mRNA Nuclear Export Factor Research Articles

Analysis of COVID-19-Associated Proteins Identifies TANK-Binding Kinase 1 As an Immunotherapeutic Target in Multiple Myeloma

Background and Rationale. Patients with multiple myeloma (MM) are at a higher risk of viral infection due to immune deficiency, as recently highlighted by the severity of COVID-19 in MM patients. However, the mechanism(s) underlying the lack of anti-viral immune response in MM are unclear. We have previously shown that there are more plasmacytoid dendritic cells (pDCs) in the bone marrow of MM patients than in the bone marrow of healthy individuals and that these dysfunctional patient pDCs suppress T and NK cell-mediated immunity. Importantly, pDCs play a key role in both recognizing viral nucleic acid and initiating anti-viral immunity via type 1 interferon (IFN) response signaling. Therefore, we analyzed the immune-pathway proteins implicated in COVID-19-host interactions [COVID-19 protein interaction map, Gordon et al, Nature 2020; 583, 459-568] to assess whether pDC-MM interactions modulate these pathways to confer immune suppression and susceptibility to COVID-19. Results. We first assessed whether the expression of human genes involved in the COVID-19-host interaction changes when ex vivo MM cells are co-cultured with patient pDCs (compared to MM cells alone). We found that 41 of these genes were differentially expressed in MM cells when exposed to pDCs (log2Fold change: ± 3.5-fold; p < 0.00001), and this expression varied widely (median (log10): -0.13 to 4.5; adj p < 0.001). Three of these genes are linked to innate immunity and are essential for IFN production in MM cells - TBK1, IRF3 (Interferon regulatory factor 3), and RAE1 (interferon-inducible mRNA nuclear export factor). Importantly, pDCs downregulated the expression of TBK1 (Log2FC: -0.5) and RAE1 (Log2FC: -0.3) and upregulated the expression of IRF3 (Log2FC: 1.5) in MM cells after coculture (compared to MM cells alone, p < 0.0001). Concordantly, analysis of publicly available gene profiling datasets on relapsed MM patients showed low levels of TBK1 and RAE1 and higher levels of IRF3 (n = 50) (Log2FC: TBK1: -0.208; RAE1: -0.286; IRF3: 0.273; vs normal; p < 0.05). Of note, TBK1 expression was positively correlated with prognosis (n = 350, p = 0.026). We found that most of the 41 genes were also expressed in pDCs (NCBI GEO Datasets based on pDC cell line CAL1 and human peripheral blood pDCs). In unstimulated pDCs, TBK1 expression was significantly lower than IRF3 and RAE1 expression (2-3-fold, p < 0.05). Yet treating pDCs with CpG-ODN type-A, which induces IFN-α expression, increased both TBK1 (adj P = 0.004) and RAE1 (adj P = 0.043) expression, without significantly altering IRF3 expression. Finally, COVID-19 relies on the host ubiquitin-proteasome system for propagation, and we found that targeting ubiquitin receptor Rpn13 with the specific inhibitor RA190 restored TBK1 expression in MM cell lines, indicating the potential clinical use of targeting Rpn13 in restoring anti-viral immune responses in MM. Conclusion. Taken together, we show that TBK1 is downregulated in pDCs, and pDCs interacting with MM cells further decrease TBK1 expression in MM cells, thereby attenuating IFN-mediated anti-viral immune response signaling. Our findings suggest that: 1) low levels of TBK1 may confer increased susceptibility of MM patients to COVID-19 and 2) targeting TBK1 may restore the anti-viral immune response and reduce COVID-19 severity in MM.

Nucleoporin TPR promotes tRNA nuclear export and protein synthesis in lung cancer cells.

The robust proliferation of cancer cells requires vastly elevated levels of protein synthesis, which relies on a steady supply of aminoacylated tRNAs. Delivery of tRNAs to the cytoplasm is a highly regulated process, but the machinery for tRNA nuclear export is not fully elucidated. In this study, using a live cell imaging strategy that visualizes nascent transcripts from a specific tRNA gene in yeast, we identified the nuclear basket proteins Mlp1 and Mlp2, two homologs of the human TPR protein, as regulators of tRNA export. TPR expression is significantly increased in lung cancer tissues and correlated with poor prognosis. Consistently, knockdown of TPR inhibits tRNA nuclear export, protein synthesis and cell growth in lung cancer cell lines. We further show that NXF1, a well-known mRNA nuclear export factor, associates with tRNAs and mediates their transport through nuclear pores. Collectively, our findings uncover a conserved mechanism that regulates nuclear export of tRNAs, which is a limiting step in protein synthesis in eukaryotes.

