mRNA Export Factor Research Articles

Chemical intervention of influenza virus mRNA nuclear export.

Influenza A viruses are human pathogens with limited therapeutic options. Therefore, it is crucial to devise strategies for the identification of new classes of antiviral medications. The influenza A virus genome is constituted of 8 RNA segments. Two of these viral RNAs are transcribed into mRNAs that are alternatively spliced. The M1 mRNA encodes the M1 protein but is also alternatively spliced to yield the M2 mRNA during infection. M1 to M2 mRNA splicing occurs at nuclear speckles, and M1 and M2 mRNAs are exported to the cytoplasm for translation. M1 and M2 proteins are critical for viral trafficking, assembly, and budding. Here we show that gene knockout of the cellular protein NS1-BP, a constituent of the M mRNA speckle-export pathway and a binding partner of the virulence factor NS1 protein, inhibits M mRNA nuclear export without altering bulk cellular mRNA export, providing an avenue to preferentially target influenza virus. We performed a high-content, image-based chemical screen using single-molecule RNA-FISH to label viral M mRNAs followed by multistep quantitative approaches to assess cellular mRNA and cell toxicity. We identified inhibitors of viral mRNA biogenesis and nuclear export that exhibited no significant activity towards bulk cellular mRNA at non-cytotoxic concentrations. Among the hits is a small molecule that preferentially inhibits nuclear export of a subset of viral and cellular mRNAs without altering bulk cellular mRNA export. These findings underscore specific nuclear export requirements for viral mRNAs and phenocopy down-regulation of the mRNA export factor UAP56. This RNA export inhibitor impaired replication of diverse influenza A virus strains at non-toxic concentrations. Thus, this screening strategy yielded compounds that alone or in combination may serve as leads to new ways of treating influenza virus infection and are novel tools for studying viral RNA trafficking in the nucleus.

Molecular basis for the exploitation of nuclear mRNA export by influenza A virus

Influenza A virus replicates in the cell nucleus and has evolved at least two mechanisms to exploit the host nuclear mRNA export pathway. First, we show that a key influenza virulence factor NS1 blocks nuclear export of host mRNAs. We identify the direct target of NS1 as the mRNA export receptor complex, NXF1‐NXT1, which is the principal receptor mediating docking and translocation of mRNAs through the nuclear pore complex via interactions with nucleoporins. We determined the crystal structure of NS1 in complex with NXF1‐NXT1 at 3.8 Å resolution. The structure reveals that NS1 prevents binding of NXF1‐NXT1 to nucleoporins, thereby inhibiting mRNA export through the nuclear pore complex into the cytoplasm for translation. Second, packaging of influenza virus genome has been suggested to require the mRNA export factor UAP56 through interaction with the influenza NP protein, but the mechanism remains elusive. Our biochemical characterization of the NP‐UAP56 interaction suggests that UAP56 primes NP for engaging with viral RNA. Together, our studies uncover the molecular basis of how influenza A virus targets nuclear mRNA export to inhibit host gene expression and promote viral proliferation.Support or Funding InformationNIH R01 GM113874, R01 AI125524, and R35 GM133743

Phosphosite Analysis of the Cytomegaloviral mRNA Export Factor pUL69 Reveals Serines with Critical Importance for Recruitment of Cellular Proteins Pin1 and UAP56/URH49.

