mRNA Export Factor Research Articles

The mRNA export factor Npl3 mediates the nuclear export of large ribosomal subunits

The nuclear export of large ribonucleoparticles is complex and requires specific transport factors. Messenger RNAs are exported through the RNA-binding protein Npl3 and the interacting export receptor Mex67. Export of large ribosomal subunits also requires Mex67; however, in this case, Mex67 binds directly to the 5S ribosomal RNA (rRNA) and does not require the Npl3 adaptor. Here, we have discovered a new function of Npl3 in mediating the export of pre-60S ribosomal subunit independently of Mex67. Npl3 interacts with the 25S rRNA, ribosomal and ribosome-associated proteins, as well as with the nuclear pore complex. Mutations in NPL3 lead to export defects of the large subunit and genetic interactions with other pre-60S export factors.

Cotranscriptional Association of mRNA Export Factor Yra1 with C-terminal Domain of RNA Polymerase II

The unique C-terminal domain (CTD) of RNA polymerase II, composed of tandem heptad repeats of the consensus sequence YSPTSPS, is subject to differential phosphorylation throughout the transcription cycle. Several RNA processing factors have been shown to bind the phosphorylated CTD and use it to localize to nascent pre-mRNA during transcription. In Saccharomyces cerevisiae, the mRNA export protein Yra1 (ALY/RNA export factor in metazoa) cotranscriptionally associates with mRNA and delivers it to the nuclear pore complex for export to the cytoplasm. Here we report that Yra1 directly binds in vitro the hyperphosphorylated form of the CTD characteristic of elongating RNA polymerase II and contains a phospho-CTD-interacting domain within amino acids 18-184, which also include an "RNA recognition motif" (RRM) (residues 77-184). Using UV cross-linking, we showed that the RRM alone binds RNA, although a larger segment extending to the C terminus (amino acids 77-226) displayed stronger RNA binding activity. Although the RRM is implicated in both RNA and CTD binding, RRM point mutations separated these two functions. Both functions are important in vivo as RNA binding-defective or CTD binding-defective versions of Yra1 engendered growth and mRNA export defects. We also report the construction and characterization of a useful new temperature-sensitive YRA1 allele (R107A/F126A). Using ChIP, we demonstrated that removing the N-terminal 76 amino acids of Yra1 (all of the phospho-CTD-interacting domain up to the RRM) results in a 10-fold decrease in Yra1 recruitment to genes during elongation. These results indicate that the phospho-CTD is likely involved directly in the cotranscriptional recruitment of Yra1.

Evolutionary development of redundant nuclear localization signals in the mRNA export factor NXF1

In human cells, the mRNA export factor NXF1 resides in the nucleoplasm and at nuclear pore complexes. Karyopherin β2 or transportin recognizes a proline-tyrosine nuclear localization signal (PY-NLS) in the N-terminal tail of NXF1 and imports it into the nucleus. Here biochemical and cellular studies to understand the energetic organization of the NXF1 PY-NLS reveal unexpected redundancy in the nuclear import pathways used by NXF1. Human NXF1 can be imported via importin β, karyopherin β2, importin 4, importin 11, and importin α. Two NLS epitopes within the N-terminal tail, an N-terminal basic segment and a C-terminal R-X(2-5)-P-Y motif, provide the majority of binding energy for all five karyopherins. Mutation of both NLS epitopes abolishes binding to the karyopherins, mislocalized NXF1 to the cytoplasm, and significantly compromised its mRNA export function. The understanding of how different karyopherins recognize human NXF1, the examination of NXF1 sequences from divergent eukaryotes, and the interactions of NXF1 homologues with various karyopherins reveals the evolutionary development of redundant NLSs in NXF1 of higher eukaryotes. Redundancy of nuclear import pathways for NXF1 increases progressively from fungi to nematodes and insects to chordates, potentially paralleling the increasing complexity in mRNA export regulation and the evolution of new nuclear functions for NXF1.

