Messenger RNAs are exported from the nucleus as large ribonucleoprotein complexes (mRNPs). To date, proteins implicated in this process

contributed equally to this work 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.

Vertebrate TAP (also called NXF1) and its yeast orthologue, Mex67p, have been implicated in the export of mRNAs from the nucleus. The TAP protein includes a noncanonical RNP-type RNA binding domain, four leucine-rich repeats, an NTF2-like domain that allows heterodimerization with p15 (also called NXT1), and a ubiquitin-associated domain that mediates the interaction with nucleoporins. Here we show that TAP belongs to an evolutionarily conserved family of proteins that has more than one member in higher eukaryotes. Not only the overall domain organization but also residues important for p15 and nucleoporin interaction are conserved in most family members. We characterize two of four human TAP homologues and show that one of them, NXF2, binds RNA, localizes to the nuclear envelope, and exhibits RNA export activity. NXF3, which does not bind RNA or localize to the nuclear rim, has no RNA export activity. Database searches revealed that although only one p15 (nxt) gene is present in the Drosophila melanogaster and Caenorhabditis elegans genomes, there is at least one additional p15 homologue (p15-2 [also called NXT2]) encoded by the human genome. Both human p15 homologues bind TAP, NXF2, and NXF3. Together, our results indicate that the TAP-p15 mRNA export pathway has diversified in higher eukaryotes compared to yeast, perhaps reflecting a greater substrate complexity. mRNAs are exported from the nucleus as large ribonucleoprotein complexes (mRNPs). To date, proteins directly implicated in this process include several nucleoporins and RNA binding proteins (hnRNPs), an RNA helicase of the DEADbox family (Dbp5), and the nuclear pore complex (NPC)-associated proteins Gle1p, TAP and Mex67p, and RAE1 (also called Gle2p) (reviewed in references 22, 28, and 32). Mex67p is essential for mRNA export in Saccharomyces cerevisiae, while RAE1 is essential for mRNA export in Schizosaccharomyces pombe (9,27,36). Their vertebrate homologues, TAP and RAE1, have also been implicated in the export of cellular mRNAs (6,8,12,15,20,31).We identified TAP as the cellular factor which is recruited by the constitutive transport element (CTE) of simian type D retroviruses to promote nuclear export of their genomic RNAs (12). In Xenopus oocytes, titration of TAP with an excess of CTE RNA prevents cellular mRNAs from exiting the nucleus (12,30,33). This suggests a role for this protein in the export of cellular mRNA.Considerable progress has been made in defining TAP structural and functional domains (see Fig. 1) and in identifying its binding partners. TAP partners include various nucleoporins (4, 17); p15 (also called NXT1), a protein related to nuclear transport factor 2 (NTF2) (7, 17); transportin, which mediates TAP nuclear import (4); and several mRNP-associated proteins, such as E1B-AP5, RAE1 (4), and members of the Yra1p/ REF family of proteins (37, 39). Binding of TAP to these mRNP-associated proteins is mediated by its N-terminal domain (residues 1 to 372) (4, 39). This domain includes a noncanonical RNP-type RNA binding domain (RBD)...

Vesicular stomatitis virus matrix protein (VSV M) has been shown to inhibit both transcription and nucleocytoplasmic transport. We have isolated a mutant form of M, termed M(D), lacking both inhibitory activities. HeLa cells expressing M, but not M(D), accumulate polyadenylated RNAs within the nucleus. Concomitantly, a fraction of M, but not of the M(D) mutant, localizes at the nuclear rim. Additionally, the nucleoporin Nup98 specifically interacts with M but not with M(D). In Nup98(-/-) cells, both the levels of M at the nuclear envelope and its inhibitory effects on host cell-directed expression of reporter genes were significantly reduced. Together, our data demonstrate that VSV M inhibits host cell gene expression by targeting a nucleoporin and primarily blocking nuclear export.

