of Pat1. The structure shows how Pat1 wraps around the C-terminal RecA domain of Dhh1, docking onto the Phe-Asp-Phe (FDF) binding site. The FDF-binding site of Dhh1 also recognizes Edc3, revealing why the binding of Pat1 and Edc3 on Dhh1 are mutually exclusive events. Using co-immunoprecipitation assays and structure-based mutants, we demonstrate that the mode of Dhh1-Pat1 recognition is conserved in humans. Pat1 and Edc3 also interfere and compete with the RNA-binding properties of Dhh1. Mapping the RNA-binding sites on Dhh1 with a crosslinking–mass spectrometry approach shows a large RNA-binding surface around the C-terminal RecA domain, including the FDF-binding pocket. The results suggest a model for how Dhh1-containing messenger ribonucleoprotein particles might be remodeled upon Pat1 and Edc3 binding.
    Full-text · Article · Jul 2013 · Nucleic Acids Research
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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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    ABSTRACT: RNA helicases are involved in all aspects of RNA metabolism. Since the helicase core is conserved between all helicases, specificity for particular cellular roles must arise from interactions with specific cofactors, which can regulate RNA binding and enzymatic activity. While recent structural studies have provided invaluable insight into some mechanisms of cofactor effects on RNA helicases, biochemical experiments must ultimately be conducted in order to validate these predictions. Here, we provide a guide for identifying helicase-specific cofactors and then studying their effects on helicase function. By measuring RNA binding and release, ATPase activity, nucleotide affinity, and unwinding and annealing activities, cofactor effects on an RNA helicase can be fully characterized.
    Full-text · Article · Jun 2012 · Methods in enzymology
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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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