Nuclear RNA Export Factor Research Articles

Cellular NS1-BP protein interacts with the mRNA export receptor NXF1 to mediate nuclear export of influenza virus M mRNAs

Influenza A viruses have eight genomic RNAs that are transcribed in the host cell nucleus. Two of the viral mRNAs undergo alternative splicing. The M1 mRNA encodes the matrix protein 1 (M1) and is also spliced into M2 mRNA, which encodes the proton channel matrix protein 2 (M2). Our previous studies have shown that the cellular NS1-binding protein (NS1-BP) interacts with the viral non-structural protein 1 (NS1) and M1 mRNA to promote M1 to M2 splicing. Another pool of NS1 protein binds the mRNA export receptor NXF1 (nuclear RNA export factor-1), leading to nuclear retention of cellular mRNAs. Here we show a series of biochemical and cell biological findings that suggest a model for nuclear export of M1 and M2 mRNAs despite the mRNA nuclear export inhibition imposed by the viral NS1 protein. NS1-BP competes with NS1 for NXF1 binding, allowing the recruitment of NXF1 to the M mRNAs after splicing. NXF1 then binds GANP (Germinal-center Associated Nuclear Protein), a member of the TRanscription and EXport complex (TREX)-2. Although both NS1 and NS1-BP remain in complex with GANP-NXF1, they dissociate once this complex docks at the nuclear pore complex (NPC), and the M mRNAs are translocated to the cytoplasm. Since this mRNA nuclear export pathway is key for expression of M1 and M2 proteins that function in viral intracellular trafficking and budding, these viral-host interactions are critical for influenza virus replication.

SUN5, a testis-specific nuclear membrane protein, participates in recruitment and export of nuclear mRNA in spermatogenesis.

SUN5, a testis-specific gene, is associated with acephalic spermatozoa syndrome (ASS). Here, we demonstrate that Sun5 is involved in mRNA export. In Sun5-knockout mice ( Sun5 -/-), poly(A) + RNA accumulates in the nuclei of germ cells, leading to reduced sperm counts, decreased sperm motility and disrupted sperm head-to-tail junctions. Additionally, in the GC-2 germ cell line with RNA interference of Sun5, heterogeneous nuclear ribonucleoproteins (hnRNPs) and poly (A) + RNA (mainly mRNA) are retained in the nucleus. Further mechanistic studies reveal that Sun5 interacts with Nxf1 (nuclear RNA export factor 1) and nucleoporin 93 (Nup93). Interference with Nup93 inhibits mRNA export. Treatment with leptomycin B to block the CRM1 pathway indicates that Sun5 regulates mRNA export through an Nxf1-dependent pathway. In Sun5 -/- mice, the binding of Nxf1 and Nup93 decreases due to loss of Sun5 function, and the process of submitting Nxf1-binding mRNPs to Nup93 is inhibited, resulting in abnormal spermatogenesis. Together, these data may elucidate a novel pathway for mRNA export in male germ cells.

HnRNPA2B1 ISGylation Regulates m6A-Tagged mRNA Selective Export via ALYREF/NXF1 Complex to Foster Breast Cancer Development.

Regulating nuclear export precisely is essential for maintaining mRNA homeostasis and impacts tumor progression. However, the mechanisms governing nuclear mRNA export remain poorly elucidated. Herein, it is revealed that the enhanced hypoxic long no-ncoding RNA (lncRNA prostate cancer associated transcript 6 (PCAT6) in breast cancer (BC) promotes the nuclear export of m6A-modified mRNAs, bolstering breast cancer stem cells (BCSCs) stemness and doxorubicin resistance. Clinically, hypoxic PCAT6 correlates with malignant BC features and poor prognosis. Mechanically, PCAT6 functions as a scaffold betweeninterferon-stimulated gene 15 (ISG15) and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1), leading to ISGylation of hnRNPA2B1, thus protecting hnRNPA2B1 from ubiquitination-mediated proteasomal degradation. Interestingly, as an m6A reader, hnRNPA2B1 selectively mediates m6A-tagged mRNAs nuclear export via the Aly/REF export factor (ALYREF)/ nuclear RNA export factor 1 (NXF1) complex, which promotes stemness-related genes expression. HnRNPA2B1 knockdown or mRNA export inhibition can result in the retention of nuclear m6A-tagged mRNA associated with stemness maintenance, which suppresses BCSCs self-renewal and effectively improves the efficacy of doxorubicin therapy. These findings demonstrate the pivotal role of m6A-modified mRNA nuclear export in BC progression, highlighting that the inhibition of m6A-tagged mRNA and its nuclear export is a potential therapeutic strategy for the amelioration of cancer chemotherapy.

