Structural and functional analysis of the interaction between the nucleoporin Nup98 and the mRNA export factor Rae1

Abstract

The export of mRNAs is a multistep process, involving the packaging of mRNAs into messenger ribonucleoprotein particles (mRNPs), their transport through nuclear pore complexes, and mRNP remodeling events prior to translation. Ribonucleic acid export 1 (Rae1) and Nup98 are evolutionarily conserved mRNA export factors that are targeted by the vesicular stomatitis virus matrix protein to inhibit host cell nuclear export. Here, we present the crystal structure of human Rae1 in complex with the Gle2-binding sequence (GLEBS) of Nup98 at 1.65 Å resolution. Rae1 forms a seven-bladed β-propeller with several extensive surface loops. The Nup98 GLEBS motif forms an ≈50-Å-long hairpin that binds with its C-terminal arm to an essentially invariant hydrophobic surface that extends over the entire top face of the Rae1 β-propeller. The C-terminal arm of the GLEBS hairpin is necessary and sufficient for Rae1 binding, and we identify a tandem glutamate element in this arm as critical for complex formation. The Rae1•Nup98^GLEBS surface features an additional conserved patch with a positive electrostatic potential, and we demonstrate that the complex possesses single-stranded RNA-binding capability. Together, these data suggest that the Rae1•Nup98 complex directly binds to the mRNP at several stages of the mRNA export pathway.

Eukaryotic cells segregate their genetic material in the nucleus, spatially separating the transcription and translation machineries. After transcription, the mRNA is spliced and packaged into a messenger ribonucleoprotein particle (mRNP). The mRNP includes mRNA export factors that provide access to the nuclear pore complex (NPC) and allow for their translocation to the cytoplasm. At the cytoplasmic face of the NPC, remodeling events result in the partial disassembly of the mRNP prior to translation (1–3).

Ribonucleic acid export 1 (Rae1) was initially discovered in Schizosaccharomyces pombe in a genetic screen for proteins involved in RNA export (4). The mammalian homolog of Rae1 was discovered independently by biochemical characterization of a rat liver nuclear envelope subfraction (5). Because the identified protein was found to be UV-cross-linked in vivo to poly(A) containing RNA it was termed mRNP41 (5). Finally, in a genetic screen in Saccharomyces cerevisiae, the homolog of Rae1 was discovered to be synthetically lethal in combination with a null mutant of the glycine-leucine-phenylalanine-glycine (GLFG) nucleoporin Nup100 and was termed GLFG lethal 2 (Gle2) (6).

The next advance in the characterization of Rae1 was the demonstration of the direct biochemical interaction between Gle2 and one of the three GLFG nucleoporins in yeast, Nup116 (7). The binding of Gle2 was mapped to a 57-residue sequence stretch in the N-terminal region of Nup116 that was termed Gle2-binding sequence (GLEBS) (7). The GLEBS motif is absent in the other two GLFG nucleoporins of S. cerevisiae, Nup100 and Nup145N (7–9). An evolutionary highly conserved GLEBS motif was also found in the only GLFG nucleoporin of vertebrates, Nup98 (10). Nup98 is a proto-oncogene that has been identified in numerous leukemogenic fusions with a variety of partner genes (11).

The Nup116 knockout yields a temperature-sensitive phenotype in which cells die at 37 °C (12). Strikingly, electron microscopic analyses revealed that at the nonpermissive temperature the NPCs were clustered on one side of the nuclear envelope and closed apparently by fusion of the nearby outer nuclear envelope membrane (12). The fusion resulted in the formation of a dome-shaped membrane seal over the cytoplasmic face of the NPC, the accumulation of export substrates, and the appearance of “herniated” nuclei (12). Subsequently, it was shown that the removal of the GLEBS motif from Nup116 was sufficient for the generation of herniated nuclei and for the loss of nuclear envelope localization of Gle2 (7). These phenotypes could be rescued by the insertion of the Nup116 GLEBS motif into Nup100 (7). Together, these data indicate that the NPC recruitment of Gle2 is essential for a normal morphology of the NPC with respect to its surrounding outer nuclear envelope membrane.

