Activation of the DExD/H-box protein Dbp5 by the nuclear-pore protein Gle1 and its coactivator InsP6 is required for mRNA export

The DExD/H-box ATPase Dbp5 is essential for nuclear mRNA export, although its precise role in this process remains poorly understood. Here, we identify the nuclear pore protein Gle1 as a cellular activator of Dbp5. Dbp5 alone is unable to stably bind RNA or effectively hydrolyse ATP under physiological conditions, but addition of Gle1 dramatically stimulates these activities. A gle1 point mutant deficient for Dbp5 stimulation in vitro displays an mRNA export defect in vivo, indicating that activation of Dbp5 is an essential function of Gle1. Interestingly, Gle1 binds directly to inositol hexakisphosphate (InsP6) and InsP6 potentiates the Gle1-mediated stimulation of Dbp5. Dominant mutations in DBP5 and GLE1 that rescue mRNA export phenotypes associated with the lack of InsP6 mimic the InsP6 effects in vitro. Our results define specific functions for Gle1 and InsP6 in mRNA export and suggest that local activation of Dbp5 at the nuclear pore is critical for mRNA export.     

Nuclear Export of Pre-Ribosomal Subunits Requires Dbp5, but Not as an RNA-Helicase as for mRNA Export

The DEAD-box RNA-helicase Dbp5/Rat8 is known for its function in nuclear mRNA export, where it displaces the export receptor Mex67 from the mRNA at the cytoplasmic side of the nuclear pore complex (NPC). Here we show that Dbp5 is also required for the nuclear export of both pre-ribosomal subunits. Yeast temperature-sensitive dbp5 mutants accumulate both ribosomal particles in their nuclei. Furthermore, Dbp5 genetically and physically interacts with known ribosomal transport factors such as Nmd3. Similar to mRNA export we show that also for ribosomal transport Dbp5 is required at the cytoplasmic side of the NPC. However, unlike its role in mRNA export, Dbp5 does not seem to undergo its ATPase cycle for this function, as ATPase-deficient dbp5 mutants that selectively inhibit mRNA export do not affect ribosomal transport. Furthermore, mutants of GLE1, the ATPase stimulating factor of Dbp5, show no major ribosomal export defects. Consequently, while Dbp5 uses its ATPase cycle to displace the export receptor Mex67 from the translocated mRNAs, Mex67 remains bound to ribosomal subunits upon transit to the cytoplasm, where it is detectable on translating ribosomes. Therefore, we propose a model, in which Dbp5 supports ribosomal transport by capturing ribosomal subunits upon their cytoplasmic appearance at the NPC, possibly by binding export factors such as Mex67. Thus, our findings reveal that although different ribonucleoparticles, mRNAs and pre-ribosomal subunits, use shared export factors, they utilize different transport mechanisms.     

The DEAD-box protein Dbp5 controls mRNA export by triggering specific RNA:protein remodeling events

Messenger RNA (mRNA) export involves the unidirectional passage of ribonucleoprotein particles (RNPs) through nuclear pore complexes (NPCs), presumably driven by the ATP-dependent activity of the DEAD-box protein Dbp5. Here we report that Dbp5 functions as an RNP remodeling protein to displace the RNA-binding protein Nab2 from RNA. Strikingly, the ADP-bound form of Dbp5 and not ATP hydrolysis is required for RNP remodeling. In vivo studies with nab2 and dbp5 mutants show that a Nab2-bound mRNP is a physiological Dbp5 target. We propose that Dbp5 functions as a nucleotide-dependent switch to control mRNA export efficiency and release the mRNP from the NPC.     