Identification of Autoantigen Markers for SARS CoV-2 Infection with Machine Learning-based Feature Selection: An Insight into COVID Symptoms

Background and Introduction:: Severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) infection has been shown to trigger autoimmunity, and the phenomenon leads to several chronic human diseases such as Type-1 diabetes, Crohn’s disease, vasculitis, Guillian-Barrė syndrome, etc. The mechanism underlying SARS CoV-2-induced autoimmune response is unknown and is an active area of interest for the researchers. Objective:: The primary objective of this study is to identify the autoantigen markers for the classification of SARS CoV-2 (COVID-19 positive and negative samples) that trigger an immune response leading to autoimmunity using a machine learning approach that provides information to obtain a more accurate diagnosis for COVID-induced diseases. Materials and Methods:: Our study reports the transcriptomic profile of the COVID patient's whole blood samples collected from 0 to 35th day of acute infection as described in the GSE215865 dataset. The binary classification algorithm from the sci-kit learn python library, namely logistic regression and random forest with 10-fold cross-validation, was applied to the processed data, followed by a selection of the 20 best gene features with recursive feature elimination from a set of 10,719 gene features to obtain the classification accuracy of 87%. Results:: The fidgetin, microtubule severing factor (FIGN), SH3 and cysteine-rich domain (STAC), Cadherin-6 (CDH6), docking protein 6 (DOK6), nuclear RNA export factor 3 (NXF3) and maternally expressed 3 (MEG3) are the autoantigens markers identified for classification of COVID-positive and negative samples. Conclusion:: The identified autoantigen markers from transcriptomic datasets using machine learning techniques provide a deeper understanding of COVID-induced diseases and may play an important role as potential diagnostic and drug targets for COVID-19.

Evidence for Viral mRNA Export from Ebola Virus Inclusion Bodies by the Nuclear RNA Export Factor NXF1.

Many negative-sense RNA viruses, including the highly pathogenic Ebola virus (EBOV), use cytoplasmic inclusion bodies (IBs) for viral RNA synthesis. However, it remains unclear how viral mRNAs are exported from these IBs for subsequent translation. We recently demonstrated that the nuclear RNA export factor 1 (NXF1) is involved in a late step in viral protein expression, i.e., downstream of viral mRNA transcription, and proposed it to be involved in this mRNA export process. We now provide further evidence for this function by showing that NXF1 is not required for translation of viral mRNAs, thus pinpointing its function to a step between mRNA transcription and translation. We further show that RNA binding of both NXF1 and EBOV NP is necessary for export of NXF1 from IBs, supporting a model in which NP hands viral mRNA over to NXF1 for export. Mapping of NP-NXF1 interactions allowed refinement of this model, revealing two separate interaction sites, one of them directly involving the RNA binding cleft of NP, even though these interactions are RNA-independent. Immunofluorescence analyses demonstrated that individual NXF1 domains are sufficient for its recruitment into IBs, and complementation assays helped to define NXF1 domains important for its function in the EBOV life cycle. Finally, we show that NXF1 is also required for protein expression of other viruses that replicate in cytoplasmic IBs, including Lloviu and Junín virus. These data suggest a role for NXF1 in viral mRNA export from IBs for various viruses, making it a potential target for broadly active antivirals. IMPORTANCE Filoviruses such as the Ebola virus (EBOV) cause severe hemorrhagic fevers with high case fatality rates and limited treatment options. The identification of virus-host cell interactions shared among several viruses would represent promising targets for the development of broadly active antivirals. In this study, we reveal the mechanistic details of how EBOV usurps the nuclear RNA export factor 1 (NXF1) to export viral mRNAs from viral inclusion bodies (IBs). We further show that NXF1 is not only required for the EBOV life cycle but also necessary for other viruses known to replicate in cytoplasmic IBs, including the filovirus Lloviu virus and the highly pathogenic arenavirus Junín virus. This suggests NXF1 as a promising target for the development of broadly active antivirals.

Understanding COVID-19 Pathogenesis: A Drug-Repurposing Effort to Disrupt Nsp-1 Binding to Export Machinery Receptor Complex.