A further advance in understanding the function of Rae1 in RNA export was made with the demonstration that a viral protein, the matrix (M) protein of the vesicular stomatitis virus (VSV), inhibited nuclear export of host cell RNA by targeting Nup98 and Rae1 (13–16). Interferon γ treatment of VSV infected cells was found to reverse the RNA export inhibition by up-regulation of Nup98 and Rae1 expression (15, 16), thereby underlining the importance of the interaction between Rae1 and Nup98 for RNA export.

Rae1 contains seven WD40 repeats and is therefore predicted to form a seven-bladed β-propeller (Fig. 1A). The β-propeller domain is a classical protein–protein interaction platform that is capable of mediating the association with several proteins (17). The interaction of Rae1 with the Nup98 GLEBS motif appears to be a critical association for RNA export. However, Rae1 can interact with other proteins, not only in the nucleus but also in the cytoplasm, and has also been reported to have several functions in the formation of mitotic spindles (18–20). Hence, Rae1 is a versatile protein that does not only function as a component of the NPC.

Fig. 1.

Structural overview of the Rae1•Nup98^GLEBS complex. (A) Domain organization of human Rae1 and human Nup98. For Rae1, the NTE (red) and the seven WD40 repeats (orange) are indicated. For Nup98, the GLEBS motif (magenta), the FG-repeat region (gray), the unstructured region (dark gray), the auto-proteolytic domain (pink), and the C-terminal 6 kDa fragment (light gray) that is removed by cotranslational proteolysis are indicated. In an alternatively spliced version, the Nup98-96 precursor, the 6 kDa fragment is replaced by Nup96, a protein that is embedded in the symmetric NPC core (Fig. S1). The arrows indicate the sites of autoproteolytic cleavage. (B) Ribbon representation of the Rae1•Nup98^GLEBS complex. The Nup98 GLEBS motif is indicated in magenta. For Rae1, the β-propeller domain (blue), the NTE (red), the 5D6A loop (green), and the 7BC loop (yellow) are indicated. A 90° rotated view is shown on the right. (C) Schematic representation of the Rae1•Nup98^GLEBS structure, colored according to B. The blades of the Rae1 β-propeller are labeled from one to seven. An asterisk indicates the Velcro-closure β-strand (17).

The molecular mechanisms that underlie the manifold functions of Rae1 remain poorly understood. In order to gain further insight into Rae1’s function in mRNA export, we have determined the atomic structure of human Rae1 in complex with the Nup98 GLEBS motif at 1.65 Å resolution. As anticipated, Rae1 forms a seven-bladed β-propeller domain that contains several distinct surface loops. The GLEBS motif forms a hairpin structure that interacts extensively with an evolutionarily conserved surface on the top face of the Rae1 β-propeller. We identify an invariant tandem glutamate element (Glu201 and Glu202) in the Nup98 GLEBS motif to be essential for complex formation with Rae1 in solution. Finally, we show that Nup116 variants that carry mutations in this element prevent Gle2 localization to the nuclear envelope and display a mild mRNA export defect in vivo. Strikingly, the Rae1•Nup98^GLEBS complex contains a highly conserved surface with a positive electrostatic potential that could represent a binding site for RNA. We demonstrate biochemically that the Rae1•Nup98^GLEBS complex binds single-stranded RNA oligonucleotides.

Results

Structure Determination.

The Nup98 polypeptide chain encodes one small structured domain at its C terminus, which possesses autoproteolytic activity and thereby facilitates the evolutionarily conserved, cotranslational cleavage of the Nup98 and Nup98-Nup96 precursor proteins (21–23) (Fig. 1A and Fig. S1). Moreover, this domain anchors Nup98 at the cytoplasmic side of the NPC by interacting with Nup88 (24). The remaining, unstructured ∼700 residue N-terminal part of Nup98 contains numerous phenylalanine-glycine (FG) repeats and the GLEBS motif that serve as docking sites for the mRNP export factors p15/TAP (for Tip-associated protein) and for Rae1, respectively (7, 10, 25–28).

The complex between full-length human Rae1 and the 57-residue GLEBS motif of Nup98 was formed by coexpression in Sf9 cells. The Rae1•Nup98^GLEBS complex crystallized in the triclinic space group P1, with four complexes in the asymmetric unit. The structure was solved by single-anomalous dispersion (SAD) using anomalous X-ray diffraction data obtained from an Os-derivative. The Rae1•Nup98^GLEBS structure was refined to 1.65 Å resolution with R_work and R_free values of 20.6% and 23.7%, respectively. For details of the crystallographic statistics, see Table S1.