Physical and genetic interactions link the yeast protein Zds1p with mRNA nuclear export

Eukaryotic gene expression requires the export of mRNA from the nucleus to the cytoplasm. The DEAD box protein Dbp5p is an essential export factor conserved from yeast to man. A fraction of Dbp5p forms a complex with nucleoporins of the cytoplasmic filaments of the nuclear pore complex. Gfd1p was identified originally as a multicopy suppressor of the rat8-2 ts allele of DBP5. Here we reported that Dbp5p and Gfd1p interact with Zds1p, a protein previously identified as a multicopy suppressor in several yeast genetic screens. By using the two-hybrid system, we showed that Zds1p interacts in vivo with both Gfd1p and Dbp5p. In vitro binding experiments revealed that Gfd1p and Dbp5p bind directly to the C-terminal part of Zds1p. In addition, ZDS1 interacted genetically with mutant alleles of genes encoding key factors in mRNA export, including DBP5 and MEX67. Furthermore, deletion of ZDS1 or of both ZDS1 and the closely related ZDS2 exacerbated the poly(A)+ export defects shown by dbp5-2 and mex67-5 mutants. We proposed that Zds1p associates with the complex formed by Dbp5p, Gfd1p, and nucleoporins at the cytosolic fibrils of the nuclear pore complex and is required for optimal mRNA export.     

Rat8p/Dbp5p is a shuttling transport factor that interacts with Rat7p/Nup159p and Gle1p and suppresses the mRNA export defect of xpo1-1 cells.

In a screen for temperature-sensitive mutants of Saccharomyces cerevisiae defective for mRNA export, we previously identified the essential DEAD-box protein Dbp5p/Rat8p and the nucleoporin Rat7p/Nup159p. Both are essential for mRNA export. Here we report that Dbp5p and Rat7p interact through their Nterminal domains. Deletion of this portion of Rat7p (Rat7pDeltaN) results in strong defects in mRNA export and eliminates association of Dbp5p with nuclear pores. Overexpression of Dbp5p completely suppressed the growth and mRNA export defects of rat7DeltaN cells and resulted in weaker suppression in cells carrying rat7-1 or the rss1-37 allele of GLE1. Dbp5p interacts with Gle1p independently of the N-terminus of Dbp5p. Dbp5p shuttles between nucleus and cytoplasm in an Xpo1p-dependent manner. It accumulates in nuclei of xpo1-1 cells and in cells with mutations affecting Mex67p (mex67-5), Gsp1p (Ran) or Ran effectors. Overexpression of Dbp5p prevents nuclear accumulation of mRNA in xpo1-1 cells, but does not restore growth, suggesting that the RNA export defect of xpo1-1 cells may be indirect. In a screen for high-copy suppressors of the rat8-2 allele of DBP5, we identified YMR255w, now called GFD1. Gfd1p is not essential, interacts with Gle1p and Rip1p/Nup42p, and is found in the cytoplasm and at the nuclear rim.     

The N-terminal domain of Nup159 forms a beta-propeller that functions in mRNA export by tethering the helicase Dbp5 to the nuclear pore

Nuclear export of mRNA in eukaryotic cells is mediated by soluble transport factors and components of the nuclear pore complex (NPC). The cytoplasmically oriented nuclear pore protein Nup159 plays a critical role in mRNA export through its conserved N-terminal domain (NTD). Here, we report the crystal structure of the Nup159 NTD, refined to 2.5 A. The structure reveals an unusually asymmetric seven-bladed beta-propeller that is structurally conserved throughout eukarya. Using structure-based conservation analysis, we have targeted specific surface residues for mutagenesis. Residue substitutions in a conserved loop of the NTD abolish in vitro binding to Dbp5, a DEAD box helicase required for mRNA export. In vivo, these mutations cause Dbp5 mislocalization and block mRNA export. These findings suggest that the Nup159 NTD functions in mRNA export as a binding platform, tethering shuttling Dbp5 molecules at the nuclear periphery and locally concentrating this mRNA remodeling factor at the cytoplasmic face of the NPC.     