Non-structural protein 1 (Nsp1) is a virulence factor found in all beta coronaviruses (b-CoVs). Recent studies have shown that Nsp1 of SARS-CoV-2 virus interacts with the nuclear export receptor complex, which includes nuclear RNA export factor 1 (NXF1) and nuclear transport factor 2-like export factor 1 (NXT1). The NXF1–NXT1 complex plays a crucial role in the transport of host messenger RNA (mRNA). Nsp1 interferes with the proper binding of NXF1 to mRNA export adaptors and its docking to the nuclear pore complex. We propose that drugs targeting the binding surface between Nsp1 and NXF1–NXT1 may be a useful strategy to restore host antiviral gene expression. Exploring this strategy forms the main goals of this paper. Crystal structures of Nsp1 and the heterodimer of NXF1–NXT1 have been determined. We modeled the docking of Nsp1 to the NXF1–NXT1 complex, and discovered repurposed drugs that may interfere with this binding. To our knowledge, this is the first attempt at drug-repurposing of this complex. We used structural analysis to screen 1993 FDA-approved drugs for docking to the NXF1–NXT1 complex. The top hit was ganirelix, with a docking score of −14.49. Ganirelix competitively antagonizes the gonadotropin releasing hormone receptor (GNRHR) on pituitary gonadotrophs, and induces rapid, reversible suppression of gonadotropin secretion. The conformations of Nsp1 and GNRHR make it unlikely that they interact with each other. Additional drug leads were inferred from the structural analysis of this complex, which are discussed in the paper. These drugs offer several options for therapeutically blocking Nsp1 binding to NFX1–NXT1, which may normalize nuclear export in COVID-19 infection.

Piwi–piRNA complexes induce stepwise changes in nuclear architecture at target loci

PIWI‐interacting RNAs (piRNAs) are germline‐specific small RNAs that form effector complexes with PIWI proteins (Piwi–piRNA complexes) and play critical roles for preserving genomic integrity by repressing transposable elements (TEs). Drosophila Piwi transcriptionally silences specific targets through heterochromatin formation and increases histone H3K9 methylation (H3K9me3) and histone H1 deposition at these loci, with nuclear RNA export factor variant Nxf2 serving as a co‐factor. Using ChEP and DamID‐seq, we now uncover a Piwi/Nxf2‐dependent target association with nuclear lamins. Hi‐C analysis of Piwi or Nxf2‐depleted cells reveals decreased intra‐TAD and increased inter‐TAD interactions in regions harboring Piwi–piRNA target TEs. Using a forced tethering system, we analyze the functional effects of Piwi–piRNA/Nxf2‐mediated recruitment of piRNA target regions to the nuclear periphery. Removal of active histone marks is followed by transcriptional silencing, chromatin conformational changes, and H3K9me3 and H1 association. Our data show that the Piwi–piRNA pathway can induce stepwise changes in nuclear architecture and chromatin state at target loci for transcriptional silencing.

Cell-free reconstitution reveals the molecular mechanisms for the initiation of secondary siRNA biogenesis in plants

Secondary small interfering RNA (siRNA) production, triggered by primary small RNA targeting, is critical for proper development and antiviral defense in many organisms. RNA-dependent RNA polymerase (RDR) is a key factor in this pathway. However, how RDR specifically converts the targets of primary small RNAs into double-stranded RNA (dsRNA) intermediates remains unclear. Here, we develop an in vitro system that allows for dissection of the molecular mechanisms underlying the production of trans-acting siRNAs, a class of plant secondary siRNAs that play roles in organ development and stress responses. We find that a combination of the dsRNA-binding protein, SUPPRESSOR OF GENE SILENCING3; the putative nuclear RNA export factor, SILENCING DEFECTIVE5, primary small RNA, and Argonaute is required for physical recruitment of RDR6 to target RNAs. dsRNA synthesis by RDR6 is greatly enhanced by the removal of the poly(A) tail, which can be achieved by the cleavage at a second small RNA-binding site bearing appropriate mismatches. Importantly, when the complementarity of the base pairing at the second target site is too strong, the small RNA-Argonaute complex remains at the cleavage site, thereby blocking the initiation of dsRNA synthesis by RDR6. Our data highlight the light and dark sides of double small RNA targeting in the secondary siRNA biogenesis.

