Nuclear export of ribosomal 60S subunits by the general mRNA export receptor Mex67-Mtr2

The yeast Mex67-Mtr2 complex and its homologous metazoan counterpart TAP-p15 operate as nuclear export receptors by binding and translocating mRNA through the nuclear pore complexes. Here, we show how Mex67-Mtr2 can also function in the nuclear export of the ribosomal 60S subunit. Biochemical and genetic studies reveal a previously unrecognized interaction surface on the NTF2-like scaffold of the Mex67-Mtr2 heterodimer, which in vivo binds to pre-60S particles and in vitro can interact with 5S rRNA. Crucial structural requirements for this binding platform are loop insertions in the middle domain of Mex67 and Mtr2, which are absent from human TAP-p15. Notably, when the positively charged amino acids in the Mex67 loop are mutated, interaction of Mex67-Mtr2 with pre-60S particles and 5S rRNA is inhibited, and 60S subunits, but not mRNA, accumulate in the nucleus. Thus, the general mRNA exporter Mex67-Mtr2 contains a distinct electrostatic interaction surface for transporting 60S preribosomal cargo.     

Nuclear mRNA Export Requires Complex Formation between Mex67p and Mtr2p at the Nuclear Pores

We have identified between Mex67p and Mtr2p a complex which is essential for mRNA export. This complex, either isolated from yeast or assembled in Escherichia coli, can bind in vitro to RNA through Mex67p. In vivo, Mex67p requires Mtr2p for association with the nuclear pores, which can be abolished by mutating either MEX67 or MTR2. In all cases, detachment of Mex67p from the pores into the cytoplasm correlates with a strong inhibition of mRNA export. At the nuclear pores, Nup85p represents one of the targets with which the Mex67p-Mtr2p complex interacts. Thus, Mex67p and Mtr2p constitute a novel mRNA export complex which can bind to RNA via Mex67p and which interacts with nuclear pores via Mtr2p.     

A versatile interaction platform on the Mex67-Mtr2 receptor creates an overlap between mRNA and ribosome export

The transport receptor Mex67-Mtr2 functions in mRNA export, and also by a loop-confined surface on the heterodimer binds to and exports pre-60S particles. We show that Mex67-Mtr2 through the same surface that recruits pre-60S particles interacts with the Nup84 complex, a structural module of the nuclear pore complex devoid of Phe-Gly domains. In vitro, pre-60S particles and the Nup84 complex compete for an overlapping binding site on the loop-extended Mex67-Mtr2 surface. Chemical crosslinking identified Nup85 as the subunit in the Nup84 complex that directly binds to the Mex67 loop. Genetic studies revealed that this interaction is crucial for mRNA export. Notably, pre-60S subunit export impaired by mutating Mtr2 or the 60S adaptor Nmd3 could be partially restored by second-site mutation in Nup85 that caused dissociation of Mex67-Mtr2 from the Nup84 complex. Thus, the Mex67-Mtr2 export receptor employs a versatile binding platform on its surface that could create a crosstalk between mRNA and ribosome export pathways.     

Nuclear export competence of pre-40S subunits in fission yeast requires the ribosomal protein Rps2

Ribosome biogenesis is an evolutionarily conserved pathway that requires ribosomal and nonribosomal proteins. Here, we investigated the role of the ribosomal protein S2 (Rps2) in fission yeast ribosome synthesis. As for many budding yeast ribosomal proteins, Rps2 was essential for cell viability in fission yeast and the genetic depletion of Rps2 caused a complete inhibition of 40S ribosomal subunit production. The pattern of pre-rRNA processing upon depletion of Rps2 revealed a reduction of 27SA₂ pre-rRNAs and the concomitant production of 21S rRNA precursors, consistent with a role for Rps2 in efficient cleavage at site A₂ within the 32S pre-rRNA. Importantly, kinetics of pre-rRNA accumulation as determined by rRNA pulse-chases assays indicated that a small fraction of 35S precursors matured into 20S-containing particles, suggesting that most 40S precursors were rapidly degraded in the absence of Rps2. Analysis of steady-state RNA levels revealed that some pre-40S particles were produced in Rps2-depleted cells, but that these precursors were retained in the nucleolus. Our findings suggest a role for Rps2 in a mechanism that monitors pre-40S export competence.     