Inositol hexakisphosphate and Gle1 activate the DEAD-box protein Dbp5 for nuclear mRNA export

Regulation of nuclear mRNA export is critical for proper eukaryotic gene expression. A key step in this process is the directional translocation of mRNA-ribonucleoprotein particles (mRNPs) through nuclear pore complexes (NPCs) that are embedded in the nuclear envelope. Our previous studies in Saccharomyces cerevisiae defined an in vivo role for inositol hexakisphosphate (InsP6) and NPC-associated Gle1 in mRNA export. Here, we show that Gle1 and InsP6 act together to stimulate the RNA-dependent ATPase activity of the essential DEAD-box protein Dbp5. Overexpression of DBP5 specifically suppressed mRNA export and growth defects of an ipk1 nup42 mutant defective in InsP6 production and Gle1 localization. In vitro kinetic analysis showed that InsP6 significantly increased Dbp5 ATPase activity in a Gle1-dependent manner and lowered the effective RNA concentration for half-maximal ATPase activity. Gle1 alone had minimal effects. Maximal InsP6 binding required both Dbp5 and Gle1. It has been suggested that Dbp5 requires unidentified cofactors. We now propose that Dbp5 activation at NPCs requires Gle1 and InsP6. This would facilitate spatial control of the remodelling of mRNP protein composition during directional transport and provide energy to power transport cycles.     

Control of mRNA Export and Translation Termination by Inositol Hexakisphosphate Requires Specific Interaction with Gle1

The unidirectional translocation of messenger RNA (mRNA) through the aqueous channel of the nuclear pore complex (NPC) is mediated by interactions between soluble mRNA export factors and distinct binding sites on the NPC. At the cytoplasmic side of the NPC, the conserved mRNA export factors Gle1 and inositol hexakisphosphate (IP₆) play an essential role in mRNA export by activating the ATPase activity of the DEAD-box protein Dbp5, promoting localized messenger ribonucleoprotein complex remodeling, and ensuring the directionality of the export process. In addition, Dbp5, Gle1, and IP₆ are also required for proper translation termination. However, the specificity of the IP₆-Gle1 interaction in vivo is unknown. Here, we characterize the biochemical interaction between Gle1 and IP₆ and the relationship to Dbp5 binding and stimulation. We identify Gle1 residues required for IP₆ binding and show that these residues are needed for IP₆-dependent Dbp5 stimulation in vitro. Furthermore, we demonstrate that Gle1 is the primary target of IP₆ for both mRNA export and translation termination in vivo. In Saccharomyces cerevisiae cells, the IP₆-binding mutants recapitulate all of the mRNA export and translation termination defects found in mutants depleted of IP₆. We conclude that Gle1 specifically binds IP₆ and that this interaction is required for the full potentiation of Dbp5 ATPase activity during both mRNA export and translation termination.     

Dbp5 — From nuclear export to translation

The DEAD-box RNA helicase Dbp5 is an essential and conserved mRNA export factor which functions in the ATP dependent remodeling of RNA/protein complexes. As such it displaces mRNA bound proteins at the cytoplasmic site of the nuclear pore complex. For the regulation of its RNA-dependent ATPase activity during late steps of nuclear transport, Dbp5 requires the nucleoporin Nup159 and its cofactors Gle1 and IP₆. In addition to its role in mRNA export, a second important function of Dbp5 was identified in translation termination, where it acts together with eRF1 once the translation machinery has reached the stop codon. Similar to mRNA export, this function also requires Gle1–IP₆, however, the counterpart of Nup159 is still missing. Potential other functions of the nucleo-cytoplasmic protein Dbp5 are discussed as well as its substrate specificity and details in its regulatory cycle that are based on recent biochemical and structural characterization. This article is part of a Special Issue entitled: The Biology of RNA helicases — Modulation for life.     

Dbp5, a DEAD-box protein required for mRNA export, is recruited to the cytoplasmic fibrils of nuclear pore complex via a conserved interaction with CAN/Nup159p.

Dbp5 is a DEAD-box protein essential for mRNA export from the nucleus in yeast. Here we report the isolation of a cDNA encoding human Dbp5 (hDbp5) which is 46% identical to yDbp5p. Like its yeast homologue, hDbp5 is localized within the cytoplasm and at the nuclear rim. By immunoelectron microscopy, the nuclear envelope-bound fraction of Dbp5 has been localized to the cytoplasmic fibrils of the nuclear pore complex (NPC). Consistent with this localization, we show that both the human and yeast proteins directly interact with an N-terminal region of the nucleoporins CAN/Nup159p. In a conditional yeast strain in which Nup159p is degraded when shifted to the nonpermissive temperature, yDbp5p dissociates from the NPC and localizes to the cytoplasm. Thus, Dbp5 is recruited to the NPC via a conserved interaction with CAN/Nup159p. To investigate its function, we generated defective hDbp5 mutants and analysed their effects in RNA export by microinjection in Xenopus oocytes. A mutant protein containing a Glu-->Gln change in the conserved DEAD-box inhibited the nuclear exit of mRNAs. Together, our data indicate that Dbp5 is a conserved RNA-dependent ATPase which is recruited to the cytoplasmic fibrils of the NPC where it participates in the export of mRNAs out of the nucleus.     