WRN modulates translation by influencing nuclear mRNA export in HeLa cancer cells

BackgroundThe Werner syndrome protein (WRN) belongs to the RecQ family of helicases and its loss of function results in the premature aging disease Werner syndrome (WS). We previously demonstrated that an early cellular change induced by WRN depletion is a posttranscriptional decrease in the levels of enzymes involved in metabolic pathways that control macromolecular synthesis and protect from oxidative stress. This metabolic shift is tolerated by normal cells but causes mitochondria dysfunction and acute oxidative stress in rapidly growing cancer cells, thereby suppressing their proliferation.ResultsTo identify the mechanism underlying this metabolic shift, we examined global protein synthesis and mRNA nucleocytoplasmic distribution after WRN knockdown. We determined that WRN depletion in HeLa cells attenuates global protein synthesis without affecting the level of key components of the mRNA export machinery. We further observed that WRN depletion affects the nuclear export of mRNAs and demonstrated that WRN interacts with mRNA and the Nuclear RNA Export Factor 1 (NXF1).ConclusionsOur findings suggest that WRN influences the export of mRNAs from the nucleus through its interaction with the NXF1 export receptor thereby affecting cellular proteostasis. In summary, we identified a new partner and a novel function of WRN, which is especially important for the proliferation of cancer cells.

Strength in Diversity: Nuclear Export of Viral RNAs.

The nuclear export of cellular mRNAs is a complex process that requires the orchestrated participation of many proteins that are recruited during the early steps of mRNA synthesis and processing. This strategy allows the cell to guarantee the conformity of the messengers accessing the cytoplasm and the translation machinery. Most transcripts are exported by the exportin dimer Nuclear RNA export factor 1 (NXF1)–NTF2-related export protein 1 (NXT1) and the transcription–export complex 1 (TREX1). Some mRNAs that do not possess all the common messenger characteristics use either variants of the NXF1–NXT1 pathway or CRM1, a different exportin. Viruses whose mRNAs are synthesized in the nucleus (retroviruses, the vast majority of DNA viruses, and influenza viruses) exploit both these cellular export pathways. Viral mRNAs hijack the cellular export machinery via complex secondary structures recognized by cellular export factors and/or viral adapter proteins. This way, the viral transcripts succeed in escaping the host surveillance system and are efficiently exported for translation, allowing the infectious cycle to proceed. This review gives an overview of the cellular mRNA nuclear export mechanisms and presents detailed insights into the most important strategies that viruses use to export the different forms of their RNAs from the nucleus to the cytoplasm.

68P Deciphering the interplay between nuclear RNA export factors and long non-coding RNAs in breast cancer metabolism

As cancer cells strongly rely on glycolysis, targeting cancer cell glucose metabolism could be a suitable approach to find novel therapeutic targets. The role of RNA export factors and long non-coding RNAs (lncRNAs) in cancer cell metabolism have not been fully uncovered yet. Therefore, we comprehensively examined the interplay between the RNA export factor paraspeckle-associated protein 1 (PAP1) and the lncRNA NEAT1 in triple negative breast cancer cell metabolism.

Germline heterozygous mutations in Nxf1 perturb RNA metabolism and trigger thrombocytopenia and lymphopenia in mice

In eukaryotic cells, messenger RNA (mRNA) molecules are exported from the nucleus to the cytoplasm, where they are translated. The highly conserved protein nuclear RNA export factor1 (Nxf1) is an important mediator of this process. Although studies in yeast and in human cell lines have shed light on the biochemical mechanisms of Nxf1 function, its contribution to mammalian physiology is less clear. Several groups have identified recurrent NXF1 mutations in chronic lymphocytic leukemia (CLL), placing it alongside several RNA-metabolism factors (including SF3B1, XPO, RPS15) whose dysregulation is thought to contribute to CLL pathogenesis. We report here an allelic series of germline point mutations in murine Nxf1. Mice heterozygous for these loss-of-function Nxf1 mutations exhibit thrombocytopenia and lymphopenia, together with milder hematological defects. This is primarily caused by cell-intrinsic defects in the survival of platelets and peripheral lymphocytes, which are sensitized to intrinsic apoptosis. In contrast, Nxf1 mutations have almost no effect on red blood cell homeostasis. Comparative transcriptome analysis of platelets, lymphocytes, and erythrocytes from Nxf1-mutant mice shows that, in response to impaired Nxf1 function, the cytoplasmic representation of transcripts encoding regulators of RNA metabolism is altered in a unique, lineage-specific way. Thus, blood cell lineages exhibit differential requirements for Nxf1-mediated global mRNA export.