A Second CRM1-Dependent Nuclear Export Signal in the Influenza A Virus NS2 Protein Contributes to the Nuclear Export of Viral Ribonucleoproteins

Influenza A virus NS2 protein, also called nuclear export protein (NEP), is crucial for the nuclear export of viral ribonucleoproteins. However, the molecular mechanisms of NEP mediation in this process remain incompletely understood. A leucine-rich nuclear export signal (NES2) in NEP, located at the predicted N2 helix of the N-terminal domain, was identified in the present study. NES2 was demonstrated to be a transferable NES, with its nuclear export activity depending on the nuclear export receptor chromosome region maintenance 1 (CRM1)-mediated pathway. The interaction between NEP and CRM1 is coordinately regulated by both the previously reported NES (NES1) and now the new NES2. Deletion of the NES1 enhances the interaction between NEP and CRM1, and deletion of the NES1 and NES2 motifs completely abolishes this interaction. Moreover, NES2 interacts with CRM1 in the mammalian two-hybrid system. Mutant viruses containing NES2 alterations generated by reversed genetics exhibit reduced viral growth and delay in the nuclear export of viral ribonucleoproteins (vRNPs). The NES2 motif is highly conserved in the influenza A and B viruses. The results demonstrate that leucine-rich NES2 is involved in the nuclear export of vRNPs and contributes to the understanding of nucleocytoplasmic transport of influenza virus vRNPs.     

Mex67p of Schizosaccharomyces pombe interacts with Rae1p in mediating mRNA export

We identified the Schizosaccharomyces pombe mex67 gene (spmex67) as a multicopy suppressor of rae1-167 nup184-1 synthetic lethality and the rae1-167 ts mutation. spMex67p, a 596-amino-acid-long protein, has considerable sequence similarity to the Saccharomyces cerevisiae Mex67p (scMex67p) and human Tap. In contrast to scMEX67, spmex67 is essential for neither growth nor nuclear export of mRNA. However, an spmex67 null mutation (Deltamex67) is synthetically lethal with the rae1-167 mutation and accumulates poly(A)(+) RNA in the nucleus. We identified a central region (149 to 505 amino acids) within spMex67p that associates with a complex containing Rae1p that complements growth and mRNA export defects of the rae1-167 Deltamex67 synthetic lethality. This region is devoid of RNA-binding, N-terminal nuclear localization, and the C-terminal nuclear pore complex-targeting regions. The (149-505)-green fluorescent protein (GFP) fusion is found diffused throughout the cell. Overexpression of spMex67p inhibits growth and mRNA export and results in the redistribution of the diffused localization of the (149-505)-GFP fusion to the nucleus and the nuclear periphery. These results suggest that spMex67p competes for essential mRNA export factor(s). Finally, we propose that the 149-505 region of spMex67p could act as an accessory factor in Rae1p-dependent transport and that spMex67p participates at various common steps with Rae1p export complexes in promoting the export of mRNA.     