Human cytomegalovirus (HCMV) encodes the viral mRNA export factor pUL69, which facilitates the cytoplasmic accumulation of mRNA via interaction with the cellular RNA helicase UAP56 or URH49. We reported previously that pUL69 is phosphorylated by cellular CDKs and the viral CDK-like kinase pUL97. Here, we set out to identify phosphorylation sites within pUL69 and to characterize their importance. Mass spectrometry-based phosphosite mapping of pUL69 identified 10 serine/threonine residues as phosphoacceptors. Surprisingly, only a few of these sites localized to the N terminus of pUL69, which could be due to the presence of additional posttranslational modifications, like arginine methylation. As an alternative approach, pUL69 mutants with substitutions of putative phosphosites were analyzed by Phos-tag SDS-PAGE. This demonstrated that serines S46 and S49 serve as targets for phosphorylation by pUL97. Furthermore, we provide evidence that phosphorylation of these serines mediates cis/trans isomerization by the prolyl isomerase Pin1, thus forming a functional Pin1 binding motif. Surprisingly, while abrogation of the Pin1 motif did not affect the replication of recombinant cytomegaloviruses, mutation of serines next to the interaction site for UAP56/URH49 strongly decreased viral replication. This was correlated with a loss of UAP56/URH49 recruitment. Intriguingly, the critical serines S13 and S15 were located within a sequence resembling the UAP56 binding motif (UBM) of cellular mRNA adaptor proteins like REF and UIF. We propose that betaherpesviral mRNA export factors have evolved an extended UAP56/URH49 recognition sequence harboring phosphorylation sites to increase their binding affinities. This may serve as a strategy to successfully compete with cellular mRNA adaptor proteins for binding to UAP56/URH49.IMPORTANCE The multifunctional regulatory protein pUL69 of human cytomegalovirus acts as a viral RNA export factor with a critical role in efficient replication. Here, we identify serine/threonine phosphorylation sites for cellular and viral kinases within pUL69. We demonstrate that the pUL97/CDK phosphosites within alpha-helix 2 of pUL69 are crucial for its cis/trans isomerization by the cellular protein Pin1. Thus, we identified pUL69 as the first HCMV-encoded protein that is phosphorylated by cellular and viral serine/threonine kinases in order to serve as a substrate for Pin1. Furthermore, our study revealed that betaherpesviral mRNA export proteins contain extended binding motifs for the cellular mRNA adaptor proteins UAP56/URH49 harboring phosphorylated serines that are critical for efficient viral replication. Knowledge of the phosphorylation sites of pUL69 and the processes regulated by these posttranslational modifications is important in order to develop antiviral strategies based on a specific interference with pUL69 phosphorylation.

Distinct effects on mRNA export factor GANP underlie neurological disease phenotypes and alter gene expression depending on intron content.

Defects in the mRNA export scaffold protein GANP, encoded by the MCM3AP gene, cause autosomal recessive early-onset peripheral neuropathy with or without intellectual disability. We extend here the phenotypic range associated with MCM3AP variants, by describing a severely hypotonic child and a sibling pair with a progressive encephalopathic syndrome. In addition, our analysis of skin fibroblasts from affected individuals from seven unrelated families indicates that disease variants result in depletion of GANP except when they alter critical residues in the Sac3 mRNA binding domain. GANP depletion was associated with more severe phenotypes compared with the Sac3 variants. Patient fibroblasts showed transcriptome alterations that suggested intron content-dependent regulation of gene expression. For example, all differentially expressed intronless genes were downregulated, including ATXN7L3B, which couples mRNA export to transcription activation by association with the TREX-2 and SAGA complexes. Our results provide insight into the molecular basis behind genotype-phenotype correlations in MCM3AP-associated disease and suggest mechanisms by which GANP defects might alter RNA metabolism.

Cellular mRNA export factor UAP56 recognizes nucleic acid binding site of influenza virus NP protein

Influenza A virus nucleoprotein (NP) is a structural component that encapsulates the viral genome into the form of ribonucleoprotein complexes (vRNPs). Efficient assembly of vRNPs is critical for the virus life cycle. The assembly route from RNA-free NP to the NP-RNA polymer in vRNPs has been suggested to require a cellular factor UAP56, but the mechanism is poorly understood. Here, we characterized the interaction between NP and UAP56 using recombinant proteins and showed that UAP56 features two NP binding sites. In addition to the UAP56 core comprised of two RecA domains, we identified the N-terminal extension (NTE) of UAP56 as a previously unknown NP binding site. In particular, UAP56-NTE recognizes the nucleic acid binding region of NP. This corroborates our observation that binding of UAP56-NTE and RNA to NP is mutually exclusive. Collectively, our results reveal the molecular basis for how UAP56 acts on RNA-free NP, and provide new insights into NP-mediated influenza genome packaging.

Into the basket and beyond: the journey of mRNA through the nuclear pore complex

The genetic information encoded in nuclear mRNA destined to reach the cytoplasm requires the interaction of the mRNA molecule with the nuclear pore complex (NPC) for the process of mRNA export. Numerous proteins have important roles in the transport of mRNA out of the nucleus. The NPC embedded in the nuclear envelope is the port of exit for mRNA and is composed of ∼30 unique proteins, nucleoporins, forming the distinct structures of the nuclear basket, the pore channel and cytoplasmic filaments. Together, they serve as a rather stationary complex engaged in mRNA export, while a variety of soluble protein factors dynamically assemble on the mRNA and mediate the interactions of the mRNA with the NPC. mRNA export factors are recruited to and dissociate from the mRNA at the site of transcription on the gene, during the journey through the nucleoplasm and at the nuclear pore at the final stages of export. In this review, we present the current knowledge derived from biochemical, molecular, structural and imaging studies, to develop a high-resolution picture of the many events that culminate in the successful passage of the mRNA out of the nucleus.