Extensive data highlight the existence of major differences in individuals' susceptibility to stress [1-4]. While genetic factors [5, 6] and exposure to early life stress [7, 8] are key components for such neurobehavioral diversity, intriguing observations revealed individual differences in response to stress in inbred mice [9-12]. This raised the possibility that other factors might be critical in stress vulnerability. A key challenge in the field is to identify non-invasively risk factors for vulnerability to stress. Here, we investigated whether behavioral factors, emerging from preexisting dominance hierarchies, could predict vulnerability to chronic stress [9, 13-16]. We applied a chronic social defeat stress (CSDS) model of depression in C57BL/6J mice to investigate the predictive power of hierarchical status to pinpoint which individuals will exhibit susceptibility to CSDS. Given that the high social status of dominant mice would be the one particularly challenged by CSDS, we predicted and found that dominant individuals were the ones showing a strong susceptibility profile as indicated by strong social avoidance following CSDS, while subordinate mice were not affected. Data from H-NMR spectroscopy revealed that the metabolic profile in the nucleus accumbens (NAc) relates to social status and vulnerability to stress. Under basal conditions, subordinates show lower levels of energy-related metabolites compared to dominants. In subordinates, but not dominants, levels of these metabolites were increased after exposure to CSDS. To the best of our knowledge, this is the first study that identifies non-invasively the origin of behavioral risk factors predictive of stress-induced depression-like behaviors associated with metabolic changes.

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The REF family of evolutionarily conserved heterogeneous ribonucleoprotein (hnRNP)-like proteins consists of one central RNPtype RNA binding domain flanked by Arg-Gly-rich regions of variable length. Members of this protein family bind directly to RNA and the mRNA export factor TAP͞Mex67p, and it has been suggested that they facilitate the recruitment of TAP͞Mex67p to cellular mRNPs. We show that the variable regions are necessary for binding of REFs to RNA and to TAP. Antibodies specific to REFs prevent their interaction with RNA in vitro. After microinjection into Xenopus oocytes, these antibodies inhibit mRNA nuclear export. This inhibition of export is observed whether or not the mRNAs are generated by splicing. The antibodies do not interfere with pre-mRNA splicing or with the nuclear export of constitutive transport element (CTE)-containing RNAs (directly mediated by TAP), so REF proteins must play a critical role in mRNA nuclear export, acting downstream of splicing and upstream of TAP͞ Mex67p. We also show that recombinant REFs stimulate directly the export of mRNAs that are otherwise exported inefficiently. Together, our data indicate that REFs are directly implicated in the export of mRNAs from the nucleus. More generally, we show that spliced and unspliced mRNAs use common export factors to reach the cytoplasm. Yra1p, a Saccharomyces cerevisiae member of the REF family, is an essential nuclear protein first identified from its RNAannealing activity (3). More recently, Yra1p was shown to be involved in the export of mRNA from the nucleus in yeast cells (2, 4). In mouse, REFs are encoded by at least three different genes (ref 1-3) and differ at multiple positions in the variable regions because of deletions and͞or amino acid changes (2). In contrast, all murine REF proteins are 98% identical in the RBD and 100% in the conserved boxes (2). The complexity of the murine subfamily is further increased by the expression of multiple splice variants (2). Murine REF1-II is generated by alternative splicing of REF1-I (also named Aly; see ref. 5) and lacks the N-terminal variable region (see Fig. 2 A), whereas murine REF2-I and REF2-II differ by one single amino acid insertion in REF2-I (Q198, ref. 2). REF1-I (Aly) was first identified as a protein interacting with LEF-1, a transcription factor that participates in the regulation of the T-cell receptor ␣ enhancer (5). In this context, it was proposed that Aly facilitates the interaction of multiple proteins in the T-cell receptor ␣ enhancer complex. In this study, we have defined the domains of REF interacting with RNA and TAP, and we have investigated the role of vertebrate REFs on the export of mRNAs from the nucleus. We show that the variable regions are required for binding of REFs to RNA and TAP. Antibodies specific to REFs, which prevent their binding to RNA in vitro, inhibit mRNA nuclear export when injected into Xenopus laevis oocytes. This export inhibition is observed whether or not the mRNAs have been generated by splicing. We show that microinjection ...

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