The Ebola Virus Nucleoprotein Recruits the Nuclear RNA Export Factor NXF1 into Inclusion Bodies to Facilitate Viral Protein Expression.

Ebola virus (EBOV) causes severe outbreaks of viral hemorrhagic fever in humans. While virus-host interactions are promising targets for antivirals, there is only limited knowledge regarding the interactions of EBOV with cellular host factors. Recently, we performed a genome-wide siRNA screen that identified the nuclear RNA export factor 1 (NXF1) as an important host factor for the EBOV life cycle. NXF1 is a major component of the nuclear mRNA export pathway that is usurped by many viruses whose life cycles include nuclear stages. However, the role of NXF1 in the life cycle of EBOV, a virus replicating in cytoplasmic inclusion bodies, remains unknown. In order to better understand the role of NXF1 in the EBOV life cycle, we performed a combination of co-immunoprecipitation and double immunofluorescence assays to characterize the interactions of NXF1 with viral proteins and RNAs. Additionally, using siRNA-mediated knockdown of NXF1 together with functional assays, we analyzed the role of NXF1 in individual aspects of the virus life cycle. With this approach we identified the EBOV nucleoprotein (NP) as a viral interaction partner of NXF1. Further studies revealed that NP interacts with the RNA-binding domain of NXF1 and competes with RNA for this interaction. Co-localization studies showed that RNA binding-deficient, but not wildtype NXF1, accumulates in NP-derived inclusion bodies, and knockdown experiments demonstrated that NXF1 is necessary for viral protein expression, but not for viral RNA synthesis. Finally, our results showed that NXF1 interacts with viral mRNAs, but not with viral genomic RNAs. Based on these results we suggest a model whereby NXF1 is recruited into inclusion bodies to promote the export of viral mRNA:NXF1 complexes from these sites. This would represent a novel function for NXF1 in the life cycle of cytoplasmically replicating viruses, and may provide a basis for new therapeutic approaches against EBOV, and possibly other emerging viruses.

FLT3-ITD Activates Cytoplasmic Drosha-Dependent Non-Canonical Mechanisms of Mir-155 Biogenesis in Acute Myeloid Leukemia