Imaging within single NPCs reveals NXF1's role in mRNA export on the cytoplasmic side of the pore.

Translocation of mRNA through the nuclear pore complex (NPC) requires interactions with different NPC regions. To determine the interactions that are crucial for effective mRNA export in living cells, we examined mRNA export within individual pores by applying various types of mRNA export blocks that stalled mRNPs at different stages of transition. Focusing on the major mRNA export factor NXF1, we found that initial mRNP binding to the NPC did not require NXF1 in the NPC, whereas release into the cytoplasm did. NXF1 localization in the NPC did not require RNA or RNA binding. Superresolution microscopy showed that NXF1 consistently occupied positions on the cytoplasmic side of the NPC. Interactions with specific nucleoporins were pinpointed using FLIM-FRET for measuring protein-protein interactions inside single NPCs, showing that Dbp5 helicase activity of mRNA release is conserved in yeast and humans. Altogether, we find that specific interactions on the cytoplasmic side of the NPC are fundamental for the directional flow of mRNA export.

SAGA DUBm-mediated surveillance regulates prompt export of stress-inducible transcripts for proteostasis

During stress, prompt export of stress-inducible transcripts is critical for cell survival. Here, we characterize a function of the SAGA (Spt-Ada-Gcn5 acetyltransferase) deubiquitylating module (DUBm) in monitoring messenger ribonucleoprotein (mRNP) biogenesis to regulate non-canonical mRNA export of stress-inducible transcripts. Our genetic and biochemical analyses suggest that there is a functional relationship between Sgf73p of DUBm and the essential mRNA export factor, Yra1p. Under physiological conditions, Sgf73p is critical for the proper chromatin localization and RNA binding of Yra1p, while also quality controlling the biogenesis of mRNPs in conjunction with the nuclear exosome exonuclease, Rrp6p. Under environmental stress, when immediate transport of stress-inducible transcripts is imperative, Sgf73p facilitates the bypass of canonical surveillance and promotes the timely export of necessary transcripts. Overall, our results show that the Sgf73p-mediated plasticity of gene expression is important for the ability of cells to tolerate stress and regulate proteostasis to survive under environmental uncertainty.

Co-transcriptional Loading of RNA Export Factors Shapes the Human Transcriptome.

SummaryDuring gene expression, RNA export factors are mainly known for driving nucleo-cytoplasmic transport. While early studies suggested that the exon junction complex (EJC) provides a binding platform for them, subsequent work proposed that they are only recruited by the cap binding complex to the 5′ end of RNAs, as part of TREX. Using iCLIP, we show that the export receptor Nxf1 and two TREX subunits, Alyref and Chtop, are recruited to the whole mRNA co-transcriptionally via splicing but before 3′ end processing. Consequently, Alyref alters splicing decisions and Chtop regulates alternative polyadenylation. Alyref is recruited to the 5′ end of RNAs by CBC, and our data reveal subsequent binding to RNAs near EJCs. We demonstrate that eIF4A3 stimulates Alyref deposition not only on spliced RNAs close to EJC sites but also on single-exon transcripts. Our study reveals mechanistic insights into the co-transcriptional recruitment of mRNA export factors and how this shapes the human transcriptome.

Drosophila Ref1/ALYREF regulates transcription and toxicity associated with ALS/FTD disease etiologies

RNA-binding proteins (RBPs) are associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the underlying disease mechanisms remain unclear. In an unbiased screen in Drosophila for RBPs that genetically interact with TDP-43, we found that downregulation of the mRNA export factor Ref1 (fly orthologue to human ALYREF) mitigated TDP-43 induced toxicity. Further, Ref1 depletion also reduced toxicity caused by expression of the C9orf72 GGGGCC repeat expansion. Ref1 knockdown lowered the mRNA levels for these related disease genes and reduced the encoded proteins with no effect on a wild-type Tau disease transgene or a control transgene. Interestingly, expression of TDP-43 or the GGGGCC repeat expansion increased endogenous Ref1 mRNA levels in the fly brain. Further, the human orthologue ALYREF was upregulated by immunohistochemistry in ALS motor neurons, with the strongest upregulation occurring in ALS cases harboring the GGGGCC expansion in C9orf72. These data support ALYREF as a contributor to ALS/FTD and highlight its downregulation as a potential therapeutic target that may affect co-existing disease etiologies.