Structure-function studies of nucleocytoplasmic transport of retroviral genomic RNA by mRNA export factor TAP

Messenger RNA export is mediated by the TAP-p15 heterodimer, which belongs to the family of NTF2-like export receptors. TAP-p15 heterodimers also bind to the constitutive transport element (CTE) present in simian type D retroviral RNAs, and mediate export of viral unspliced RNAs to the host cytoplasm. We have solved the crystal structure of the RNA recognition and leucine-rich repeat motifs of TAP bound to one symmetrical-half of CTE RNA. L-shaped conformations of protein and RNA are involved in a mutual molecular embrace on complex formation. We have monitored the impact of structure-guided mutations on binding affinities in vitro and transport assays in vivo. Our studies define the principles by which CTE RNA subverts the mRNA export receptor TAP, thereby facilitating nuclear export of viral genomic RNAs, and more generally, provide insights on cargo RNA recognition by mRNA export receptors.

MRNA export and cancer

Studies in the past several years highlight important features of the messenger RNA (mRNA) export process. For instance, groups of mRNAs acting in the same biochemical processes can be retained or exported in a coordinated manner thereby impacting on specific biochemistries and ultimately on cell physiology. mRNAs can be transported by either bulk export pathways involving NXF1/TAP or more specialized pathways involving chromosome region maintenance 1 (CRM1). Studies on primary tumor specimens indicate that many common and specialized mRNA export factors are dysregulated in cancer including CRM1, eukaryotic translation initiation factor 4E (eIF4E), HuR, nucleoporin 88, REF/Aly, and THO. This positions these pathways as potential therapeutic targets. Recently, specific targeting of the eIF4E-dependent mRNA export pathway in a phase II proof-of-principle trial with ribavirin led to impaired eIF4E-dependent mRNA export correlating with clinical responses including remissions in leukemia patients. Here, we provide an overview of these mRNA export pathways and highlight their relationship to cancer.

The Dbp5 cycle at the nuclear pore complex during mRNA export II: nucleotide cycling and mRNP remodeling by Dbp5 are controlled by Nup159 and Gle1

Essential messenger RNA (mRNA) export factors execute critical steps to mediate directional transport through nuclear pore complexes (NPCs). At cytoplasmic NPC filaments, the ATPase activity of DEAD-box protein Dbp5 is activated by inositol hexakisphosphate (IP(6))-bound Gle1 to mediate remodeling of mRNA-protein (mRNP) complexes. Whether a single Dbp5 executes multiple remodeling events and how Dbp5 is recycled are unknown. Evidence suggests that Dbp5 binding to Nup159 is required for controlling interactions with Gle1 and the mRNP. Using in vitro reconstitution assays, we found here that Nup159 is specifically required for ADP release from Dbp5. Moreover, Gle1-IP(6) stimulates ATP binding, thus priming Dbp5 for RNA loading. In vivo, a dbp5-R256D/R259D mutant with reduced ADP binding bypasses the need for Nup159 interaction. However, NPC spatial control is important, as a dbp5-R256D/R259D nup42Δ double mutant is temperature-sensitive for mRNA export. Further analysis reveals that remodeling requires a conformational shift to the Dbp5-ADP form. ADP release factors for DEAD-box proteins have not been reported previously and reflect a new paradigm for regulation. We propose a model wherein Nup159 and Gle1-IP(6) regulate Dbp5 cycles by controlling its nucleotide-bound state, allowing multiple cycles of mRNP remodeling by a single Dbp5 at the NPC.

RNA export factor RAE1 contributes to NUP98-HOXA9-mediated leukemogenesis

Chromosomal translocations involving chimeric fusions of the nucleoporin NUP98 protein have often been described in acute myelogenous leukemia (AML). All the fusion proteins have an identical NUP98 N terminus, which contains the GLEBS motif for interaction with the mRNA export factor RAE1 and FG repeats that associate with the transcription factors HDAC1 and p300. It is virtually unknown whether these interaction partners affect leukemogenesis. We previously showed that RAE1 depletion caused aneuploidy, which enhanced tumorigenesis. We speculated that RAE1 may also be directly involved in NUP98 fusion-mediated leukemogenesis. We show here that RNA interference (RNAi)-mediated knockdown of NUP98 caused severe chromosome segregation defects and disrupted RAE1 but not HDAC1 expression and localization. Next, we performed rescue experiments to confirm that the RAE1-NUP98 complex orchestrates proper chromosome segregation. Interestingly, we found diverse behaviors of NUP98 and the leukemogenic fusion protein NUP98-HOXA9 throughout the cell cycle. Strikingly, in NUP98-HOXA9-transfected cells, RAE1 protein were reduced and mis-localized. Our cellular interpretations were further confirmed by NUP98-HOXA9 transgenic mice and the NUP98-HOXA9 AML patient. These data suggest that RAE1 orchestrates NUP98-mediated leukemogenesis and raise the possibility that targeting this negative feedback loop may provide a new strategy for the therapy of aggressive leukemias.