Analysis of Sars-Cov-2-Associated Proteins Identify Tank-Binding Kinase-1 As an Immunotherapeutic Target in Multiple Myeloma

Background and Rationale Patients with multiple myeloma (MM) are at a higher risk of viral infection due to immune deficiency. Importantly, recent studies highlighted the severity of COVID-19 in MM. To date, however, the mechanism(s) underlying the lack of anti-viral immune response in MM are unclear. Plasmacytoid dendritic cells (pDCs) play a key role in both recognition of viral nucleic acid and initiating anti-viral activity via type 1 interferon (IFN) response signaling. We showed that interactions of dysfunctional pDCs with MM cells, T cells, and NK cells confer immune suppression in the host-MM bone marrow (BM) microenvironment (Chauhan et al, Cancer Cell 2009; 16: p309; Ray et al, Leukemia 2015; 29: p1441). Here, we analyzed the immune-pathway proteins implicated in Covid-19-host interactions [Gordon et al, Nature 2020; 583, p459] to assess whether pDC-MM interactions modulate these pathways to confer immune suppression and susceptibility to COVID-19. We identified the TLR pathway serine/threonine kinase TBK1 (TANK-binding kinase 1), involved in type I IFN induction, as a potential immunotherapeutic target in MM. Moreover, Covid-19 relies on host-Ubiquitin-proteasome-system for propagation, and we found that targeting ubiquitin receptor Rpn13 with a specific inhibitor RA190 restores TBK1 expression in MM.Methods For our studies, we examined SARS-CoV-2-human protein-protein interactions (PPIs) maps (Gordon et al, 2020). pDC-MM co-cultures, and RNAseq using next- generation sequencing (NGS): Purified MM patient pDCs were cocultured with autologous MM cells (1pDC/5MM) for 48h, followed by separation of MM cells from pDCs using FACS. Total RNA from MM cells was subjected to RNAseq analysis using Illumina NGS. Raw sequence data were analyzed to generate differential expression (DEseq2). Linear model (Limma) and its GUI (Glimma) were also used. Statistical significance: log2FC (fold change) values in coculture vs control, with a False Discovery Rate value of <0.05, was considered significant (CI > 95). The heatmap analysis was done using Morpheus software (Broad Institute, MIT). MM patient and pDCs data used for bioinformatics were from the NCBI GEO.Results A total of 41 genes involved in the Covid-19 host-pathogen immune interactions are also differentially expressed in MM after coculture with pDCs. (log2Fold change: ± 3.5-fold; p < 0.00001; pDC-MM vs MM alone). The gene expression widely varies in pDC-MM vs MM [Median (log10): -0.13 to 4.5; adj p < 0.001]. We identified 3 specific innate immunity-linked genes TBK1, IRF3 (Interferon regulatory factor 3), and RAE1 (interferon-inducible mRNA nuclear export factor) which are essential for IFN production in MM. Importantly, pDCs decrease TBK1 (Log2FC: -0.5) and RAE1 (Log2FC: -0.3) as well as induce IRF3 (Log2FC: 1.5) in MM (p < 0.0001). Analysis of publicly available gene profiling datasets on relapsed MM patient showed low levels of TBK1 and RAE1 and higher IRF3 (n = 50) [Log2FC: TBK1: -0.208; RAE1: -0.286; IRF3: 0.273; vs normal; p < 0.05). Of note, low TBK1 expression correlates with poor survival in MM patients (n = 350) and elevated TBK1 correlates with a better prognosis (p = 0.026). Similar analysis showed that most of 41 genes involved in the Covid-19 host-pathogen immune interactions are expressed in pDCs. In unstimulated pDCs, TBK1 expression is significantly lower than IRF3 and RAE1 (2-3-fold, p < 0.05). In functional studies, treatment of pDCs with IFN-α activating CpG-ODN type-A increases both TBK1 (adj P = 0.004) and RAE1 (adj P = 0.043), without significantly altering IRF3 expression. Taken together, we show that TBK1 is downregulated in pDCs, and pDCs-MM interactions further decreases TBK1 in MM, thereby attenuating IFN-mediated anti-viral immune response signaling in MM. Finally, we found that blockade of proteasome-mediated protein degradation via inhibition of Ubiquitin receptor Rpn13 upregulates TBK-1 expression, indicating potential clinical use of targeting Rpn13 in restoring anti-viral immune responses in MM.Conclusion We found that pDC-MM interactions downregulate TBK1, which in turn reduces IFN response signaling essential to generate an effective anti-viral immune response in the MM BM microenvironment. Our findings suggest that: 1) low levels of TBK1 may confer increased susceptibility of MM patients to COVID-19; and 2) targeting TBK1 may restore anti-viral immune response and reduce COVID-19 severity in MM.DisclosuresChauhan:consultant to Stemline Therapeutics, Inc., and Equity owner in C4 Therapeutics.: Consultancy, Other: Equity owner in C4 Therapeutics.; Oncopeptide AB: Consultancy. Anderson:Janssen: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees.

