Nuclear Export Activity Research Articles

The eukaryotic translation initiation factor eIF4E reprograms alternative splicing

Aberrant splicing is typically attributed to splice‐factor (SF) mutation and contributes to malignancies including acute myeloid leukemia (AML). Here, we discovered a mutation‐independent means to extensively reprogram alternative splicing (AS). We showed that the dysregulated expression of eukaryotic translation initiation factor eIF4E elevated selective splice‐factor production, thereby impacting multiple spliceosome complexes, including factors mutated in AML such as SF3B1 and U2AF1. These changes generated a splicing landscape that predominantly supported altered splice‐site selection for ~800 transcripts in cell lines and ~4,600 transcripts in specimens from high‐eIF4E AML patients otherwise harboring no known SF mutations. Nuclear RNA immunoprecipitations, export assays, polysome analyses, and mutational studies together revealed that eIF4E primarily increased SF production via its nuclear RNA export activity. By contrast, eIF4E dysregulation did not induce known SF mutations or alter spliceosome number. eIF4E interacted with the spliceosome and some pre‐mRNAs, suggesting its direct involvement in specific splicing events. eIF4E induced simultaneous effects on numerous SF proteins, resulting in a much larger range of splicing alterations than in the case of mutation or dysregulation of individual SFs and providing a novel paradigm for splicing control and dysregulation.

Cytokine-induced transcriptional memory is evident in the kinetics of transcriptional bursts.

During the onset of cytokine-induced transcriptional memory, chromatin-associated proteins, modifications of DNA, and histones act in concert to control gene expression and maintain transcriptional states over several generations. This process is well-exemplified by the transient and amplified expression of pro-inflammatory GBP gene products, which have been shown to exhibit cytokine-induced transcriptional memory under the control of cohesin. However, the effects of transcriptional memory on the transcriptional kinetics of GBP genes remain largely unexplored. Here, we characterize the first-order transcriptional kinetics of GBP5 by modeling ensemble snapshots of nascent and mature RNA transcript counts acquired using fluorescence in-situ hybridization. Our model rigorously infers kinetic parameters for a multi-state model of promoter activation, transcription, and mature RNA nuclear export during cytokine induction, and illustrates the kinetic effects of cytokine-induced transcriptional memory. To perform robust parameter inference and prevent overfitting, we utilize a Sequential Monte Carlo (SMC) algorithm to compute the full posterior distribution on the first-order rate constants. Bayesian model selection reveals modifications to the GBP5 transcriptional state space in HeLa cells primed with interferon-gamma.

C‐terminal deletion‐induced condensation sequesters AID from IgH targets in immunodeficiency

In activated B cells, activation‐induced cytidine deaminase (AID) generates programmed DNA lesions required for antibody class switch recombination (CSR), which may also threaten genome integrity. AID dynamically shuttles between cytoplasm and nucleus, and the majority stays in the cytoplasm due to active nuclear export mediated by its C‐terminal peptide. In immunodeficient‐patient cells expressing mutant AID lacking its C‐terminus, a catalytically active AID‐delC protein accumulates in the nucleus but nevertheless fails to support CSR. To resolve this apparent paradox, we dissected the function of AID‐delC proteins in the CSR process and found that they cannot efficiently target antibody genes. We demonstrate that AID‐delC proteins form condensates both in vivo and in vitro, dependent on its N‐terminus and on a surface arginine‐rich patch. Co‐expression of AID‐delC and wild‐type AID leads to an unbalanced nuclear AID‐delC/AID ratio, with AID‐delC proteins able to trap wild‐type AID in condensates, resulting in a dominant‐negative phenotype that could contribute to immunodeficiency. The co‐condensation model of mutant and wild‐type proteins could be an alternative explanation for the dominant‐negative effect in genetic disorders.

Novel insights into the pathogenesis of DYT1 dystonia from induced patient-derived neurons.

Novel insights into the pathogenesis of DYT1 dystonia from induced patient-derived neurons.

The RanBP2/RanGAP1-SUMO complex gates β-arrestin2 nuclear entry to regulate the Mdm2-p53 signaling axis.