Association of mRNA export factor Yra1 with the C‐terminal Domain of RNA Polymerase II: a mechanism for cotranscriptional recruitment

The unique C‐terminal domain (CTD) of RNA polymerase II (Pol II) is composed of tandem heptad repeats of the consensus sequence YSPTSPS. This domain is subject to differential phosphorylation throughout the transcription cycle, mainly on serines 2 and 5 of the heptad. RNA processing factors have been shown to bind the CTD to localize to nascent pre‐mRNA during transcription. In S. cerevisiae, the mRNA export protein Yra1 (ALY/REF in metazoa) cotranscriptionally associates with mRNA and delivers it to the nuclear pore complex for export to the cytoplasm. Here we report that Yra1 directly binds in vitro to the elongating or hyperphosphorylated form of the CTD. Using truncated versions of Yra1, we have determined that amino acids 18–184, which includes its RNA Recognition Motif (RRM), are involved in phosphoCTD binding. Although the RRM is involved in CTD binding, RRM point mutations that produce defects in RNA binding and mRNA export do not affect CTD binding. In addition, posttranslational modifications in the N‐ and C‐termini of Yra1 abrogate RNA binding but not CTD binding. Using Yra1 truncations and point mutants, the coordinated recruitment of mRNA packaging and export factors in yeast will be investigated in vivo. This study was supported by NIH grant GM040505 to ALG.

Recruitment of cyclin-dependent kinase 9 to nuclear compartments during cytomegalovirus late replication: importance of an interaction between viral pUL69 and cyclin T1

Cyclin-dependent protein kinases (CDKs) are important regulators of cellular processes and are functionally integrated into the replication of human cytomegalovirus (HCMV). Recently, a regulatory impact of CDK activity on the viral mRNA export factor pUL69 was shown. Here, specific aspects of the mode of interaction between CDK9/cyclin T1 and pUL69 are described. Intracellular localization was studied in the presence of a novel selective CDK9 inhibitor, R22, which exerts anti-cytomegaloviral activity in vitro. A pronounced R22-induced formation of nuclear speckled aggregation of pUL69 was demonstrated. Multi-labelling confocal laser-scanning microscopy revealed that CDK9 and cyclin T1 co-localized perfectly with pUL69 in individual speckles. The effects were similar to those described recently for the broad CDK inhibitor roscovitine. Co-immunoprecipitation and yeast two-hybrid analyses showed that cyclin T1 interacted with both CDK9 and pUL69. The interaction region of pUL69 for cyclin T1 could be attributed to aa 269-487. Moreover, another component of CDK inhibitor-induced speckled aggregates was identified with RNA polymerase II, supporting earlier reports that strongly suggested an association of pUL69 with transcription complexes. Interestingly, when using a UL69-deleted recombinant HCMV, no speckled aggregates were formed by CDK inhibitor treatment. This indicated that pUL69 is the defining component of aggregates and generally may represent a crucial viral interactor of cyclin T1. In conclusion, these data emphasize that HCMV inter-regulation with CDK9/cyclin T1 is at least partly based on a pUL69-cylin T1 interaction, thus contributing to the importance of CDK9 for HCMV replication.

Nuclear Tau, a Key Player in Neuronal DNA Protection

Tau, a neuronal protein involved in neurodegenerative disorders such as Alzheimer disease, which is primarily described as a microtubule-associated protein, has also been observed in the nuclei of neuronal and non-neuronal cells. However, the function of the nuclear form of Tau in neurons has not yet been elucidated. In this work, we demonstrate that acute oxidative stress and mild heat stress (HS) induce the accumulation of dephosphorylated Tau in neuronal nuclei. Using chromatin immunoprecipitation assays, we demonstrate that the capacity of endogenous Tau to interact with neuronal DNA increased following HS. Comet assays performed on both wild-type and Tau-deficient neuronal cultures showed that Tau fully protected neuronal genomic DNA against HS-induced damage. Interestingly, HS-induced DNA damage observed in Tau-deficient cells was completely rescued after the overexpression of human Tau targeted to the nucleus. These results highlight a novel role for nuclear Tau as a key player in early stress response.