Expanding Clinical Presentations Due to Variations in THOC2 mRNA Nuclear Export Factor.

Multiple TREX mRNA export complex subunits (e.g., THOC1, THOC2, THOC5, THOC6, THOC7) have now been implicated in neurodevelopmental disorders (NDDs), neurodegeneration and cancer. We previously implicated missense and splicing-defective THOC2 variants in NDDs and a broad range of other clinical features. Here we report 10 individuals from nine families with rare missense THOC2 variants including the first case of a recurrent variant (p.Arg77Cys), and an additional individual with an intragenic THOC2 microdeletion (Del-Ex37-38). Ex vivo missense variant testing and patient-derived cell line data from current and published studies show 9 of the 14 missense THOC2 variants result in reduced protein stability. The splicing-defective and deletion variants result in a loss of small regions of the C-terminal THOC2 RNA binding domain (RBD). Interestingly, reduced stability of THOC2 variant proteins has a flow-on effect on the stability of the multi-protein TREX complex; specifically on the other NDD-associated THOC subunits. Our current, expanded cohort refines the core phenotype of THOC2 NDDs to language disorder and/or ID, with a variable severity, and disorders of growth. A subset of affected individuals’ has severe-profound ID, persistent hypotonia and respiratory abnormalities. Further investigations to elucidate the pathophysiological basis for this severe phenotype are warranted.

The Sac3 TPR-like region in the Saccharomyces cerevisiae TREX-2 complex is more extensive but independent of the CID region.

Transcription-export complex 2 (TREX-2 complex) facilitates the localization of actively transcribing genes to the nuclear periphery and also functions to contribute to the generation of export-competent mRNPs through interactions with the general mRNA nuclear export factor Mex67:Mtr2. The TREX-2 complex is based on a Sac3 scaffold to which Thp1, Sem1, Cdc31, and Sus1 bind. TREX-2 can be subdivided into two modules: one, in which Thp1 and Sem1 bind to the Sac3M region (residues ∼100–551), and the other in which Cdc31 and two Sus1 chains bind to the Sac3CID region (residues ∼710–805). Complementary structural analyses using X-ray crystallography, electron microscopy, and small-angle X-ray scattering of the Saccharomyces cerevisiae TREX-2 complex, expressed using Baculovirus-infected Sf9 cells, have indicated that the TPR-like repeats of the Sac3M region extend considerably further towards the N-terminus than previously thought, and also indicate that this region and Sac3CID:Sus1:Cdc31 region of the S. cerevisiae complex are structurally independent. Although the density visible accounted for only ∼100 kDa, a 5.3 Å resolution cryo-EM reconstruction was obtained of the M-region of TREX-2 that showed an additional three putative α-helices extending towards the Sac3 N-terminus and these helices were also seen in a 4.9 Å resolution structure obtained by X-ray crystallography.Summary statementWe describe the expression, purification and structural characterization of the S. cerevisiae TREX-2 complex and demonstrate that the Sac3 TPR-like repeats are more extensive than previously thought and that the M- and CID-regions do not appear to have a defined spatial orientation.