MiRNAs are small noncoding RNAs that control gene expression by binding to cognate sites in the 3′ untranslated region (3′ UTR) of target messenger RNAs (mRNAs). MiRNAs are transcribed as primary (pri) mRNAs and processed into precursor (pre) miRNAs by nuclear DROSHA before being exported into the cytoplasm via the Exportin 5 (XPO5)/RAN-GTP complex. Once in the cytoplasm, pre-miRNAs are further processed by Dicer into mature miRNAs. The FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is one of the most common mutations in acute myeloid leukemia (AML) and confers growth and survival advantages to leukemia blasts. Among miRNAs, miR-155 has emerged as one of the most significantly upregulated in the FLT3-ITD+ AML and has been shown to play a pivotal role in uncontrolled blast hyperproliferation and survival. Herein, we report a previously unrecognized activity of FLT3-ITD that leads to deregulation of miR-155 expression by providing the evidence for the existence of FLT3-dependent non-canonical mechanisms of miRNA biogenesis. We initially observed that FLT3-ITD blocked the biogenesis of intronic miRNAs via inhibition of the "gatekeeper" XPO5/RAN-GTP complex that allow nucleus-to-cytoplasm pre-miRNA transport. By using in vitro phosphorylation assay with [32P]-ATP labeling, we demonstrated that FLT3-ITD phosphorylates a Sprouty related EVH1 domain-containing protein 1 (SPRED1), and that phospho-SPRED1 in turn inhibited the XPO5/RAN-GTP complex thereby halting transportation of pre-miRNAs from the nucleus to cytoplasm. This resulted in a decrease of several intronic miRNAs involved in normal hematopoiesis (i.e., miR-29b, miR-181a, miR-146b, miR-126). Accordingly, knocking down of SPRED1 expression in FLT3-ITD+ AML cells resulted in increased production of mature intronic miRNAs. Since the XPO5/RAN-GTP complex is the main gatekeeper for miRNA biogenesis, these results appeared in contradiction with the miR-155 upregulation which has been invariably observed in FLT3-ITD+ AML blasts. Differently from intronic miRNAs, miR-155 is hosted at the genomic site of long non-coding RNA (lnc-RNA) and we therefore hypothesized that it follows a different path of biogenesis. In fact forced expression of FLT3-ITD in lin-Sca1+kit+ (LSK) cells decreased intronic miRNA biogenesis (i.e., miR-126) and increase production of lnc-RNA hosted miRNAs (i.e., miR-155). Accordingly, we demonstrated that upon FLT3-ITD activation, AKT phosphorylated DDX3X, a DEAD-box RNA helicase protein thereby reducing its ability to bind heterogeneous nuclear Ribonucleoprotein U (hnRNP U), a component of the hnRNP complex associated with pre-mRNA splicing. Consequently, in FLT3-ITD cells, BIC-155 splicing is decreased. In fact, we measured significant increased levels of phospho-DDX3X and unspliced BIC-155 in primary FLT3-ITD+ blasts compared with FLT3-ITD- AML blasts. In consistent, DDX3X knock-down (KO) in FLT3-ITD- cells decreased BIC-155 splicing whereas DDX3X re-expression reversed these effects. The excess of unspliced BIC-155 RNA then bound to nuclear RNA export factor 1 (NXF1), a shuttle protein that transports poly A+ RNAs from the nucleus to the cytoplasm. In consistent, NXF1 KO blocked unspliced BIC-155 nucleus-to-cytoplasm transportation. In FLT3-ITD+ blasts, once in the cytoplasm, the unspliced BIC-155 RNA was then processed by cytoplasmic isoforms of DROSHA, as demonstrated using immunostaining, RNA Immunoprecipitation (RIP), and other gain- and loss-of-function experiments. Furthermore, overexpression of cytoplasmic DROSHA but not nuclear DROSHA isoform increased the processing of unspliced BIC-155 to mature miR-155. None of the intronic miRNAs studies interacted with cytoplasmic DROSHA. The bound of BIC-155 RNA with cytoplasmic DROSHA was unique of FLT3-ITD+ blasts, as it was not observed in FLT3-ITD- blasts. Thus, our results indicate a two-fold activity of FLT3-ITD that leads to decreased levels of intronic miRNAs via XPO5/RAN-GTP blockage and upregulation of other lnc-RNA hosted miRNAs via cytoplasmic DROSHA activation. The net result is suppression of intronic miRNAs that participates to the regulation of normal hematopoiesis and upregulation of lnc-RNA-hosted miRNA, especially miR-155 that contribute to aberrant blast hyperproliferation in the FLT3-ITD AML phenotype (Figure 1). Disclosures No relevant conflicts of interest to declare.

Nuclear RNA export factor variant initiates piRNA-guided co-transcriptional silencing.

The PIWI-interacting RNA (piRNA) pathway preserves genomic integrity by repressing transposable elements (TEs) in animal germ cells. Among PIWI-clade proteins in Drosophila, Piwi transcriptionally silences its targets through interactions with cofactors, including Panoramix (Panx) and forms heterochromatin characterized by H3K9me3 and H1. Here, we identified Nxf2, a nuclear RNA export factor (NXF) variant, as a protein that forms complexes with Piwi, Panx, and p15. Panx-Nxf2-P15 complex formation is necessary in the silencing by stabilizing protein levels of Nxf2 and Panx. Notably, ectopic targeting of Nxf2 initiates co-transcriptional repression of the target reporter in a manner independent of H3K9me3 marks or H1. However, continuous silencing requires HP1a and H1. In addition, Nxf2 directly interacts with target TE transcripts in a Piwi-dependent manner. These findings suggest a model in which the Panx-Nxf2-P15 complex enforces the association of Piwi with target transcripts to trigger co-transcriptional repression, prior to heterochromatin formation in the nuclear piRNA pathway. Our results provide an unexpected connection between an NXF variant and small RNA-mediated co-transcriptional silencing.

Structural basis for influenza virus NS1 protein block of mRNA nuclear export.