PCID2 influences BRCA1/BARD1 Localization and Centrosome Duplication through its functions in Nuclear Protein and mRNA Export

Nuclear protein export has an emerging role in the regulation of centrosome duplication, a function that involves interaction of centrosomal proteins with the Crm1 nuclear export factor. The protein PCID2, while originally identified as an mRNA export factor, is also known to function in Crm1 mediated protein export, with PCID2 siRNA knockdown slowing the rate of export. In addition, PCID2 has been localized to the centrosome. Despite the fact that PCID2 is poised to be a cell cycle regulator through potential combination of it's centrosomal and nuclear export roles, the function of PCID2 at these locations has not been well characterized. We sought to elucidate a potential link between these roles of by exploring PCID2's involvement in the export of particular cargo known to transit between these the nucleus and centrosome. Our studies focus on BRCA1 and BARD1 as potential PCID2 nuclear to centrosomal cargo, as these DNA damage repair proteins transit from the nucleus to the centrosome in a Crm1 dependent manner, particularly in times of cellular stress. We found that knockdown of PCID2 caused a 10% increase in the nuclear retention of BRCA1, with a concomitant 40‐fold decrease in centrosomes exhibiting BRCA1 localization. These results suggest a role for PCID2 in the export of the BRCA1 protein from the nucleus and in it's subsequent localization to the centrosome. Conversely, BARD1 nuclear levels decreased by nearly 20% in the absence of PCID2, which was accompanied by a 40‐fold decrease in BARD1 positive centrosomes. These effects appear to stem from a 50% drop in BARD1 protein levels in PCID2 knockdown cells, suggesting a potential role for PCID2 in the export of BARD1 mRNA rather than protein, with failure to export contributing to loss in protein expression. Additionally, we discovered that altered transport of BRCA1/BARD1 due to PCID2 knockdown had an unexpected effect on centrosome duplication. Rather than the increase in centrosome number seen in the absence of the BRCA1/BARD1 interacting protein OLA1, PCID2 knockdown led to a four‐fold reduction in cells with an aberrant number of centrosomes (3 or more) as visualized by yTubulin staining. This variation is potentially due to the use of non‐breast cancer cells in this work. Our ongoing studies seek to identify CRM1‐PCID2‐BRCA1 export complexes and determining the effect of PCID2 knockdown on the CRM1‐BRCA1 interaction. In addition, we are investigating the role of PCID2 in BARD1 mRNA export. Determining how PCID2 influences the cellular localization of the BRCA1/BARD1 complex will help to elucidate the mechanism of PCID2's effect on centrosome duplication and therefore determine it's potential impacts on cell cycle regulation and tumor development.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Iws1 and Spt6 Regulate Trimethylation of Histone H3 on Lysine 36 through Akt Signaling and are Essential for Mouse Embryonic Genome Activation

The mRNA processing and export factor, Iws1, interacts with the histone H3/H4 chaperone, Spt6 (Supt6 in mouse gene ontology) and recruits the lysine methyltransferase, Setd2, to chromatin to regulate H3K36me3. This recruitment is known to be crucial for pre-mRNA splicing and Iws1 has been shown to interact with REF1/Aly to mediate mRNA export. However, the role of this complex has not yet been examined in embryonic development. Here, we show that knockdown of either Iws1 or Supt6 blocked embryo development, primarily at the 8/16-cell stage, indicating that Iws1 and Supt6 are crucial for mouse preimplantation development. In the knockdown embryos, we observed decreases in pre-mRNA splicing, mRNA export and the expression of the lineage-specific transcription factor, Nanog. We found that either Iws1 or Supt6 are required for H3K36 trimethylation and that concurrent knockdown of both Iws1 and Supt6 blocks embryonic development at the 2-cell stage. We show that H3K36me3 is modulated by the Pi3k/Akt pathway, as inhibition of this pathway reduced the global level of H3K36me3 while activation of the pathway increased the level of this modification in 2-cell embryos. We observed that Iws1 interacts with nuclear Akt in early embryos, and herein propose that Akt modulates H3K36me3 through interaction with Iws1. Together, our results indicate that the Iws1 and Supt6 play crucial roles in embryonic genome activation, lineage specification, and histone modification during mouse early development.