Mdm2 antagonizes the tumor suppressor p53. Targeting the Mdm2-p53 interaction represents an attractive approach for the treatment of cancers with functional p53. Investigating mechanisms underlying Mdm2-p53 regulation is therefore important. The scaffold protein β-arrestin2 (β-arr2) regulates tumor suppressor p53 by counteracting Mdm2. β-arr2 nucleocytoplasmic shuttling displaces Mdm2 from the nucleus to the cytoplasm resulting in enhanced p53 signaling. β-arr2 is constitutively exported from the nucleus, via a nuclear export signal, but mechanisms regulating its nuclear entry are not completely elucidated. β-arr2 can be SUMOylated, but no information is available on how SUMO may regulate β-arr2 nucleocytoplasmic shuttling. While we found β-arr2 SUMOylation to be dispensable for nuclear import, we identified a non-covalent interaction between SUMO and β-arr2, via a SUMO interaction motif (SIM), that is required for β-arr2 cytonuclear trafficking. This SIM promotes association of β-arr2 with the multimolecular RanBP2/RanGAP1-SUMO nucleocytoplasmic transport hub that resides on the cytoplasmic filaments of the nuclear pore complex. Depletion of RanBP2/RanGAP1-SUMO levels result in defective β-arr2 nuclear entry. Mutation of the SIM inhibits β-arr2 nuclear import, its ability to delocalize Mdm2 from the nucleus to the cytoplasm and enhanced p53 signaling in lung and breast tumor cell lines. Thus, a β-arr2 SIM nuclear entry checkpoint, coupled with active β-arr2 nuclear export, regulates its cytonuclear trafficking function to control the Mdm2-p53 signaling axis.

Structural Mimicry Drives HIV-1 Rev-Mediated HERV-K Expression

Expression of the Human Endogenous Retrovirus Type K (HERV-K), the youngest and most active HERV, has been associated with various cancers and neurodegenerative diseases. As in all retroviruses, a fraction of HERV-K transcripts is exported from the nucleus in unspliced or incompletely spliced forms to serve as templates for translation of viral proteins. In a fraction of HERV-K loci (Type 2 proviruses), nuclear export of the unspliced HERV-K mRNA appears to be mediated by a cis-acting signal on the mRNA, the RcRE, and the protein Rec—these are analogous to the RRE-Rev system in HIV-1. Interestingly, the HIV-1 Rev protein is able to mediate the nuclear export of the HERV-K RcRE, contributing to elevated HERV-K expression in HIV-infected patients. We aimed to understand the structural basis for HIV Rev-HERV-K RcRE recognition. We examined the conformation of the RcRE RNA in solution using small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM). We found that the 433-nt long RcRE can assume folded or extended conformations as observed by AFM. SAXS analysis of a truncated RcRE variant revealed an “A”-shaped topological structure similar to the one previously reported for the HIV-1 RRE. The effect of the overall topology was examined using several deletion variants. SAXS and biochemical analyses demonstrated that the “A” shape is necessary for efficient Rev-RcRE complex formation in vitro and nuclear export activity in cell culture. The findings provide insight into the mechanism of HERV-K expression and a structural explanation for HIV-1 Rev-mediated expression of HERV-K in HIV-infected patients. ImportanceExpression of the human endogenous retrovirus type K (HERV-K) has been associated with various cancers and autoimmune diseases. Nuclear export of both HIV-1 and HERV-K mRNAs is dependent on the interaction between a small viral protein (Rev in HIV-1 and Rec in HERV-K) and a region on the mRNA (RRE in HIV-1 and RcRE in HERV-K). HIV-1 Rev is able to mediate the nuclear export of RcRE-containing HERV-K mRNAs, which contributes to elevated production of HERV-K proteins in HIV-infected patients. We report the solution conformation of the RcRE RNA—the first three-dimensional topological structure for a HERV molecule—and find that the RcRE resembles the HIV-1 nuclear export signal, RRE. The finding reveals the structural basis for the increased HERV-K expression observed in HIV-infected patients. Elevated HERV expression, mediated by HIV infection or other stressors, can have various HERV-related biological consequences. The findings provide structural insight for regulation of HERV-K expression.

Features of Nuclear Export Signals of NS2 Protein of Influenza D Virus.

Emerging influenza D viruses (IDVs), the newest member in the genus Orthomyxovirus family, which can infect and transmit in multiple mammalian species as its relatives the influenza A viruses (IAVs). Additional studies of biological characteristics of IDVs are needed; here, we studied the characteristics of IDV nonstructural protein 2 (NS2), which shares the lowest homology to known influenza proteins. First, we generated reassortant viruses via reverse genetics to analyze the segment compatibility and gene interchangeability between IAVs and IDVs. Next, we investigated the locations and exact sequences of nuclear export signals (NESs) of the IDV NS2 protein. Surprisingly, three separate NES regions were found to contribute to the nuclear export of an eGFP fusion protein. Alanine scanning mutagenesis identified critical amino acid residues within each NES, and co-immunoprecipitation experiments demonstrated that their nuclear export activities depend on the CRM1-mediated pathway, particularly for the third NES (136-146aa) of IDV NS2. Interestingly, the third NES was important for the interaction of NS2 protein with CRM1. The findings in this study contribute to the understanding of IDV NS2 protein’s role during nucleocytoplasmic transport of influenza viral ribonucleoprotein complexes (vRNPs) and will also facilitate the development of novel anti-influenza drugs targeting nuclear export signals of IDV NS2 protein.