Influenza viruses antagonize key immune defense mechanisms via the virulence factor NS1 protein. A major NS1 strategy is to block nuclear export of host mRNAs including those encoding immune factors1–3; however, the direct cellular target of NS1 and the mechanism of host mRNA export inhibition are not known. Here, we identify the target of NS1 as the mRNA export receptor NXF1•NXT1, which is the principal receptor to mediate docking and translocation of mRNAs through the nuclear pore complex by interacting with nucleoporins4,5. 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. We demonstrate that a mutant influenza virus deficient in binding NXF1•NXT1 does not block host mRNA export and is attenuated. This attenuation is marked by the release of mRNAs encoding immune factors from the nucleus. Together, our study uncovers the molecular basis of a major nuclear function of influenza NS1 protein that causes potent blockage of host gene expression and contributes to inhibition of host immunity.

Anti-cancer effect of dung beetle glycosaminoglycans on melanoma

BackgroundDung beetle glycosaminoglycan is known to possess anti-aging activities. However, its anti-cancer mechanisms are not fully elucidated yet. The objective of this study was to evaluate the anti-cancer effect of insect-derived polymer dung beetle glycosaminoglycan (GAG) after intraperitoneally injecting it to melanoma mice induced by B16F10 cells.MethodsTo determine molecular mechanism involved in the anti-cancer effect of dung beetle GAG, its origin N-glycan under 3KD Dalton was assayed for melanoma cell cytotoxicity. Quantitative comparisons of adhesive molecule on extracellular matrix and activities of tissue inhibitor of metalloprotease 2 (TIMP-2) were also investigated. In vivo anti-cancer effect of dung beetle GAG on solid tumor size, survival time and gene-expression profiles was also assayed using B10F10 melanoma mice model. Mice with induced melanoma were then treated with Catharsius molossus (dung beetle) GAG (CaG) at 5 mg/kg for 8 weeks to investigate its anti-cancer effects compared to bumblebee (Bombus ignitus) queen glycosaminoglycan (IQG) and Huechys sanguinea glycosaminoglycan (HEG).ResultsThese N-glycans derived from these GAG were composed of many linear heparinoid polysaccharides, polymers with hexose and N-acetylhexose. Adminstration with these GAGs increased survival time and decreased melanoma sizes in mice, in accordance with their inhibitory effects on cell growth ratio of melanoma B16F10. In addition, treatment with N-glycans derived from theses glycosaminoglycan increased activities of TIMP-2 in HMVEC cells pretreated with TNF-alpha and in melanoma cells, suggesting that they had anti-inflammatory and anticancer activities. In DNA microarray results, compared to control, CaG treated mouse group showed upregulation of 192 genes including collagen,typeI,alpha1 (Col1a1), consistent with the highly increased in vitro extracellular matrix (ECM) adhesion on collagen 1 and up-regulation of heparanase (Hpse). After treatment with CaG, a total of 152 genes were down-regulated, including nuclear RNA export factor (Nxf3) and hyaluronan proteoglycan link protein1 (Hapln1).ConclusionsGlycosaminoglycan, CaG can strengthen ECM by increasing activity of TIMP-2 and adhesion activity on collagen known to inhibit changes of ECM, leading to tumor cell invasion and progression.

Integrated analysis of microarray data to identify the genes critical for the rupture of intracranial aneurysm.

Intracranial aneurysm (IA) is a localized dilation of the blood vessel. The present study was designed to explore the mechanisms of rupture of IA. GSE13353 (including 11 ruptured and 8 unruptured IA samples) and GSE15629 (including 8 ruptured and 6 unruptured IA samples) were downloaded from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) identified using limma and MetaDE packages were merged, and a protein-protein interaction (PPI) network analysis was performed using Cytoscape software. Pathway enrichment analysis was performed for the nodes of the PPI network using the fisher algorithm. The 100 most prominent genes in the network were designated candidate genes and a hierarchical clustering analysis was performed. The tune.svm function of e1071 package was used to construct a support vector machine (SVM) classifier, and the Candidate Cancer Gene Database was applied to analyze the characterization of gene-associated cancer. Furthermore, the genes involved in the SVM classifier were assessed via principal component analysis (PCA). In the ruptured samples, 1,292 DEGs and 1,029 DEGs separately were identified by limma and MetaDE packages. The 100 most prominent genes in the network included fibronectin 1 (FN1), amyloid β (A4) precursor protein (APP), nuclear RNA export factor 1 (NXF1) and signal transducer and activator of transcription 3 (STAT3). Pathway enrichment analysis identified that toll-like receptor 3 (TLR3) was enriched in the Toll-like receptor signaling pathway. A total of 15 genes (including FN1) were used to construct the SVM classifier. NXF1 was identified to be associated with Nervous System Cancer. PCA revealed that APP, NXF1 and STAT3 were the 3 principal components. TLR3, FN1, APP, NXF1 and STAT3 may affect the rupture of IA.