A Role for the M9 Transport Signal of hnRNP A1 in mRNA Nuclear Export

Among the nuclear proteins associated with mRNAs before their export to the cytoplasm are the abundant heterogeneous nuclear (hn) RNPs. Several of these contain the M9 signal that, in the case of hnRNP A1, has been shown to be sufficient to signal both nuclear export and nuclear import in cultured somatic cells. Kinetic competition experiments are used here to demonstrate that M9-directed nuclear import in Xenopus oocytes is a saturable process. Saturating levels of M9 have, however, no effect on the import of either U snRNPs or proteins carrying a classical basic NLS. Previous work demonstrated the existence of nuclear export factors specific for particular classes of RNA. Injection of hnRNP A1 but not of a mutant protein lacking the M9 domain inhibited export of mRNA but not of other classes of RNA. This suggests that hnRNP A1 or other proteins containing an M9 domain play a role in mRNA export from the nucleus. However, the requirement for M9 function in mRNA export is not identical to that in hnRNP A1 protein transport.The transport of macromolecules between the nucleus and cytoplasm is a bi-directional process. The best understood aspect is the import of nuclear proteins that carry a basic nuclear localization signal (NLS)¹ like the simple NLS found in SV-40 T antigen or the bipartite NLS found in nucleoplasmin (Dingwall and Laskey, 1991). Proteins of this class are recognized by the heterodimeric importin receptor, composed of importin α and importin β (for review see Powers and Forbes, 1994; Melchior and Gerace, 1995; Görlich and Mattaj, 1996). The NLS binds directly to the importin α subunit. The importin NLS protein complex docks at the cytoplasmic face of the nuclear pore complex in an energy-independent manner (Newmeyer and Forbes, 1988; Richardson et al., 1988). Subsequently, the small GTPase Ran/TC4 (Melchior et al., 1993; Moore and Blobel, 1993) and a protein of unknown function named variously pp15, p10, or NTF2 (Moore and Blobel, 1994; Paschal and Gerace, 1995) are required for translocation of the NLS-containing complex through the nuclear pore complex.A second major class of imported macromolecules are the uracil rich small nuclear (U sn) RNPs. They do not have a basic NLS but instead have a bipartite nuclear targeting signal. This is composed of an essential signal formed when the Sm core proteins bind to the U snRNA and an additional signal, the trimethyl-guanosine (m₃G) cap, which depending on the cell type or the U snRNA is either essential or required for optimal U snRNP import efficiency (Fischer and Lührmann, 1990; Hamm et al., 1990; Fischer et al., 1993). Kinetic competition experiments have supported the conclusion that U snRNPs require different limiting factors than do NLS-containing proteins for their import and that U snRNPs do not bind to importin α (Fischer et al., 1991, 1993; Michaud and Goldfarb, 1991; van Zee et al., 1993). There is also preliminary evidence that additional different receptors may be required for the nuclear uptake of other RNA species (Michaud and Goldfarb, 1992).Similarly, RNA export from the nucleus relies on recognition of the RNA or RNP export substrates by saturable factors (Zasloff, 1983; Bataillé et al., 1990; Jarmolowski et al., 1994). As for import, evidence for the existence of RNA class-specific export receptors has been obtained from kinetic competition experiments (Jarmolowski et al., 1994). Two RNA-binding proteins have been directly shown to function in RNA export, a nuclear cap binding protein complex in the case of U snRNAs (Izaurralde et al., 1995a ) and the HIV-1 Rev protein in the case of RNAs containing a rev response element (Fischer et al., 1994, 1995). In the case of mRNAs, the best candidates for export mediators are the heterogeneous nuclear (hn) RNP proteins (for review see Piñol-Roma and Dreyfuss, 1993; Izaurralde and Mattaj, 1995).About 20 different hnRNP proteins have been characterized in vertebrate cells (for review see Dreyfuss et al., 1993). The association of hnRNP proteins with mRNA in the nucleus and the cytoplasm suggests that they may regulate and/or facilitate different aspects of gene expression. The possibility that hnRNP proteins might be directly involved in the nucleocytoplasmic trafficking of mRNA molecules was suggested by the observation that several hnRNP proteins, including A1, A2, D, E, I, and K shuttle continuously and rapidly between the nucleus and the cytoplasm and are associated with mRNA in both compartments (Piñol-Roma and Dreyfuss 1992, 1993; Michael et al., 1995a , b). Of these, the best studied example is hnRNP A1. An A1-like hnRNP protein has been shown by immunoelectron microscopy to be associated with a specific mRNA in transit to the cytoplasm through the nuclear pore complex in the insect Chironomus tentans (Visa et al., 1996a ). In mammalian cells, the amount of A1 which is in constant flux between nucleus and cytoplasm is striking. It has been estimated that at least 120,000 molecules of A1 are exported to the cytoplasm per minute but then rapidly reimported such that the steady state localization of A1 is nuclear (Michael et al., 1995a ). Taken together, these results suggest that A1 and other shuttling hnRNP proteins such as A2, D, E, I, and K could play a significant role in the transport of mRNA from the nucleus to the cytoplasm.One key in understanding how hnRNPs may facilitate mRNA export is to determine the signals that mediate their shuttling, i.e., their import into and exit from the nucleus. The nucleocytoplasmic transport of A1 has been recently studied in detail, and the signals that mediate shuttling have been identified (Michael et al., 1995b ; Siomi and Dreyfuss, 1995; Weighardt et al., 1995). Nuclear import of A1 is determined by a 38-amino acid sequence, termed M9, located near the COOH terminus of the protein between amino acids 268 and 305. Its fusion to cytoplasmic reporter proteins such as pyruvate kinase resulted in rapid import of the fusion protein into the nucleus (Siomi and Dreyfuss, 1995). However, the A1 NLS has no sequence similarity to classical protein NLSs such as that of SV-40 large T antigen or nucleoplasmin (Siomi and Dreyfuss, 1995).Surprisingly, M9 also acts as a nuclear export signal (NES). In heterokaryon shuttling assays this domain is necessary and sufficient to allow the export of heterologous proteins, such as the nucleoplasmin core domain (NPLc), which are normally retained in the nucleus (Michael et al., 1995b ). Thus, M9 alone can account for the shuttling of A1. Other hnRNPs such as A2 and B1 bear sequences with striking similarities to M9 (Siomi and Dreyfuss, 1995). Mutagenesis experiments indicate that the NES and NLS activities of M9 are either identical or overlapping as mutants which block M9 NLS activity also abolish NES activity (Michael et al., 1995b ). It is therefore possible that M9 is recognized in the nucleus and the cytoplasm by the same receptor.The second category of NES described was first found in the HIV-1 Rev protein and the inhibitor of protein kinase A (Fischer et al., 1995; Wen et al., 1995; Bogerd et al., 1996; for review see Gerace, 1995). These short, leucine-rich NES sequences bear no relationship to the primary sequence of M9. Furthermore, saturation of the export factor recognized by the Rev NES has no effect on mRNA export (Fischer et al., 1995). A model for mRNA export has been postulated on the basis of the hnRNP data described above. In this model, NES/NLS containing hnRNPs bind in the nucleus to mRNA molecules and deliver them, via the export pathway they access, to the cytoplasm. In the cytoplasm these hnRNP proteins dissociate from the mRNA and return to the nucleus. To further test this model we have analyzed the transport of hnRNP A1 and mRNA in Xenopus laevis oocytes. The oocyte offers a unique opportunity to manipulate specific import or export pathways, like that accessed by M9, and examine the effect on mRNA nuclear export. By using this approach we show here that M9 is, as in somatic cells, a functional NLS in oocytes. Moreover, competition studies indicate that M9 defines a novel class of NLS, since saturation of the M9- mediated import pathway does not interfere with the two previously identified import pathways used by classical NLS-bearing proteins or m₃G-capped-spliceosomal U snRNPs. Injection of an excess of hnRNP A1 but not of a mutant form of the protein lacking the M9 domain, resulted in a specific inhibition of mRNA export, demonstrating that the M9 domain is recognized by a saturable component of the mRNA export machinery. The export of other cellular RNAs such as U snRNAs and tRNA was, in contrast, not affected. Further analysis of mutant hnRNP A1 proteins provides evidence that M9 recognition during mRNA export differs from its recognition during protein transport.     