Structural Basis for the Recognition of Cellular mRNA Export Factor REF by Herpes Viral Proteins HSV-1 ICP27 and HVS ORF57

The herpesvirus proteins HSV-1 ICP27 and HVS ORF57 promote viral mRNA export by utilizing the cellular mRNA export machinery. This function is triggered by binding to proteins of the transcription-export (TREX) complex, in particular to REF/Aly which directs viral mRNA to the TAP/NFX1 pathway and, subsequently, to the nuclear pore for export to the cytoplasm. Here we have determined the structure of the REF-ICP27 interaction interface at atomic-resolution and provided a detailed comparison of the binding interfaces between ICP27, ORF57 and REF using solution-state NMR. Despite the absence of any obvious sequence similarity, both viral proteins bind on the same site of the folded RRM domain of REF, via short but specific recognition sites. The regions of ICP27 and ORF57 involved in binding by REF have been mapped as residues 104–112 and 103–120, respectively. We have identified the pattern of residues critical for REF/Aly recognition, common to both ICP27 and ORF57. The importance of the key amino acid residues within these binding sites was confirmed by site-directed mutagenesis. The functional significance of the ORF57-REF/Aly interaction was also probed using an ex vivo cytoplasmic viral mRNA accumulation assay and this revealed that mutants that reduce the protein-protein interaction dramatically decrease the ability of ORF57 to mediate the nuclear export of intronless viral mRNA. Together these data precisely map amino acid residues responsible for the direct interactions between viral adaptors and cellular REF/Aly and provide the first molecular details of how herpes viruses access the cellular mRNA export pathway.

The RNA Binding Motif Protein 15B (RBM15B/OTT3) Is a Functional Competitor of Serine-Arginine (SR) Proteins and Antagonizes the Positive Effect of the CDK11p110-Cyclin L2α Complex on Splicing

Here, we report the identification of the RNA binding motif protein RBM15B/OTT3 as a new CDK11(p110) binding partner that alters the effects of CDK11 on splicing. RBM15B was initially identified as a binding partner of the Epstein-Barr virus mRNA export factor and, more recently, as a cofactor of the nuclear export receptor NXF1. In this study, we found that RBM15B co-elutes with CDK11(p110), cyclin L2α, and serine-arginine (SR) proteins, including SF2/ASF, in a large nuclear complex of ∼1-MDa molecular mass following size exclusion chromatography. Using co-immunoprecipitation experiments and in vitro pulldown assays, we mapped two distinct domains of RBM15B that are essential for its direct interaction with the N-terminal extension of CDK11(p110), cyclin L2α, and SR proteins such as 9G8 and SF2/ASF. Finally, we established that RBM15B is a functional competitor of the SR proteins SF2/ASF and 9G8, inhibits formation of the functional spliceosomal E complex, and antagonizes the positive effect of the CDK11(p110)-cyclin L2α complex on splicing both in vitro and in vivo.

Characterization of the betaherpesviral pUL69 protein family reveals binding of the cellular mRNA export factor UAP56 as a prerequisite for stimulation of nuclear mRNA export and for efficient viral replication.