The mRNA Export Receptor NXF1 Coordinates Transcriptional Dynamics, Alternative Polyadenylation, and mRNA Export

Alternative polyadenylation (APA) produces mRNA isoforms with different 3' UTR lengths. Previous studies indicated that 3' end processing and mRNA export are intertwined in gene regulation. Here, we show that mRNA export factors generally facilitate usage of distal cleavage and polyadenylation sites (PASs), leading to long 3' UTR isoform expression. By focusing on the export receptor NXF1, which exhibits the most potent effect on APA in this study, we reveal several gene features that impact NXF1-dependent APA, including 3' UTR size, gene size, and AT content. Surprisingly, NXF1 downregulation results in RNA polymerase II (Pol II) accumulation at the 3' end of genes, correlating with its role in APA regulation. Moreover, NXF1 cooperates with CFI-68 to facilitate nuclear export of long 3' UTR isoform with UGUA motifs. Together, our work reveals important roles of NXF1 in coordinating transcriptional dynamics, 3' end processing, and nuclear export of long 3' UTR transcripts, implicating NXF1 as a nexus of gene regulation.

The UAP56-Interacting Export Factors UIEF1 and UIEF2 Function in mRNA Export.

In eukaryotes, the regulated transport of mRNAs from the nucleus to the cytosol through nuclear pore complexes represents an important step in the expression of protein-coding genes. In plants, the mechanism of nucleocytosolic mRNA transport and the factors involved are poorly understood. The Arabidopsis (Arabidopsis thaliana) genome encodes two likely orthologs of UAP56-interacting factor, which acts as mRNA export factor in mammalian cells. In yeast and plant cells, both proteins interact directly with the mRNA export-related RNA helicase UAP56 and the interaction was mediated by an N-terminal UAP56-binding motif. Accordingly, the two proteins were termed UAP56-INTERACTING EXPORT FACTOR1 and 2 (UIEF1/2). Despite lacking a known RNA-binding motif, recombinant UIEF1 interacted with RNA, and the C-terminal part of UIEF1 mainly contributed to the RNA interaction. Mutation of UIEF1, UIEF2, or both in the double-mutant 2xuief caused modest growth defects. A cross between the 2xuief and 4xaly (defective in the four ALY1-4 mRNA export factors) mutants produced the sextuple mutant 4xaly 2xuief, which displayed more severe growth impairment than the 4xaly plants. Developmental defects including delayed bolting and reduced seed set were observed in the 4xaly but not the 2xuief plants. Analysis of the cellular distribution of polyadenylated mRNAs revealed more pronounced nuclear mRNA accumulation in 4xaly 2xuief than in 2xuief and 4xaly cells. In conclusion, the results indicate that UIEF1 and UIEF2 act as mRNA export factors in plants and that they cooperate with ALY1-ALY4 to mediate efficient nucleocytosolic mRNA transport.

It's Not the Destination, It's the Journey: Heterogeneity in mRNA Export Mechanisms.

The process of creating a translation-competent mRNA is highly complex and involves numerous steps including transcription, splicing, addition of modifications, and, finally, export to the cytoplasm. Historically, much of the research on regulation of gene expression at the level of the mRNA has been focused on either the regulation of mRNA synthesis (transcription and splicing) or metabolism (translation and degradation). However, in recent years, the advent of new experimental techniques has revealed the export of mRNA to be a major node in the regulation of gene expression, and numerous large-scale and specific mRNA export pathways have been defined. In this chapter, we will begin by outlining the mechanism by which most mRNAs are homeostatically exported ("bulk mRNA export"), involving the recruitment of the NXF1/TAP export receptor by the Aly/REF and THOC5 components of the TREX complex. We will then examine various mechanisms by which this pathway may be controlled, modified, or bypassed in order to promote the export of subset(s) of cellular mRNAs, which include the use of metazoan-specific orthologs of bulk mRNA export factors, specific cis RNA motifs which recruit mRNA export machinery via specific trans-acting-binding factors, posttranscriptional mRNA modifications that act as "inducible" export cis elements, the use of the atypical mRNA export receptor, CRM1, and the manipulation or bypass of the nuclear pore itself. Finally, we will discuss major outstanding questions in the field of mRNA export heterogeneity and outline how cutting-edge experimental techniques are providing new insights into and tools for investigating the intriguing field of mRNA export heterogeneity.