Structural Characterization of the Chaetomium thermophilum TREX-2 Complex and its Interaction with the mRNA Nuclear Export Factor Mex67:Mtr2

SummaryThe TREX-2 complex integrates mRNA nuclear export into the gene expression pathway and is based on a Sac3 scaffold to which Thp1, Sem1, Sus1, and Cdc31 bind. TREX-2 also binds the mRNA nuclear export factor, Mex67:Mtr2, through the Sac3 N-terminal region (Sac3N). Here, we characterize Chaetomium thermophilum TREX-2, show that the in vitro reconstituted complex has an annular structure, and define the structural basis for interactions between Sac3, Sus1, Cdc31, and Mex67:Mtr2. Crystal structures show that the binding of C. thermophilum Sac3N to the Mex67 NTF2-like domain (Mex67NTF2L) is mediated primarily through phenylalanine residues present in a series of repeating sequence motifs that resemble those seen in many nucleoporins, and Mlp1 also binds Mex67:Mtr2 using a similar motif. Deletion of Sac3N generated growth and mRNA export defects in Saccharomyces cerevisiae, and we propose TREX-2 and Mlp1 function to facilitate export by concentrating mature messenger ribonucleoparticles at the nuclear pore entrance.

The principal mRNA nuclear export factor NXF1:NXT1 forms a symmetric binding platform that facilitates export of retroviral CTE-RNA.

The NXF1:NXT1 complex (also known as TAP:p15) is a general mRNA nuclear export factor that is conserved from yeast to humans. NXF1 is a modular protein constructed from four domains (RRM, LRR, NTF2-like and UBA domains). It is currently unclear how NXF1:NXT1 binds transcripts and whether there is higher organization of the NXF1 domains. We report here the 3.4 Å resolution crystal structure of the first three domains of human NXF1 together with NXT1 that has two copies of the complex in the asymmetric unit arranged to form an intimate domain-swapped dimer. In this dimer, the linkers between the NXF1 LRR and NTF2-like domains interact with NXT1, generating a 2-fold symmetric platform in which the RNA-binding RRM, LRR and NTF2-like domains are arranged on one face. In addition to bulk transcripts, NXF1:NXT1 also facilitates the export of unspliced retroviral genomic RNA from simple type-D retroviruses such as SRV-1 that contain a constitutive transport element (CTE), a cis-acting 2-fold symmetric RNA stem–loop motif. Complementary structural, biochemical and cellular techniques indicated that the formation of a symmetric RNA binding platform generated by dimerization of NXF1:NXT1 facilitates the recognition of CTE-RNA and promotes its nuclear export.

Genome-wide in silico screen for CCCH-type zinc finger proteins of Trypanosoma brucei, Trypanosoma cruzi and Leishmania major

BackgroundCCCH type zinc finger proteins are RNA binding proteins with regulatory functions at all stages of mRNA metabolism. The best-characterized member, tritetraproline (TTP), binds to AU rich elements in 3' UTRs of unstable mRNAs, mediating their degradation. In kinetoplastids, CCCH type zinc finger proteins have been identified as being involved in the regulation of the life cycle and possibly the cell cycle. To date, no systematic listing of CCCH proteins in kinetoplastids is available.ResultsWe have identified the complete set of CCCH type zinc finger proteins in the available genomes of the kinetoplastid protozoa Trypanosoma brucei, Trypanosoma cruzi and Leishmania major. One fifths (20%) of all CCCH motifs fall into non-conventional classes and many had not been previously identified. One third of all CCCH proteins have more than one CCCH motif, suggesting multivalent RNA binding. One third have additional recognizable domains. The vast majority are unique to Kinetoplastida or to a subgroup within. Two exceptions are of interest: the putative orthologue of the mRNA nuclear export factor Mex67 and a 3'-5' exoribonuclease restricted to Leishmania species. CCCH motifs are absent from these proteins in other organisms and might be unique, novel features of the Kinetoplastida homologues. Of the others, several have a predicted, and in one case experimentally confirmed, connection to the ubiquitination pathways, for instance a HECT-type E3 ubiquitin ligase. The total number of kinetoplastid CCCH proteins is similar to the number in higher eukaryotes but lower than in yeast. A comparison of the genomic loci between the Trypanosomatidae homologues provides insight into both the evolution of the CCCH proteins as well as the CCCH motifs.ConclusionThis study provides the first systematic listing of the Kinetoplastida CCCH proteins. The number of CCCH proteins with more then one CCCH motif is larger than previously estimated, due to the identification of non-conventional CCCH motifs. Experimental approaches are now necessary to examine the functions of the many unique CCCH proteins as well as the function of the putative Mex67 and the Leishmania 3'-5' exoribonuclease.