Using a Simple Cellular Assay to Map NES Motifs in Cancer-Related Proteins, Gain Insight into CRM1-Mediated NES Export, and Search for NES-Harboring Micropeptides.

The nuclear export receptor CRM1 (XPO1) recognizes and binds specific sequence motifs termed nuclear export signals (NESs) in cargo proteins. About 200 NES motifs have been identified, but over a thousand human proteins are potential CRM1 cargos, and most of their NESs remain to be identified. On the other hand, the interaction of NES peptides with the “NES-binding groove” of CRM1 was studied in detail using structural and biochemical analyses, but a better understanding of CRM1 function requires further investigation of how the results from these in vitro studies translate into actual NES export in a cellular context. Here we show that a simple cellular assay, based on a recently described reporter (SRVB/A), can be applied to identify novel potential NESs motifs, and to obtain relevant information on different aspects of CRM1-mediated NES export. Using cellular assays, we first map 19 new sequence motifs with nuclear export activity in 14 cancer-related proteins that are potential CRM1 cargos. Next, we investigate the effect of mutations in individual NES-binding groove residues, providing further insight into CRM1-mediated NES export. Finally, we extend the search for CRM1-dependent NESs to a recently uncovered, but potentially vast, set of small proteins called micropeptides. By doing so, we report the first NES-harboring human micropeptides.

Stella protein facilitates DNA demethylation by disrupting the chromatin association of the RING finger–type E3 ubiquitin ligase UHRF1

Stella is a maternal gene required for oogenesis and early embryogenesis. Stella overexpression in somatic cells causes global demethylation. As we have recently shown, Stella sequesters nuclear ubiquitin-like with PHD and RING finger domains 1 (UHRF1), a RING finger-type E3 ubiquitin ligase essential for DNA methylation mediated by DNA methyltransferase 1 and triggers global demethylation. Here, we report an overexpressed mutant Stella protein without nuclear export activity surprisingly retained its ability to cause global demethylation. By combining biochemical interaction assays, isothermal titration calorimetry, immunostaining, and live-cell imaging with fluorescence recovery after photobleaching, we found that Stella disrupts UHRF1's association with chromatin by directly binding to the plant homeodomain of UHRF1 and competing for the interaction between UHRF1 and the histone H3 tail. Consistently, overexpression of Stella mutants that do not directly interact with UHRF1 fails to cause genome-wide demethylation. In the presence of nuclear Stella, UHRF1 could not bind to chromatin and exhibited increased dynamics in the nucleus. Our results indicate that Stella employs a multilayered mechanism to achieve robust UHRF1 inhibition, which involves the dissociation from chromatin and cytoplasmic sequestration of UHRF1.

Comprehensive analysis of nuclear export of herpes simplex virus type 1 tegument proteins and their Epstein-Barr virus orthologs.

Morphogenesis of herpesviral virions is initiated in the nucleus but completed in the cytoplasm. Mature virions contain more than 25 tegument proteins many of which perform both nuclear and cytoplasmic functions suggesting they shuttle between these compartments. While nuclear import of herpesviral proteins was shown to be crucial for viral propagation, active nuclear export and its functional impact are still poorly understood. To systematically analyze nuclear export of tegument proteins present in virions of Herpes simplex virus type 1 (HSV1) and Epstein‐Barr virus (EBV), the Nuclear EXport Trapped by RAPamycin (NEX‐TRAP) was applied. Nine of the 22 investigated HSV1 tegument proteins including pUL4, pUL7, pUL11, pUL13, pUL21, pUL37d11, pUL47, pUL48 and pUS2 as well as 2 out of 6 EBV orthologs harbor nuclear export activity. A functional leucine‐rich nuclear export sequence (NES) recognized by the export factor CRM1/Xpo1 was identified in six of them. The comparison between experimental and bioinformatic data indicates that experimental validation of predicted NESs is required. Mutational analysis of the pUL48/VP16 NES revealed its importance for herpesviral propagation. Together our data suggest that nuclear export is an important feature of the herpesviral life cycle required to co‐ordinate nuclear and cytoplasmic processes.