Structural–functional interactions of NS1-BP protein with the splicing and mRNA export machineries for viral and host gene expression

The influenza virulence factor NS1 protein interacts with the cellular NS1-BP protein to promote splicing and nuclear export of the viral M mRNAs. The viral M1 mRNA encodes the M1 matrix protein and is alternatively spliced into the M2 mRNA, which is translated into the M2 ion channel. These proteins have key functions in viral trafficking and budding. To uncover the NS1-BP structural and functional activities in splicing and nuclear export, we performed proteomics analysis of nuclear NS1-BP binding partners and showed its interaction with constituents of the splicing and mRNA export machineries. NS1-BP BTB domains form dimers in the crystal. Full-length NS1-BP is a dimer in solution and forms at least a dimer in cells. Mutations suggest that dimerization is important for splicing. The central BACK domain of NS1-BP interacts directly with splicing factors such as hnRNP K and PTBP1 and with the viral NS1 protein. The BACK domain is also the site for interactions with mRNA export factor Aly/REF and is required for viral M mRNA nuclear export. The crystal structure of the C-terminal Kelch domain shows that it forms a β-propeller fold, which is required for the splicing function of NS1-BP. This domain interacts with the polymerase II C-terminal domain and SART1, which are involved in recruitment of splicing factors and spliceosome assembly, respectively. NS1-BP functions are not only critical for processing a subset of viral mRNAs but also impact levels and nuclear export of a subset of cellular mRNAs encoding factors involved in metastasis and immunity.

PRP4KA, a Putative Spliceosomal Protein Kinase, Is Important for Alternative Splicing and Development in Arabidopsis thaliana.

Splicing of precursor messenger RNAs (pre-mRNAs) is an essential step in the expression of most eukaryotic genes. Both constitutive splicing and alternative splicing, which produces multiple messenger RNA (mRNA) isoforms from a single primary transcript, are modulated by reversible protein phosphorylation. Although the plant splicing machinery is known to be a target for phosphorylation, the protein kinases involved remain to be fully defined. We report here the identification of pre-mRNA processing 4 (PRP4) KINASE A (PRP4KA) in a forward genetic screen based on an alternatively spliced GFP reporter gene in Arabidopsis thaliana (Arabidopsis). Prp4 kinase is the first spliceosome-associated kinase shown to regulate splicing in fungi and mammals but it has not yet been studied in plants. In the same screen we identified mutants defective in SAC3A, a putative mRNA export factor that is highly coexpressed with PRP4KA in Arabidopsis Whereas the sac3a mutants appear normal, the prp4ka mutants display a pleiotropic phenotype featuring atypical rosettes, late flowering, tall final stature, reduced branching, and lowered seed set. Analysis of RNA-sequencing data from prp4ka and sac3a mutants identified widespread and partially overlapping perturbations in alternative splicing in the two mutants. Quantitative phosphoproteomic profiling of a prp4ka mutant detected phosphorylation changes in several serine/arginine-rich proteins, which regulate constitutive and alternative splicing, and other splicing-related factors. Tests of PRP4KB, the paralog of PRP4KA, indicated that the two genes are not functionally redundant. The results demonstrate the importance of PRP4KA for alternative splicing and plant phenotype, and suggest that PRP4KA may influence alternative splicing patterns by phosphorylating a subset of splicing regulators.

Epstein-Barr Virus-Induced Nodules on Viral Replication Compartments Contain RNA Processing Proteins and a Viral Long Noncoding RNA.