The Rae1•Nup98^GLEBS complex elutes as a single peak in size-exclusion chromatography, corresponding to a heterodimer. Analytical ultracentrifugation determined the molecular weight to be 45.4 ± 7.5 kDa, confirming the single monomeric state with a 1∶1 stoichiometry in solution (theoretical molecular weight of 48.4 kDa, Fig. S2). Hence, in the following text, we focus on the description of the Rae1•Nup98^GLEBS heterodimer structure.

Structural Overview.

The polypeptide chain of Rae1 can be divided into a 30-residue N-terminal extended peptide segment (NTE) followed by a canonical seven-bladed β-propeller domain (17) (Fig. 1 B and C). The β-propeller domain contains a central tunnel along its pseudo-seven-fold axis and is decorated by several long surface loops. In particular, the interblade connector 5D6A and the 7BC loop project outward from the top face of the β-propeller domain.

The Nup98 GLEBS motif folds into an N-terminal 10-residue coil region followed by two β-strands, β1 and β2, that are connected via a 13-residue linker segment to the C-terminal α-helix αA (Fig. 1B). Overall, the GLEBS motif forms an ≈50 Å long hairpin structure, in which the antiparallel β-strands, β1 and β2, the “β-tongue,” form the kink of the hairpin. No density is observed for five residues of the β1-β2 connector that form the tip of the β-tongue. Hence, these residues have been omitted from the final model.

The Nup98 GLEBS hairpin binds to the top face of the Rae1 β-propeller domain and extends across the entire surface. The GLEBS motif is anchored to the Rae1 β-propeller primarily via two key interactions: (i) β2 of the GLEBS β-tongue interacts with the 5D6A interblade connector, extending the β-tongue with an additional parallel β-strand 5D′. (ii) the N-terminal tip of the GLEBS helix αA inserts into the central tunnel of the Rae1 β-propeller. The Rae1-Nup98^GLEBS interaction is reinforced by the protruding Rae1 7BC loop that binds to a hydrophobic pocket, which is formed by the coil regions and helix αA of the GLEBS motif. In total, 2,900 Å² of surface area are buried between Rae1 and the Nup98 GLEBS motif, involving 38 and 29 residues of Rae1 and the GLEBS motif, respectively.

Interestingly, two crystal contacts that link adjacent Rae1•Nup98^GLEBS complexes in the asymmetric unit involve two FG-repeat-like sequence stretches of the Rae1 NTE that bind to hydrophobic pockets in the surface of neighboring Rae1 molecules (Fig. S3). These hydrophobic pockets may provide transient binding sites for the FG-repeats of Nup98 or other FG-nucleoporins.

Surface Properties.

Many β-propeller domains allow for the simultaneous attachment of several binding partners (17). Rae1 was identified as a component of the mRNA export machinery and has been found to directly interact with poly(A)-containing mRNA in cross-linking experiments conducted in HeLa cells (5). Therefore, we analyzed the surface properties of Rae1 and the Rae1•Nup98^GLEBS complex to identify potential, additional interaction sites.

Rae1 features two large and essentially invariant surface patches (Fig. 2 and Fig. S4). The first patch is primarily hydrophobic and extends over the entire top face of the β-propeller domain. This surface is utilized for the binding of the Nup98 GLEBS motif. The second patch is located on the side of the β-propeller, contiguous to the first patch, and is formed by surface residues in blades five and six. This surface area has a highly positive electrostatic potential. Strikingly, the binding of the Nup98 GLEBS places the β-tongue directly adjacent to this conserved surface patch, generating a cradle with a positive surface potential at its deepest site. The properties of this second surface patch are consistent with those with a potential RNA-binding capability.

Fig. 2.