Feature genes in metastatic breast cancer identified by MetaDE and SVM classifier methods.

The aim of the present study was to investigate the feature genes in metastatic breast cancer samples. A total of 5 expression profiles of metastatic breast cancer samples were downloaded from the Gene Expression Omnibus database, which were then analyzed using the MetaQC and MetaDE packages in R language. The feature genes between metastasis and non-metastasis samples were screened under the threshold of P<0.05. Based on the protein-protein interactions (PPIs) in the Biological General Repository for Interaction Datasets, Human Protein Reference Database and Biomolecular Interaction Network Database, the PPI network of the feature genes was constructed. The feature genes identified by topological characteristics were then used for support vector machine (SVM) classifier training and verification. The accuracy of the SVM classifier was then evaluated using another independent dataset from The Cancer Genome Atlas database. Finally, function and pathway enrichment analyses for genes in the SVM classifier were performed. A total of 541 feature genes were identified between metastatic and non-metastatic samples. The top 10 genes with the highest betweenness centrality values in the PPI network of feature genes were Nuclear RNA Export Factor 1, cyclin-dependent kinase 2 (CDK2), myelocytomatosis proto-oncogene protein (MYC), Cullin 5, SHC Adaptor Protein 1, Clathrin heavy chain, Nucleolin, WD repeat domain 1, proteasome 26S subunit non-ATPase 2 and telomeric repeat binding factor 2. The cyclin-dependent kinase inhibitor 1A (CDKN1A), E2F transcription factor 1 (E2F1), and MYC interacted with CDK2. The SVM classifier constructed by the top 30 feature genes was able to distinguish metastatic samples from non-metastatic samples [correct rate, specificity, positive predictive value and negative predictive value >0.89; sensitivity >0.84; area under the receiver operating characteristic curve (AUROC) >0.96]. The verification of the SVM classifier in an independent dataset (35 metastatic samples and 143 non-metastatic samples) revealed an accuracy of 94.38% and AUROC of 0.958. Cell cycle associated functions and pathways were the most significant terms of the 30 feature genes. A SVM classifier was constructed to assess the possibility of breast cancer metastasis, which presented high accuracy in several independent datasets. CDK2, CDKN1A, E2F1 and MYC were indicated as the potential feature genes in metastatic breast cancer.

Anti‐cancer effect and gene expression profiling of melanoma induced mice treated with dung beetle glycosaminoglycan

Dung beetle (Catharsius molossus) glycosaminoglycan (CaG) possessed anti‐cancer activities of elongation in survival time showing reduced melanoma tumor sizes in treated CaG melanoma induced mice. Thus microarray analyses were performed with a rat 28K cDNA clone set array to identify the gene‐expression profiles for C57BL/6N mice treated with the CaG, 5 mg/kg, over a month for investigation of anti‐cancer effect compared with queen of Bombus ignitus glycosaminoglycan (IQG, 5 mg/kg). CaG5 or IQG5 produced meaningful anti‐cancer effect showing inhibition of melanoma cell growth in XTT assay. In addition, treated theses glycosaminoglycan increased tissue inhibitor of metalloprotease 2 (TIMP‐2) activities in HMVEC cells pretreated by TNF‐alpha or melanoma cells. CaG5 treated rat group, compared to control, showed that 192 genes including collagen, type XII, alpha1 (Col12a1), and hemoglobin, beta adult minor chain (Hbb‐bt), amino levulinic acid synthase2, erythroid (Alas2) and heparanase (Hpse) were up‐regulated and 152 genes including nuclear RNA export factor (Nxf3), hyaluronan and proteoglycan link protein1 (Hapln1), G protein‐coupled receptor17 (Gpr17) and myelin protein zero (Mpz) were down‐regulated. Data suggest collagen, type XII, alpha1, was up‐regulated, and nuclear RNA export factor (Nxf3) were down‐regulated by CaG5 treatment indicating to be potential therapeutic markers for anti‐cancer agentSupport or Funding InformationThis work was supported by the basic research of National Academy of Agricultural Science, Rural Development Administration, PJ011853.