UL69 of human cytomegalovirus (HCMV) encodes a pleiotropic transactivator protein and has a counterpart in every member of the Herpesviridae family thus far sequenced. However, little is known about the conservation of the functions of the nuclear phosphoprotein pUL69 in the homologous proteins of other betaherpesviruses. Therefore, eukaryotic expression vectors were constructed for pC69 of chimpanzee cytomegalovirus, pRh69 of rhesus cytomegalovirus, pM69 of murine cytomegalovirus, pU42 of human herpesvirus 6, and pU42 of elephant endotheliotropic herpesvirus. Indirect immunofluorescence experiments showed that all pUL69 homologs expressed by these vectors were localized to the cell nucleus. Coimmunoprecipitation experiments identified homodimerization as a conserved feature of all homologs, whereas heterodimerization with pUL69 was restricted to its closer relatives. Further analyses demonstrated that pC69 and pRh69 were the only two homologs that functioned, like pUL69, as viral-mRNA export factors. As we had reported recently that nucleocytoplasmic shuttling and interaction with the cellular DExD/H-box helicases UAP56 and URH49 were prerequisites for the nuclear-mRNA export activity of pUL69, the homologs were characterized with regard to these properties. Heterokaryon assays demonstrated nucleocytoplasmic shuttling for all homologs, and coimmunoprecipitation and mRNA export assays revealed that the interaction of UAP56 and/or URH49 with pC69 or pRh69 was required for mRNA export activity. Moreover, characterization of HCMV recombinants harboring mutations within the N-terminal sequence of pUL69 revealed a strong replication defect of viruses expressing pUL69 variants that were deficient in UAP56 binding. In summary, homodimerization and nucleocytoplasmic shuttling activity were identified as conserved features of betaherpesviral pUL69 homologs. UAP56 binding was shown to represent a unique characteristic of members of the genus Cytomegalovirus that is required for efficient replication of HCMV.

Interchromatin granule clusters of the scorpionfly oocytes contain poly(A)+ RNA, heterogeneous ribonucleoproteins A/B and mRNA export factor NXF1

IGCs (interchromatin granule clusters), or nuclear speckles, are one of the most universal subnuclear organelles of eukaryotic cells. We have used insect oocytes to study the possible association of poly(A)+ RNA and some factors involved in mRNA export with IGCs. Oogenesis of the mecopteran, Panorpa communis, used as a model object, is characterized by a strict cessation of oocyte genome transcription activity towards the end of oocyte growth. Our previous studies on P. communis oocyte nuclei have shown that oocyte IGC counterparts in this species are very unusual, both in morphology and molecular composition, compared with the typical IGCs of mammalian somatic cells traditionally used as a model system. We have now used microinjections of 2'-O-Me(U)₂₂ probes conjugated with the fluorochrome TAMRA to localize poly(A)+ RNA in IGCs. RNA export proteins were also detected by immunofluorescent/confocal and immunogold labelling electron microscopy. We found that poly(A)+ RNA, hnRNPs A/B and NXF1 mRNA export factors are located in IGCs regardless of the transcriptional status of the nucleus. Our data support the idea of IGCs as universal and conserved nuclear domains that serve not only as splicing factor reservoirs, but also take part in mRNA retention and export.

The Mitogen-Activated Protein Kinase Slt2 Regulates Nuclear Retention of Non-Heat Shock mRNAs during Heat Shock-Induced Stress

Cellular adaptation to environmental stress conditions requires rapid and specific changes in gene expression. During heat shock, most polyadenylated mRNAs are retained in the nucleus, whereas the export of heat shock-induced mRNAs is allowed. Although essential mRNA export factors are known, the precise mechanism for regulating transport is not fully understood. Here we find that during heat shock in Saccharomyces cerevisiae, the mRNA-binding protein Nab2 is phosphorylated on threonine 178 and serine 180 by the mitogen-activated protein (MAP) kinase Slt2/Mpk1. Slt2 is required for nuclear poly(A(+)) mRNA accumulation upon heat shock, and thermotolerance is decreased in a nup42 nab2-T178A/S180A mutant. Coincident with phosphorylation, Nab2 and Yra1 colocalize in nuclear foci with Mlp1, a protein involved in mRNA retention. Nab2 nuclear focus formation and Nab2 phosphorylation are independent, suggesting that heat shock induces multiple cellular alterations that impinge upon transport efficiency. Under normal conditions, we find that the mRNA export receptor Mex67 and Nab2 directly interact. However, upon heat shock stress, Mex67 does not localize to the Mlp1 nuclear foci, and its association with Nab2 complexes is reduced. These results reveal a novel mechanism by which the MAP kinase Slt2 and Mlp1 control mRNA export factors during heat shock stress.

A Screening Method Tuned for mRNA Processing Factors in Human Cells by Evaluation of the Luciferase Reporter Activity and the Subcellular Distribution of Bulk Poly(A)+RNA

Screening of mRNA export factors in Saccharomyces cerevisiae and Drosophila melanogaster has identified a number of mRNA processing factors involved in multiple mRNA processing steps. However, only limited information is available on human cells. Here we established a screening system searching for mRNA processing factors in human cells by combining the luciferase reporter system and fluorescence in situ hybridization, which evaluates the nuclear/cytoplasmic distribution of bulk poly(A)+ RNA. This system makes it possible to search for the compounds affecting mRNA processing from the various resources.