Surface properties of Rae1 and the Rae1•Nup98^GLEBS complex. (A) Surface renditions of Rae1 and Rae1•Nup98^GLEBS. The Rae1 surface that mediates the association with the Nup98 GLEBS motif is colored in magenta; the remainder is colored in blue. In the Rae1•Nup98^GLEBS complex, the Rae1 and the Nup98 GLEBS motif surfaces are colored in blue and light magenta, respectively (Right). As a reference, a ribbon representation of the Rae1•Nup98^GLEBS complex is shown and corresponds to the orientation of the left panel. The black line in the left panel indicates the location of the side view surface. (B) The surface representations of Rae1 and the Rae1•Nup98^GLEBS complex are colored according to multispecies sequence alignments (Fig. 4A and Fig. S3). The conservation at each position is mapped onto the surface and shaded in a color gradient from yellow (60% similarity) to red (100% identity). (C) Rae1 and Rae1•Nup98^GLEBS surface renditions, colored according to the electrostatic potential, ranging from red (-10 k_B T/e) to blue (+10 k_B T/e).

Structural Comparison.

Several proteins that carry stretches of high-sequence homology to the Nup98 GLEBS motif have been identified (29). Among these proteins are the human spindle checkpoint components Bub1 and BubR1 (in yeast Mad3) that bind to the cell cycle arrest protein Bub3 (30, 31). Together with other components, these proteins form an inhibitory complex at the kinetochore that prevents the progression of the cell cycle (32). Rae1 has been found to be capable of forming complexes with the two GLEBS-like motifs of Bub1 and BubR1 (29). Therefore, we compared our Rae1•Nup98^GLEBS structure to the previously determined structures of Bub3 in complex with the GLEBS-like sequences of Bub1 and Mad3 (33).

On the primary sequence level, human Rae1 and yeast Bub3 are 21% identical (Fig. S5). Comparison of the Rae1•Nup98^GLEBS and Bub3•Mad3^GLEBS structures reveals that Rae1 and Bub3 both form seven-bladed β-propellers (Fig. 3). 245 equivalent Cα atoms of the two β-propeller cores superimpose with an rmsd of ≈1.4 Å. The GLEBS-like Bub3 binding sequences of Bub1 and Mad3 are composed of 36 and 42 residues, respectively, about 20 residues shorter than the Nup98 GLEBS motif. The Bub3•Bub1 and Bub3•Mad3 structures reveal that the binding mode of the GLEBS-like sequences of Bub1 and Mad3 are similar to that of the Nup98 GLEBS motif, involving the 5D6A and 7BC loops on the top surface of the β-propeller. However, the shorter GLEBS-like sequences are composed of only the C-terminal arm of the Nup98 GLEBS hairpin (GLEBS-C) and lack the N-terminal arm (GLEBS-N). The GLEBS-C is composed of β2, the αA helix, and a linker segment between these two secondary structure elements that varies in length among the three sequence motifs. Whereas the 5D6A loop forms a similar interaction with the β2 strand in all three cases, the interaction of the 7BC loop with the three GLEBS motifs differs substantially. The lack of the GLEBS-N in the Bub1 and Mad3 sequence motifs is compensated by a longer 7BC loop of Bub3 that partially covers and stabilizes the shorter GLEBS-like sequences.

Fig. 3.

Structural comparison between the Rae1•Nup98 and Bub3•Mad3 complexes. The superposition of the Rae1 and Bub3 β-propeller domains is shown in the right panel. Bub3 and Mad3 are colored in gray and pink, respectively. The 5D6A interblade connectors and 7BC loops of the Rae1 and Bub3 β-propellers are indicated in different shades of green and yellow, respectively. A 90° rotated view is shown in the lower panel.

Rae1-Nup98^GLEBS Interface.

The edge of the Nup98 GLEBS hairpin binds to the top face of the Rae1 β-propeller domain, primarily involving the residues in the GLEBS-C arm. This result is in line with a multispecies sequence alignment that confirms that the majority of the conserved residues are located in the GLEBS-C segment (Fig. 4A). The GLEBS-C arm forms two primary interactions with Rae1: β2 forms a short parallel β-sheet with 5D′ of the 5D6A interblade connector loop, and helix αA dips into the central tunnel of the Rae1 β-propeller (Fig. 1B). Whereas the residues in the β2 strand are only moderately conserved, consistent with the formation of a main-chain, hydrogen-bond network, the majority of the residues in helix αA are involved in numerous side-chain contacts and are invariant from yeast to human (Fig. 4B). Specifically, the N terminus of helix αA is anchored at the central tunnel of Rae1 by two key salt bridges that are formed between E201 and E202 of the Nup98 GLEBS, and R216 and R172 of Rae1, respectively. Moreover, helix αA makes several additional highly conserved interactions with Rae1, involving R204, D207, and Y208. The interactions of the GLEBS-N arm involve the formation of the three-stranded β-sheet between Rae1 and the Nup98 GLEBS motif. In addition, D171 of the GLEBS-N arm is clamped by R239 and K258 of Rae1, forming an additional evolutionarily conserved salt bridge between the two molecules. Finally, the interaction between Rae1 and the Nup98 GLEBS is reinforced by Rae1’s 7BC loop that binds to the open end of the GLEBS hairpin, involving helix αA and the two coil regions.