Structural Requirements for the Ubiquitin-associated Domain of the mRNA Export Factor Mex67 to Bind Its Specific Targets, the Transcription Elongation THO Complex Component Hpr1 and Nucleoporin FXFG Repeats

The ubiquitin-associated (UBA) domain of the principal Saccharomyces cerevisiae mRNA nuclear export factor, Mex67, can bind both nuclear pore protein (nucleoporin) FG repeats and Hpr1, a component of the TREX.THO complex that functions to link transcription and export. Using fluorescence resonance energy transfer-based assays, we show here that Hpr1 and the FG repeats interact with overlapping binding sites on the Mex67 UBA domain. We present the solution structure of the Mex67 UBA domain (UBA-Mex67) complexed with a FXFG nucleoporin peptide and define residues engaged in the interaction and those involved in the FXFG-induced conformational change. We show by NMR titration that the binding of Hpr1 produces analogous changes in chemical shifts in similar regions of the UBA domain. Together the data presented here indicate that both Hpr1 and FXFG nucleoporins may bind in a similar way to the UBA-Mex67 domain. However, whereas binding of Hpr1 allows UBA-Mex67 to interact with tetra-ubiquitin, the complex between UBA-Mex67 and FXFG is unable to bind mono- or tetra-ubiquitin, suggesting that both substrate binding and also the nature of the substrate may influence the affinity of the UBA-Mex67 domain for ubiquitin.

Fragile X mental retardation protein FMRP and the RNA export factor NXF2 associate with and destabilize Nxf1 mRNA in neuronal cells.

Fragile X syndrome is caused by the inactivation of the X-linked FMR1 gene, leading to the loss of its encoded protein FMRP. Although macroorchidism and defects in neuronal architecture and function have been associated with lack of FMRP, the exact molecular mechanism underlying this disease remains unclear. We have reported previously that in the brain and testis of mice, FMRP specifically interacts with a distinct mRNA nuclear export factor NXF2 but not with its close relative NXF1, a ubiquitously expressed essential mRNA nuclear export factor. This interaction marked NXF2 as a putative functional partner of FMRP. Here, we demonstrate by immunoprecipitation and quantitative real-time RT-PCR that, in cultured mouse neuronal cells, both FMRP and NXF2 are present in Nxf1 mRNA-containing ribonucleoprotein particles. Further, we show that expression of NXF2 leads to the destabilization of Nxf1 mRNA and that this effect is abolished when Fmr1 expression is reduced by siRNA, arguing that both proteins collaborate to exert this effect. Importantly, these findings correlate well with our observations that in both mouse hippocampal neurons and male germ cells where the expression of FMRP and NXF2 is most prominent, the expression of NXF1 is relatively poorly expressed. Our studies thus identify Nxf1 mRNA as a likely biologically relevant in vivo target of both FMRP and NXF2 and implicate FMRP, in conjunction with NXF2, as a posttranscriptional regulator of a major mRNA export factor. Such regulation may prove important in the normal development and function of neurons as well as of male germ cells.

Fragile X mental retardation protein controls trailer hitch expression and cleavage furrow formation in Drosophila embryos

During the cleavage stage of animal embryogenesis, cell numbers increase dramatically without growth, and a shift from maternal to zygotic genetic control occurs called the midblastula transition. Although these processes are fundamental to animal development, the molecular mechanisms controlling them are poorly understood. Here, we demonstrate that Drosophila fragile X mental retardation protein (dFMRP) is required for cleavage furrow formation and functions within dynamic cytoplasmic ribonucleoprotein (RNP) bodies during the midblastula transition. dFMRP is observed to colocalize with the cytoplasmic RNP body components Maternal expression at 31B (ME31B) and Trailer Hitch (TRAL) in a punctate pattern throughout the cytoplasm of cleavage-stage embryos. Complementary biochemistry demonstrates that dFMRP does not associate with polyribosomes, consistent with their reported exclusion from many cytoplasmic RNP bodies. By using a conditional mutation in small bristles (sbr), which encodes an mRNA nuclear export factor, to disrupt the normal cytoplasmic accumulation of zygotic transcripts at the midblastula transition, we observe the formation of giant dFMRP/TRAL-associated structures, suggesting that dFMRP and TRAL dynamically regulate RNA metabolism at the midblastula transition. Furthermore, we show that dFMRP associates with endogenous tral mRNA and is required for normal TRAL protein expression and localization, revealing it as a previously undescribed target of dFMRP control. We also show genetically that tral itself is required for cleavage furrow formation. Together, these data suggest that in cleavage-stage Drosophila embryos, dFMRP affects protein expression by controlling the availability and/or competency of specific transcripts to be translated.