The yeast hnRNP-Like proteins Yra1p and Yra2p participate in mRNA export through interaction with Mex67p

Yra1p is an essential nuclear protein which belongs to the evolutionarily conserved REF (RNA and export factor binding proteins) family of hnRNP-like proteins. Yra1p contributes to mRNA export in vivo and directly interacts with RNA and the shuttling mRNP export receptor Mex67p in vitro. Here we describe a second nonessential Saccharomyces cerevisiae family member, called Yra2p, which is able to complement a YRA1 deletion when overexpressed. Like other REF proteins, Yra1p and Yra2p consist of two highly conserved N- and C-terminal boxes and a central RNP-like RNA-binding domain (RBD). These conserved regions are separated by two more variable regions, N-vr and C-vr. Surprisingly, the deletion of a single conserved box or the deletion of the RBD in Yra1p does not affect viability. Consistently, neither the conserved N and C boxes nor the RBD is required for Mex67p and RNA binding in vitro. Instead, the N-vr and C-vr regions both interact with Mex67p and RNA. We further show that Yra1 deletion mutants which poorly interact with Mex67p in vitro affect the association of Mex67p with mRNP complexes in vivo and are paralleled by poly(A)(+) RNA export defects. These observations support the idea that Yra1p promotes mRNA export by facilitating the recruitment of Mex67p to the mRNP.     