Fig. 4.

The tandem glutamate element of the Nup98 GLEBS motif is essential for the interaction with Rae1. (A) Multispecies sequence alignment of the Nup98 GLEBS motif. The overall sequence conservation at each position is shaded in a color gradient from yellow (40% similarity) to red (100% identity) using the Blosum62 weighting algorithm (40). The numbering of the residues and the secondary structure are according to human Nup98. The secondary structure is indicated above the sequence as green arrows (β-strands), blue rectangles (α-helices), gray lines (coil regions), and gray dots (disordered residues). Dots below the sequence indicate residues involved in Rae1 binding (magenta and green). Residues that are shown in B are highlighted with magenta dots. Asterisks indicate the positions of the invariant Glu201 and Glu202 of the tandem glutamate element. (B) Details of the interaction between Rae1 and the Nup98 GLEBS motif. The ribbon representation is colored according to Fig. 1B. The inset illustrates the position of the tandem glutamate element and is expanded on the right. (C) Analysis of the interaction between Rae1 and GST-Nup98 GLEBS fragments and mutants. The C-terminal arm of the Nup98 GLEBS (GLEBS-C) is necessary and sufficient for Rae1 binding. A double mutant in Nup98 GLEBS, E201K/E202K, abolishes the Rae1-Nup98^GLEBS interaction.

The Nup98 GLEBS motif and the GLEBS-like sequences of Mad3 and Bub1 are evolutionarily very divergent, preventing a reliable alignment. In fact, the only conserved sequence feature between the three structures refers to the two key salt bridges that involve the tandem glutamate element and anchor helix αA at the tunnel in the Rae1 β-propeller. All remaining interactions only impose a loose restraint on the sequence.

Biochemical Analysis of the Interaction Between Rae1 and the Nup98 GLEBS Motif.

The Nup98 GLEBS motif contains a unique N-terminal region (GLEBS-N) that is not found in the GLEBS-like sequences of Bub1 and Mad3. Based on the structural observation that the GLEBS-N segment only contributes to a minor extent to the interaction with Rae1, we analyzed whether a “minimal” Nup98 GLEBS region, as defined by the structural comparison with the Bub3•Bub1 and Bub3•Mad3 structures, would be sufficient for complex formation with Rae1. Indeed, in a pull-down experiment, we found that the GLEBS-C arm is necessary and sufficient for Rae1 binding, whereas the GLEBS-N region is dispensable, consistent with the crystallographic findings (Fig. 4C). Our result is also in agreement with previous in vivo experiments in which the overexpression of a similar GLEBS-C fragment (residues 181–224) in HtTA cells is as sufficient to induce a comparable mRNA export defect as is the entire GLEBS motif (residues 150–224) (10).

We then tested if the tandem glutamate element which is a shared structural feature between the GLEBS motifs of Nup98, Mad3, and Bub1 is essential for complex formation between Nup98 and Rae1. As predicted, the charge-reversal mutations E201K and E202K abolish the interaction between the two proteins. This result is in concurrence with previous mutagenesis experiments that identified the salt bridge formed between E382 of Mad3 and R197 of Bub3 as critical for complex formation in this system (33).

Rae1•Nup98^GLEBS Binds to RNA in Vitro.