Nup159 Weakens Gle1 Binding to Dbp5 But Does Not Accelerate ADP Release

Dbp5, DDX19 in humans, is an essential DEAD-box protein involved in mRNA export, which has also been linked to other cellular processes, including rRNA export and translation. Dbp5 ATPase activity is regulated by several factors, including RNA, the nucleoporin proteins Nup159 and Gle1, and the endogenous small-molecule inositol hexakisphosphate (InsP₆). To better understand how these factors modulate Dbp5 activity and how this modulation relates to in vivo RNA metabolism, a detailed characterization of the Dbp5 mechanochemical cycle in the presence of those regulators individually or together is necessary. In this study, we test the hypothesis that Nup159 controls the ADP-bound state of Dbp5. In addition, the contributions of Mg²⁺ to the kinetics and thermodynamics of ADP binding to Dbp5 were assessed. Using a solution based in vitro approach, Mg²⁺ was found to slow ADP and ATP release from Dbp5 and increased the overall ADP and ATP affinities, as observed with other NTPases. Furthermore, Nup159 did not accelerate ADP release, while Gle1 actually slowed ADP release independent of Mg²⁺. These findings are not consistent with Nup159 acting as a nucleotide exchange factor to promote ADP release and Dbp5 ATPase cycling. Instead, in the presence of Nup159, the interaction between Gle1 and ADP-bound Dbp5 was found to be reduced by ~ 18-fold, suggesting that Nup159 alters the Dbp5–Gle1 interaction to aid Gle1 release from Dbp5.     

Nup42 and IP6 coordinate Gle1 stimulation of Dbp5/DDX19B for mRNA export in yeast and human cells

The mRNA lifecycle is driven through spatiotemporal changes in the protein composition of mRNA particles (mRNPs) that are triggered by RNA‐dependent DEAD‐box protein (Dbp) ATPases. As mRNPs exit the nuclear pore complex (NPC) in Saccharomyces cerevisiae, this remodeling occurs through activation of Dbp5 by inositol hexakisphosphate (IP₆)‐bound Gle1. At the NPC, Gle1 also binds Nup42, but Nup42's molecular function is unclear. Here we employ the power of structure‐function analysis in S. cerevisiae and human (h) cells, and find that the high‐affinity Nup42‐Gle1 interaction is integral to Dbp5 (hDDX19B) activation and efficient mRNA export. The Nup42 carboxy‐terminal domain (CTD) binds Gle1/hGle1B at an interface distinct from the Gle1‐Dbp5/hDDX19B interaction site. A nup42‐CTD/gle1‐CTD/Dbp5 trimeric complex forms in the presence of IP₆. Deletion of NUP42 abrogates Gle1‐Dbp5 interaction, and disruption of the Nup42 or IP₆ binding interfaces on Gle1/hGle1B leads to defective mRNA export in S. cerevisiae and human cells. In vitro, Nup42‐CTD and IP₆ stimulate Gle1/hGle1B activation of Dbp5 and DDX19B recombinant proteins in similar, nonadditive manners, demonstrating complete functional conservation between humans and S. cerevisiae. Together, a highly conserved mechanism governs spatial coordination of mRNP remodeling during export. This has implications for understanding human disease mutations that perturb the Nup42‐hGle1B interaction.      