Functional Analysis of Tpr: Identification of Nuclear Pore Complex Association and Nuclear Localization Domains and a Role in mRNA Export

Tpr is a 270-kD coiled-coil protein localized to intranuclear filaments of the nuclear pore complex (NPC). The mechanism by which Tpr contributes to the structure and function of the nuclear pore is currently unknown. To gain insight into Tpr function, we expressed the full-length protein and several subdomains in mammalian cell lines and examined their effects on nuclear pore function. Through this analysis, we identified an NH₂-terminal domain that was sufficient for association with the nucleoplasmic aspect of the NPC. In addition, we unexpectedly found that the acidic COOH terminus was efficiently transported into the nuclear interior, an event that was apparently mediated by a putative nuclear localization sequence. Ectopic expression of the full-length Tpr caused a dramatic accumulation of poly(A)⁺ RNA within the nucleus. Similar results were observed with domains that localized to the NPC and the nuclear interior. In contrast, expression of these proteins did not appear to affect nuclear import. These data are consistent with a model in which Tpr is tethered to intranuclear filaments of the NPC by its coiled coil domain leaving the acidic COOH terminus free to interact with soluble transport factors and mediate export of macromolecules from the nucleus.     

Nuclear export and cytoplasmic processing of precursors to the 40S ribosomal subunits in mammalian cells

It is generally assumed that, in mammalian cells, preribosomal RNAs are entirely processed before nuclear exit. Here, we show that pre-40S particles exported to the cytoplasm in HeLa cells contain 18S rRNA extended at the 3' end with 20-30 nucleotides of the internal transcribed spacer 1. Maturation of this pre-18S rRNA (which we named 18S-E) involves a cytoplasmic protein, the human homolog of the yeast kinase Rio2p, and appears to be required for the translation competence of the 40S subunit. By tracking the nuclear exit of this precursor, we have identified the ribosomal protein Rps15 as a determinant of preribosomal nuclear export in human cells. Interestingly, inhibition of exportin Crm1/Xpo1 with leptomycin B strongly alters processing of the 5'-external transcribed spacer, upstream of nuclear export, and reveals a new cleavage site in this transcribed spacer. Completion of the maturation of the 18S rRNA in the cytoplasm, a feature thought to be unique to yeast, may prevent pre-40S particles from initiating translation with pre-mRNAs in eukaryotic cells. It also allows new strategies for the study of preribosomal transport in mammalian cells.     

Comparative Analysis of Seven Viral Nuclear Export Signals (NESs) Reveals the Crucial Role of Nuclear Export Mediated by the Third NES Consensus Sequence of Nucleoprotein (NP) in Influenza A Virus Replication

The assembly of influenza virus progeny virions requires machinery that exports viral genomic ribonucleoproteins from the cell nucleus. Currently, seven nuclear export signal (NES) consensus sequences have been identified in different viral proteins, including NS1, NS2, M1, and NP. The present study examined the roles of viral NES consensus sequences and their significance in terms of viral replication and nuclear export. Mutation of the NP-NES3 consensus sequence resulted in a failure to rescue viruses using a reverse genetics approach, whereas mutation of the NS2-NES1 and NS2-NES2 sequences led to a strong reduction in viral replication kinetics compared with the wild-type sequence. While the viral replication kinetics for other NES mutant viruses were also lower than those of the wild-type, the difference was not so marked. Immunofluorescence analysis after transient expression of NP-NES3, NS2-NES1, or NS2-NES2 proteins in host cells showed that they accumulated in the cell nucleus. These results suggest that the NP-NES3 consensus sequence is mostly required for viral replication. Therefore, each of the hydrophobic (Φ) residues within this NES consensus sequence (Φ1, Φ2, Φ3, or Φ4) was mutated, and its viral replication and nuclear export function were analyzed. No viruses harboring NP-NES3 Φ2 or Φ3 mutants could be rescued. Consistent with this, the NP-NES3 Φ2 and Φ3 mutants showed reduced binding affinity with CRM1 in a pull-down assay, and both accumulated in the cell nucleus. Indeed, a nuclear export assay revealed that these mutant proteins showed lower nuclear export activity than the wild-type protein. Moreover, the Φ2 and Φ3 residues (along with other Φ residues) within the NP-NES3 consensus were highly conserved among different influenza A viruses, including human, avian, and swine. Taken together, these results suggest that the Φ2 and Φ3 residues within the NP-NES3 protein are important for its nuclear export function during viral replication.     