Rae1 has been found to be cross-linked to poly(A)-containing mRNA after the UV irradiation of HeLa cells (5). The analysis of the Rae1•Nup98^GLEBS surface reveals an evolutionarily conserved basic surface patch that supports the previously proposed RNA-binding capability. Therefore, we analyzed whether the Rae1•Nup98^GLEBS complex is able to bind to a degenerate, decameric, single-stranded RNA oligonucleotide using an electrophoretic mobility shift assay (Fig. 5). Indeed, the Rae1•Nup98^GLEBS heterodimer shifts the RNA band to lower mobility in a concentration-dependent manner, indicating the formation of a Rae1•Nup98^GLEBS•RNA complex. This result demonstrates that Rae1•Nup98^GLEBS directly binds to single-stranded RNA with an approximate dissociation constant in the low micromolar range.

Fig. 5.

Rae1•Nup98^GLEBS binds RNA in vitro. An electrophoretic mobility shift assay was carried out, with a constant amount of degenerate 10-mer RNA oligonucleotide (2 μM) and increasing concentrations of the Rae1•Nup98^GLEBS complex, as indicated. The RNA oligonucleotide was visualized with SYBR Gold nucleic acid gel stain. The estimated dissociation constant of the interaction is in the low micromolar range.

In Vivo Analysis of the Interaction Between Gle2 and Nup116.

A wealth of data implicate the Rae1•Nup98^GLEBS complex in mRNA export (4–10, 16, 28). By taking advantage of the structural information, we examined the consequences of disrupting the Rae1•Nup98^GLEBS complex in vivo using yeast as a model system (Fig. 6). Deletion of the GLEBS motif in Nup116 (the yeast homolog of Nup98) results in a temperature-sensitive growth defect, as well as in the mislocalization of Gle2 (the yeast homolog of Rae1). This observation is in agreement with previous findings that the nuclear envelope localization of Gle2 is dependent on the Nup116 GLEBS motif (7). We further tested the growth and localization phenotypes of the Nup116 E154K/E155K double mutant, which is equivalent to the Nup98 tandem glutamate element mutant (E201K/E202K) that abolishes the interaction with Rae1 in vitro (Fig. 4C). Similar to the Nup116 ΔGLEBS mutant, this yeast Nup116 mutant results in a temperature-sensitive growth phenotype and the loss of Gle2 nuclear envelope staining. These data demonstrate that the mutation of two key glutamate residues of the Nup116 GLEBS motif abolishes the Gle2-Nup116 interaction in vivo.

Fig. 6.

In vivo analysis of the Gle2-Nup116^GLEBS interaction. (A) Yeast growth assay performed using nup116Δ, Gle2-GFP cells transformed with the indicated mCherry (mCh)-Nup116 constructs. Ten-fold serial dilutions were spotted on SD-LEU plates and grown for 2–3 days at the indicated temperatures. (B) In vivo localization of Gle2-GFP and mCh-Nup116 carried out at 30 °C. In the presence of full-length mCh-Nup116, Gle2-GFP is enriched at the nuclear envelope. The ΔGLEBS and the tandem glutamate element (E154K/E155K) Nup116 mutants result in a dispersed staining of Gle2-GFP throughout the cell with no enrichment at the nuclear envelope. (Scale bar: 5 μm.)

Based on our observation that the Rae1•Nup98^GLEBS heterodimer binds single-stranded RNA in solution, we hypothesized that the disruption of the Gle2•Nup116^GLEBS complex may result in mRNA export defects in yeast. To characterize the function of the Gle2-Nup116 interaction in mRNA export, we analyzed whether Nup116 mutants that fail to localize Gle2 to the nuclear envelope also result in the nuclear retention of poly(A) mRNA by fluorescence in situ hybridization (FISH), using an Alexa647-labeled oligo dT₅₀ probe (Fig. S6). In a typical wild-type population, 1.7 ± 0.9% of the cells display a marked nuclear FISH signal, while 6.4 ± 2.2% of the cells are stained in the Nup116 knockout strain. The introduction of the Nup116 ΔGLEBS or Nup116 E154K/E155K mutants to the Nup116 knockout strains shows a nuclear mRNA accumulation in 6.1 ± 0.9% and 4.2 ± 1.0% of the cells, respectively. These data demonstrate that the disruption of the Gle2-Nup116 interaction results in a modest, but detectable total poly(A) mRNA export defect.