Facilitated transport and diffusion take distinct spatial routes through the nuclear pore complex

Transport across the nuclear envelope is regulated by nuclear pore complexes (NPCs). Much is understood about the factors that shuttle and control the movement of cargos through the NPC, but less has been resolved about the translocation process itself. Various models predict how cargos move through the channel; however, direct observation of the process is missing. Therefore, we have developed methods to accurately determine cargo positions within the NPC. Cargos were instantly trapped in transit by high-pressure freezing, optimally preserved by low-temperature fixation and then localized by immunoelectron microscopy. A statistical modelling approach was used to identify cargo distribution. We found import cargos localized surprisingly close to the edge of the channel, whereas mRNA export factors were at the very centre of the NPC. On the other hand, diffusion of GFP was randomly distributed. Thus, we suggest that spatially distinguished pathways exist within the NPC. Deletion of specific FG domains of particular NPC proteins resulted in collapse of the peripheral localization and transport defects specific to a certain karyopherin pathway. This further confirms that constraints on the route of travel are biochemical rather than structural and that the peripheral route of travel is essential for facilitated import.

Binding of the Human Cytomegalovirus (HCMV) Tegument Protein UL69 to UAP56/URH49 Is Not Required for Efficient Replication of HCMV

The human cytomegalovirus (HCMV) tegument protein UL69 is important for efficient viral replication at low multiplicities of infection. Several molecular mechanisms by which UL69 contributes to HCMV replication have been proposed, including UL69's ability to interact with the mRNA export factors UAP56 and URH49 to facilitate the shuttling of viral mRNAs from the nuclei of infected cells. Using a UL69 viral mutant that is unable to bind UAP56 and URH49, we demonstrated that UL69's interaction with UAP56 or URH49 does not contribute to the growth phenotype associated with the UL69 deletion mutant.

Structural Basis for the Function of the Saccharomyces cerevisiae Gfd1 Protein in mRNA Nuclear Export

Following transcription, mRNA is processed, packaged into messenger ribonucleoprotein (mRNP) particles, and transported through nuclear pores (NPCs) to the cytoplasm. At the NPC cytoplasmic face, Dbp5 mediates mRNP remodeling and mRNA export factor dissociation, releasing transcripts for translation. In Saccharomyces cerevisiae, the conserved poly(A) RNA-binding protein, Nab2, facilitates NPC targeting of transcripts and also modulates poly(A) tail length. Dbp5 removes Nab2 from mRNPs at the cytoplasmic face of the pore and, importantly, a Nab2 RNA-binding mutant suppresses the thermosensitive rat8-2 (dbp5) mutant. GFD1 is a multicopy suppressor of rat8-2 (dbp5), and Gfd1 interacts physically with both Dbp5 and the Nab2 N-terminal domain (Nab2-N). Here, we present a structural and functional analysis of the Gfd1/Nab2-N interaction. Crystallography, supported by solution NMR, shows that Gfd1 residues 126–150 form an α-helix when bound to Nab2-N. Engineered Nab2-N and Gfd1 mutants that inhibit this interaction in vitro were used to probe its function in vivo using the genetic interaction between GFD1 and NAB2. Although GFD1 is not essential for viability, its deletion severely impairs growth of rat8-2 (dbp5) cells. Moreover, although Gfd1 overexpression suppresses rat8-2 (dbp5), Gfd1 mutants that do not bind Nab2 only partially suppress rat8-2 (dbp5). Furthermore, rat8-2 (dbp5) cells that express nab2-Y34A, in which binding to Gfd1 is impaired, show a synthetic growth phenotype and nuclear accumulation of poly(A) RNA. These data support the importance of the Gfd1/Nab2 interaction for Dbp5 activity and provide further molecular details of the interactions that facilitate Dbp5-mediated mRNP remodeling in the terminal step of mRNA export.

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

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 A resolution. Rae1 forms a seven-bladed beta-propeller with several extensive surface loops. The Nup98 GLEBS motif forms an approximately 50-A-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 beta-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.