Profound alterations in host cell nuclear architecture accompany the lytic phase of Epstein-Barr virus (EBV) infection. Viral replication compartments assemble, host chromatin marginalizes to the nuclear periphery, cytoplasmic poly(A)-binding protein translocates to the nucleus, and polyadenylated mRNAs are sequestered within the nucleus. Virus-induced changes to nuclear architecture that contribute to viral host shutoff (VHS) must accommodate selective processing and export of viral mRNAs. Here we describe additional previously unrecognized nuclear alterations during EBV lytic infection in which viral and cellular factors that function in pre-mRNA processing and mRNA export are redistributed. Early during lytic infection, before formation of viral replication compartments, two cellular pre-mRNA splicing factors, SC35 and SON, were dispersed from interchromatin granule clusters, and three mRNA export factors, Y14, ALY, and NXF1, were depleted from the nucleus. During late lytic infection, virus-induced nodular structures (VINORCs) formed at the periphery of viral replication compartments. VINORCs were composed of viral (BMLF1 and BGLF5) and cellular (SC35, SON, SRp20, and NXF1) proteins that mediate pre-mRNA processing and mRNA export. BHLF1 long noncoding RNA was invariably found in VINORCs. VINORCs did not contain other nodular nuclear cellular proteins (PML or coilin), nor did they contain viral proteins (BRLF1 or BMRF1) found exclusively within replication compartments. VINORCs are novel EBV-induced nuclear structures. We propose that EBV-induced dispersal and depletion of pre-mRNA processing and mRNA export factors during early lytic infection contribute to VHS; subsequent relocalization of these pre-mRNA processing and mRNA export proteins to VINORCs and viral replication compartments facilitates selective processing and export of viral mRNAs.IMPORTANCE In order to make protein, mRNA transcribed from DNA in the nucleus must enter the cytoplasm. Nuclear export of mRNA requires correct processing of mRNAs by enzymes that function in splicing and nuclear export. During the Epstein-Barr virus (EBV) lytic cycle, nuclear export of cellular mRNAs is blocked, yet export of viral mRNAs is facilitated. Here we report the dispersal and dramatic reorganization of cellular (SC35, SON, SRp20, Y14, ALY, and NXF1) and viral (BMLF1 and BGLF5) proteins that play key roles in pre-mRNA processing and export of mRNA. These virus-induced nuclear changes culminate in formation of VINORCs, novel nodular structures composed of viral and cellular RNA splicing and export factors. VINORCs localize to the periphery of viral replication compartments, where viral mRNAs reside. These EBV-induced changes in nuclear organization may contribute to blockade of nuclear export of host mRNA, while enabling selective processing and export of viral mRNA.

Nuclear egress of TDP-43 and FUS occurs independently of Exportin-1/CRM1

TDP-43 and FUS are nuclear proteins with multiple functions in mRNA processing. They play key roles in ALS (amyotrophic lateral sclerosis) and FTD (frontotemporal dementia), where they are partially lost from the nucleus and aggregate in the cytoplasm of neurons and glial cells. Defects in nucleocytoplasmic transport contribute to this pathology, hence nuclear import of both proteins has been studied in detail. However, their nuclear export routes remain poorly characterized and it is unclear whether aberrant nuclear export contributes to TDP-43 or FUS pathology. Here we show that predicted nuclear export signals in TDP-43 and FUS are non-functional and that both proteins are exported independently of the export receptor CRM1/Exportin-1. Silencing of Exportin-5 or the mRNA export factor Aly/REF, as well as mutations that abrogate RNA-binding do not impair export of TDP-43 and FUS. However, artificially enlarging TDP-43 or FUS impairs their nuclear egress, suggesting that they could leave the nucleus by passive diffusion. Finally, we found that inhibition of transcription causes accelerated nuclear egress of TDP-43, suggesting that newly synthesized RNA retains TDP-43 in the nucleus, limiting its egress into the cytoplasm. Our findings implicate reduced nuclear retention as a possible factor contributing to mislocalization of TDP-43 in ALS/FTD.

The RNA Exosome Adaptor ZFC3H1 Functionally Competes with Nuclear Export Activity to Retain Target Transcripts

SummaryMammalian genomes are promiscuously transcribed, yielding protein-coding and non-coding products. Many transcripts are short lived due to their nuclear degradation by the ribonucleolytic RNA exosome. Here, we show that abolished nuclear exosome function causes the formation of distinct nuclear foci, containing polyadenylated (pA+) RNA secluded from nucleocytoplasmic export. We asked whether exosome co-factors could serve such nuclear retention. Co-localization studies revealed the enrichment of pA+ RNA foci with “pA-tail exosome targeting (PAXT) connection” components MTR4, ZFC3H1, and PABPN1 but no overlap with known nuclear structures such as Cajal bodies, speckles, paraspeckles, or nucleoli. Interestingly, ZFC3H1 is required for foci formation, and in its absence, selected pA+ RNAs, including coding and non-coding transcripts, are exported to the cytoplasm in a process dependent on the mRNA export factor AlyREF. Our results establish ZFC3H1 as a central nuclear pA+ RNA retention factor, counteracting nuclear export activity.