The catalytic subunit of DNA polymerase δ is a nucleocytoplasmic shuttling protein

The DNA polymerase δ catalytic subunit (PolD1) is a highly conserved protein with established functions in both the nucleus and the cytoplasm: whereas PolD1 participates in the replication and repair of nuclear DNA, it plays a role in the control of cytoplasmic microtubule growth by directly acting on microtubule-nucleator γ-tubulin ring complexes. Here, we show that PolD1 shuttles between the nucleus and the cytoplasm. PolD1 harbors two nuclear localization signals that mediate the active transport of PolD1 to the nucleus; conversely, PolD1 is exported from the nucleus by the exportin CRM1-dependent mechanism, a major nuclear-export pathway that mediates the export of various cargos. These findings suggest that the nucleocytoplasmic distribution of PolD1 is influenced by both the nuclear import and export activities of the protein.

WDR20 regulates shuttling of the USP12 deubiquitinase complex between the plasma membrane, cytoplasm and nucleus

The human deubiquitinases USP12 and USP46 are very closely related paralogs with critical functions as tumor suppressors. The catalytic activity of these enzymes is regulated by two cofactors: UAF1 and WDR20. USP12 and USP46 show nearly 90% amino acid sequence identity and share some cellular activities, but have also evolved non-overlapping functions. We hypothesized that, correlating with their functional divergence, the subcellular localization of USP12 and USP46 might be differentially regulated by their cofactors. We used confocal and live microscopy analyses of epitope-tagged proteins to determine the effect of UAF1 and WDR20 on the localization of USP12 and USP46. We found that WDR20 differently modulated the localization of the DUBs, promoting recruitment of USP12, but not USP46, to the plasma membrane. Using site-directed mutagenesis, we generated a large set of USP12 and WDR20 mutants to characterize in detail the mechanisms and sequence determinants that modulate the subcellular localization of the USP12/UAF1/WDR20 complex. Our data suggest that the USP12/UAF1/WDR20 complex dynamically shuttles between the plasma membrane, cytoplasm and nucleus. This shuttling involved active nuclear export mediated by the CRM1 pathway, and required a short N-terminal motif (1MEIL4) in USP12, as well as a novel nuclear export sequence (450MDGAIASGVSKFATLSLHD468) in WDR20. In conclusion, USP12 and USP46 have evolved divergently in terms of cofactor binding-regulated subcellular localization. WDR20 plays a crucial role in as a “targeting subunit” that modulates CRM1-dependent shuttling of the USP12/UAF1/WDR20 complex between the plasma membrane, cytoplasm and nucleus.

PO-143 Frequent loss of annexin A1 expression in head and neck squamous cell carcinomas by transcriptional regulatory mechanisms

IntroductionColorectal cancer (CRC) is amongst most frequent tumour types worldwide. Survivin, the smallest member of the inhibitors of apoptosis (IAP) family, is a well-established resistance marker, highly expressed in all tumour types including CRC. The aim of our study was to ascertain whether cellular localization of Survivin might affect the response of CRC cells to the first-line treatment with the topoisomerase I inhibitor irinotecan (IT).Material and methodsThe methods used were DNA cloning, site-directed mutagenesis, transfection and generation of stable clones, immune-fluorescence, colony-forming assays, flow cytometry, ELISA, PCR-based homologous recombination (HR) repair activity assays, western blot analysis, γH2AX foci formation and chromosomal aberration assays.Results and discussionsIn order to examine the effects of Survivin expression and localization on the response to IT, we transfected the p53-proficient CRC cell line (SW48) with the recombinant vector expressing Survivin-GFP fusion protein mainly localised to the cytoplasm. We also generated stable SW48 clones mutated in the nuclear export sequence (NES) of Survivin leading to its retention and accumulation in the nucleus. The proper localization of the fusion protein variants (∼42 kDa) i.e. the functional Survivin-GFP nuclear shuttling by passive diffusion and the active nuclear export were proven by leptomycin B (nuclear export inhibitor), leading to accumulation of the fusion protein in the nucleus. In colony-forming assays the striking difference between the clones was observed: the Survivin-NES-GFP clone was hypersensitive towards IT as compared to the Survivin-GFP clone or parental cells. Flow-cytometric cell-cycle distribution analyses showed that IT induced a prolonged G2-arrest (without induction of senescence) and a higher apoptotic frequency in the Survivin-NES clone, as compared to the parental cell line and the Survivin-GFP clone. Most interesting, the NES clone showed a low repair efficiency of DNA double-strand breaks (DSBs) by HR and lots of aberrant mitoses with a high clastogenic frequency, underlying the onset of mitotic catastrophe.ConclusionCytoplasm-localised Survivin with a normal shuttling function enhanced reproductive survival upon IT exposure and showed a reduced amount of IT-induced DSBs, probably supporting the repair by HR, while the export-impaired NES-Survivin led to an extremely toxic response towards IT. Therefore, Survivin localization could be a prognostic marker for topoisomerase I inhibitor-based CRC therapy.