Functional Characterization of Nuclear Localization and Export Signals in Hepatitis C Virus Proteins and Their Role in the Membranous Web

The hepatitis C virus (HCV) is a positive strand RNA virus of the Flavivirus family that replicates in the cytoplasm of infected hepatocytes. Previously, several nuclear localization signals (NLS) and nuclear export signals (NES) have been identified in HCV proteins, however, there is little evidence that these proteins travel into the nucleus during infection. We have recently shown that nuclear pore complex (NPC) proteins (termed nucleoporins or Nups) are present in the membranous web and are required during HCV infection. In this study, we identify a total of 11 NLS and NES sequences in various HCV proteins. We show direct interactions between HCV proteins and importin α5 (IPOA5/kapα1), importin β3 (IPO5/kap β3), and exportin 1 (XPO1/CRM1) both in-vitro and in cell culture. These interactions can be disrupted using peptides containing the specific NLS or NES sequences of HCV proteins. Moreover, using a synchronized infection system, we show that these peptides inhibit HCV infection during distinct phases of the HCV life cycle. The inhibitory effects of these peptides place them in two groups. The first group binds IPOA5 and inhibits infection during the replication stage of HCV life cycle. The second group binds IPO5 and is active during both early replication and early assembly. This work delineates the entire life cycle of HCV and the active involvement of NLS sequences during HCV replication and assembly. Given the abundance of NLS sequences within HCV proteins, our previous finding that Nups play a role in HCV infection, and the relocation of the NLS double-GFP reporter in HCV infected cells, this work supports our previous hypothesis that NPC-like structures and nuclear transport factors function in the membranous web to create an environment conducive to viral replication.     

Targeting XPO1-Dependent Nuclear Export in Cancer

Biochemistry (Moscow) - Nucleocytoplasmic transport of macromolecules is tightly regulated in eukaryotic cells. XPO1 is a transport factor responsible for the nuclear export of several hundred...     

Role of S65, Q67, I68, and Y69 residues in homotetrameric R67 dihydrofolate reductase

R67 dihydrofolate reductase (DHFR) shares no sequence or structural homology with chromosomal DHFRs. This enzyme arose recently in response to the clinical use of the antibacterial drug trimethoprim. R67 DHFR is a homotetramer possessing a single active site pore. A high-resolution crystal structure shows the homotetramer possesses exact 222 symmetry [Narayana, N., et al. (1995) Nat. Struct. Biol. 2, 1018-1025]. This symmetry dictates four symmetry-related binding sites must exist for each substrate as well as each cofactor. Isothermal titration calorimetry studies, however, indicate only two molecules bind: either two dihydrofolate molecules, two NADPH molecules, or one substrate and one cofactor [Bradrick, T. D., et al. (1996) Biochemistry 35, 11414-11424]. The latter is the productive ternary complex. To evaluate the role of S65, Q67, I68, and Y69 residues, located near the center of the active site pore, site-directed mutagenesis was performed. One mutation in the gene creates four mutations per active site pore which typically result in large cumulative effects. Steady state kinetic data indicate the mutants have altered K(m) values for both cofactor and substrate. For example, the Y69F R67 DHFR displays an 8-fold increase in the K(m) for dihydrofolate and a 20-fold increase in the K(m) for NADPH. Residues involved in ligand binding in R67 DHFR display very little, if any, specificity, consistent with their possessing dual roles in binding. These results support a model where R67 DHFR utilizes an unusual "hot spot" binding surface capable of binding both ligands and indicate this enzyme has adopted a novel yet simple approach to catalysis.