The fragile X mental retardation protein interacts with a distinct mRNA nuclear export factor NXF2

Loss of fragile X mental retardation protein, FMRP, causes the fragile X syndrome. Highly expressed in the brain and testis, FMRP has been implicated in the transport and translation of specific mRNAs. Here we show that FMRP and the mRNA nuclear export factor NXF2 co-express in the mouse male germ cells and hippocampal neurons and that FMRP associates with NXF2 but not with its close relative NXF1. We thus hypothesize that FMRP and NXF2 may act in concert to promote the nucleocytoplasmic transport of specific mRNAs in male germ cells and neurons.

The mRNA Nuclear Export Factor Hpr1 Is Regulated by Rsp5-mediated Ubiquitylation

Ubiquitin conjugation and in particular two distinct HECT ubiquitin ligases, Rsp5p and Tom1p, have been shown to participate in the regulation of mRNA export in Saccharomyces cerevisiae. The identification of the ubiquitin ligase substrates represents a major challenge in understanding how this modification may modulate mRNA export. Here, we identified Hpr1p, a member of the THO/TREX (transcription/export) complex that couples mRNA transcription to nuclear export as a target of the ubiquitin-proteasome pathway. Hpr1p degradation is enhanced at high temperature and appears linked to on-going RNA-polymeraseII-mediated transcription. Interestingly, the stability of the other THO complex components is not affected under these conditions indicating that Hpr1p turnover could control the formation of the THO/TREX complex and consequently mRNA export. Using in vivo and in vitro approaches we demonstrate that Rsp5p is responsible for the ubiquitylation of Hpr1p that also involves the ubiquitin-conjugating enzyme Ubc4p. Thus, Hpr1p represents the first nuclear export factor regulated by ubiquitylation, strongly suggesting that this post-translational modification participates in the coordination of transcription and mRNA export processes.

Structural Basis for the Recognition of a Nucleoporin FG Repeat by the NTF2-like Domain of the TAP/p15 mRNA Nuclear Export Factor

TAP-p15 heterodimers have been implicated in the export of mRNAs through nuclear pore complexes (NPCs). We report a structural analysis of the interaction domains of TAP and p15 in a ternary complex with a Phe-Gly (FG) repeat of an NPC component. The TAP-p15 heterodimer is structurally similar to the homodimeric transport factor NTF2, but unlike NTF2, it is incompatible with either homodimerization or Ran binding. The NTF2-like heterodimer functions as a single structural unit in recognizing an FG repeat at a hydrophobic pocket present only on TAP and not on p15. This FG binding site interacts synergistically with a second site at the C terminus of TAP to mediate mRNA transport through the pore. In general, our findings suggest that FG repeats bind with a similar conformation to different classes of transport factors.

NXF5, a novel member of the nuclear RNA export factor family, is lost in a male patient with a syndromic form of mental retardation

Background: Although X-linked mental retardation (XLMR) affects 2%–3% of the human population, little is known about the underlying molecular mechanisms. Recent interest in this topic led to the identification of several genes for which mutations result in the disturbance of cognitive development.Results: We identified a novel gene that is interrupted by an inv(X)(p21.1;q22) in a male patient with a syndromic form of mental retardation. Molecular analysis of both breakpoint regions did not reveal an interrupted gene on Xp, but identified a novel nuclear RNA export factor (NXF) gene cluster, Xcen-NXF5-NXF2-NXF4-NXF3-Xqter, in which NXF5 is split by the breakpoint, leading to its functional nullisomy. The predicted NXF5 protein shows high similarity with the central part of the presumed mRNA nuclear export factor TAP/NXF1. Functional analysis of NXF5 demonstrates binding to RNA as well as to the RNA nuclear export-associated protein p15/NXT. In contrast to TAP/NXF1, overexpression studies localized NXF5 in the form of granules in the cell body and neurites of mature hippocampal neurons, suggesting a role in mRNA transport. The two newly identified mouse nxf homologs, nxf-a and nxf-b, which also map on X, show highest mRNA levels in the brain.Conclusions: A novel member of the nuclear RNA export factor family is absent in a male patient with a syndromic form of mental retardation. Although we did not find direct evidence for the involvement of NXF5 in MR, the gene could be involved in development, possibly through a process in mRNA metabolism in neurons.