The DEAD-box protein Dbp5p is required to dissociate Mex67p from exported mRNPs at the nuclear rim

Eukaryotic mRNAs are exported from the nucleus to the cytoplasm as complex mRNA-protein particles (mRNPs), and translocation through the nuclear pore complex (NPC) is accompanied by extensive structural changes of the mRNP. We have tested the hypothesis that the DEAD-box ATPase Dbp5p is required for such an mRNP rearrangement. In dbp5 mutant cells, the mRNA export receptor Mex67p accumulates on mRNA. This aberrant accumulation of Mex67p with RNA and the cold-sensitive growth phenotype of a dbp5 allele are suppressed by a mex67 mutation. Moreover, Mex67 bound mRNA accumulates at the nuclear rim in a temperature-sensitive dbp5 mutant when the nuclear exosome is impaired. Importantly, although accumulation of Mex67p-containing mRNPs is also observed when a nuclear basket component is mutated, these mRNPs still contain the nuclear export factor Yra1p. In contrast, the dbp5-trapped mRNPs lack Yra1p. We propose that Dbp5p's function is specifically required to displace Mex67p from exported mRNPs, thus terminating export.     

The DExH/D box protein HEL/UAP56 is essential for mRNA nuclear export in Drosophila.

Dbp5 is the only member of the DExH/D box family of RNA helicases that is directly implicated in the export of messenger RNAs from the nucleus of yeast and vertebrate cells. Dbp5 localizes in the cytoplasm and at the cytoplasmic face of the nuclear pore complex (NPC). In an attempt to identify proteins present in a highly enriched NPC fraction, two other helicases were detected: RNA helicase A (RHA) and UAP56. This suggested a role for these proteins in nuclear transport. Contrary to expectation, we show that the Drosophila homolog of Dbp5 is not essential for mRNA export in cultured Schneider cells. In contrast, depletion of HEL, the Drosophila homolog of UAP56, inhibits growth and results in a robust accumulation of polyadenylated RNAs within the nucleus. Consequently, incorporation of [35S]methionine into newly synthesized proteins is inhibited. This inhibition affects the expression of both heat-shock and non-heat-shock mRNAs, as well as intron-containing and intronless mRNAs. In HeLa nuclear extracts, UAP56 preferentially, but not exclusively, associates with spliced mRNAs carrying the exon junction complex (EJC). We conclude that HEL is essential for the export of bulk mRNA in Drosophila. The association of human UAP56 with spliced mRNAs suggests that this protein might provide a functional link between splicing and export.     

The nucleoporin Gle1 activates DEAD-box protein 5 (Dbp5) by promoting ATP binding and accelerating rate limiting phosphate release

The DEAD-box protein Dbp5 is essential for RNA export, which involves regulation by the nucleoporins Gle1 and Nup159 at the cytoplasmic face of the nuclear pore complex (NPC). Mechanistic understanding of how these nucleoporins regulate RNA export requires analyses of the intrinsic and activated Dbp5 ATPase cycle. Here, kinetic and equilibrium analyses of the Saccharomyces cerevisiae Gle1-activated Dbp5 ATPase cycle are presented, indicating that Gle1 and ATP, but not ADP-P_i or ADP, binding to Dbp5 are thermodynamically coupled. As a result, Gle1 binds Dbp5-ATP > 100-fold more tightly than Dbp5 in other nucleotide states and Gle1 equilibrium binding of ATP to Dbp5 increases >150-fold via slowed ATP dissociation. Second, Gle1 accelerated Dbp5 ATPase activity by increasing the rate-limiting P_i release rate constant ∼20-fold, which remains rate limiting. These data show that Gle1 activates Dbp5 by modulating ATP binding and P_i release. These Gle1 activities are expected to facilitate ATPase cycling, ensuring a pool of ATP bound Dbp5 at NPCs to engage RNA during export. This work provides a mechanism of Gle1-activation of Dbp5 and a framework to understand the joint roles of Gle1, Nup159, and other nucleoporins in regulating Dbp5 to mediate RNA export and other Dbp5 functions in gene expression.     

Gle1 does double duty

During nuclear export, Gle1 (the nuclear-pore-associated mRNA export factor) activates the DEAD-box protein Dbp5 to remodel exported mRNA-protein complexes on the cytoplasmic face of the nuclear pore complex. In this issue, Bolger et al. (2008) now report additional roles for Gle1 in translation initiation and termination.