Discussion

The Rae1•Nup98 complex has distinct and versatile roles at different stages of the cell cycle, including the nuclear export of mRNAs and the regulation of the cell cycle progression (4–10, 18–20, 28, 34). We have determined an atomic model that resolves the interaction of Rae1 with the GLEBS motif of Nup98. The analysis of our structure allowed for the identification of a tandem glutamate element in the GLEBS motif that is critical for the interaction with Rae1 in vitro, as well as for the nuclear envelope localization of Gle2 (the yeast homolog of Rae1) in vivo. Moreover, our structure reveals an evolutionarily conserved surface patch with a positive surface potential. Strikingly, the Rae1•Nup98^GLEBS heterodimer is capable of directly binding to single-stranded RNA oligonucleotides.

In the export of nuclear mRNA to the cytoplasm, the mRNA is organized into an mRNP with the help of mRNA binding proteins, including mRNA export factors, such as the p15/TAP heterodimer (1–3). The targeting of the mature mRNP to the NPC is facilitated by the interaction of the general mRNA export factor p15/TAP with Nup98 (26). Photobleaching experiments in living cells have yielded a residence time for Nup98 of about 3 h (35). This finding, when correlated with the mRNP’s average “dwell” time of several 100 ms at the NPC (36), suggests that Nup98 stays associated with the NPC and conducts the sequential export of many mRNPs. Nup98 is attached to the cytoplasmic side of the NPC with its C-terminal domain, whereas the remainder of the protein is unstructured (24). This architecture allows Nup98 to remain attached at the cytoplasmic side of the NPC, while simultaneously reaching into the interior of the nucleus with its flexible N-terminal unstructured region. Our finding that the Rae1•Nup98^GLEBS heterodimer is capable of RNA binding in solution suggests that Nup98 may recruit mRNPs to the NPC not only by binding to p15/TAP (via its FG repeats) (26), but also by directly interacting with the RNA (via its association with Rae1).

The next step in mRNP export is the translocation through the central channel of the NPC. This transit is followed by mRNP remodeling, whereby proteins bound to nuclear mRNA are dissociated and replaced by cytoplasmic mRNA binding proteins (1–3). Key factors in this process are the DEAD-box helicase hDbp5 (also known as DDX19), which is anchored to the cytoplasmic filament nucleoporin Nup214 and the hDbp5 ATPase activating protein Gle1 (37–39). The weak RNA-binding affinity of the Rae1•Nup98 heterodimer may also allow the complex to be involved in this process by temporarily covering and thereby protecting naked mRNA stretches prior to translation. However, the Rae1•Nup98-RNA interaction may constitute only one of many redundant mechanisms to protect the RNA, which would explain our observation that disruption of the Gle2•Nup116 complex in yeast only yields a modest accumulation of poly(A) mRNA in the nucleus. An alternative explanation would be that the Gle2•Nup116-mediated export pathway is utilized only by a subfraction of mRNAs. In this context, Gle2 may function as an mRNP recruitment factor that allows for the preferential export of a distinct mRNA subset by providing an additional binding site to Nup116. In fact, Nup100 and Nup145N are two Nup116 homologs that lack a GLEBS motif and may allow for the Gle2-independent, but Mtr2/Mex67 (the yeast homolog of p15/TAP)-mediated mRNP export.

The structure of the Rae1•Nup98^GLEBS complex reported here provides an important piece in the mRNA export puzzle. Many more structures of nucleoporins that constitute the mRNA export machinery at the cytoplasmic face of the NPC remain to be determined. Together, these structures are expected to ultimately provide a detailed, mechanistic understanding of the various processes that accompany mRNA export and occur prior to translation.

Methods

The details of protein expression, purification, crystallization, structure determination, protein interaction analysis, yeast experiments, and RNA-binding assay are described in the SI Text published online. In short, Rae1 and the Nup98 GLEBS motif were coexpressed in Sf9 insect cells using the pFastbac Dual baculovirus system (Invitrogen) (Table S2). The Rae1•Nup98^GLEBS complex was purified using several chromatographic techniques. X-ray diffraction data were collected at the General Medicine and Cancer Institutes Collaborative Access Team (GM/CA-CAT) beamline 23ID-B at the Advanced Photon Source, Argonne National Laboratory. The structure was solved by SAD, using data obtained from OsO₄-derivatized crystals. Data collection and refinement statistics are summarized in Table S1.