Correlation of CRM1-NES affinity with nuclear export activity.

CRM1 (Exportin1/XPO1) exports hundreds of broadly functioning protein cargoes out of the cell nucleus by binding to their classical nuclear export signals (NESs). The 8- to 15-amino-acid-long NESs contain four to five hydrophobic residues and are highly diverse in both sequence and CRM1-bound structure. Here we examine the relationship between nuclear export activities of 24 different NES peptides in cells and their CRM1-NES affinities. We found that binding affinity and nuclear export activity are linearly correlated for NESs with dissociation constants (Kds) between tens of nanomolar to tens of micromolar. NESs with Kds outside this range have significantly reduced nuclear export activities. These include two unusually tight-binding peptides, one from the nonstructural protein 2 of murine minute virus (MVM NS2) and the other a mutant of the protein kinase A inhibitor (PKI) NES. The crystal structure of CRM1-bound MVM NS2NES suggests that extraordinarily tight CRM1 binding arises from intramolecular contacts within the NES that likely stabilizes the CRM1-bound conformation in free peptides. This mechanistic understanding led to the design of two novel peptide inhibitors that bind CRM1 with picomolar affinity.

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.

Association of USP10 with G3BP2 Inhibits p53 Signaling and Contributes to Poor Outcome in Prostate Cancer.

Ubiquitin-specific protease 10 (USP10) is known to deubiquitylate its target proteins, mainly to enhance their stabilities. USP10 maintains p53 protein levels and controls epigenetic changes induced by the androgen receptor (AR). GTPase-activating protein-binding protein 2 (G3BP2), an androgen-responsive gene, is known as the main component of stress granules (SG) that interacts with USP10 in SGs. This study explores the roles of USP10 in prostate cancer progression in p53, G3BP2, and AR signaling. Using chromatin immunoprecipitation (ChIP) and sequence analysis, it was found that USP10 is transcriptionally induced with AR recruitment to an intronic region. Furthermore, USP10 regulates androgen-mediated signaling and cell growth. USP10 maintained G3BP2 protein stability by reducing polyubiquitylation. G3BP2-dependent growth activation and p53 nuclear export that reduced p53 signaling were repressed by USP10 knockdown. Clinically, USP10 was expressed primarily in the cytoplasm of prostate cancer tissues. High levels of USP10 expression were strongly correlated with high levels of AR, G3BP2, and p53 in the cytoplasm. High expression of USP10 was significantly associated with poor prognosis of patients with prostate cancer. Taken together, USP10 has a repressive effect on p53 signaling for cell growth by regulating G3BP2 expression. These findings highlight an important oncogenic aspect of USP10 through its modulation of the p53-G3BP2 complex and AR signaling in prostate cancer.Implications: These findings elucidate the oncogenic role of USP10 in prostate cancer through an increase in G3BP2 protein that inhibits p53 activity, in addition to the promotion of AR signaling. Mol Cancer Res; 16(5); 846-56. ©2018 AACR.

Gastrin Induces Nuclear Export and Proteasome Degradation of Menin in Enteric Glial Cells

The multiple endocrine neoplasia, type 1 (MEN1) locus encodes the nuclear protein and tumor suppressor menin. MEN1 mutations frequently cause neuroendocrine tumors such as gastrinomas, characterized by their predominant duodenal location and local metastasis at time of diagnosis. Diffuse gastrin cell hyperplasia precedes the appearance of MEN1 gastrinomas, which develop within submucosal Brunner's glands. We investigated how menin regulates expression of the gastrin gene and induces generation of submucosal gastrin-expressing cell hyperplasia. Primary enteric glial cultures were generated from the VillinCre:Men1FL/FL:Sst-/- mice or C57BL/6 mice (controls), with or without inhibition of gastric acid by omeprazole. Primary enteric glial cells from C57BL/6 mice were incubated with gastrin and separated into nuclear and cytoplasmic fractions. Cells were incubated with forskolin and H89 to activate or inhibit protein kinase A (a family of enzymes whose activity depends on cellular levels of cyclic AMP). Gastrin was measured in blood, tissue, and cell cultures using an ELISA. Immunoprecipitation with menin or ubiquitin was used to demonstrate post-translational modification of menin. Primary glial cells were incubated with leptomycin b and MG132 to block nuclear export and proteasome activity, respectively. We obtained human duodenal, lymph node, and pancreatic gastrinoma samples, collected from patients who underwent surgery from 1996 through 2007 in the United States or the United Kingdom. Enteric glial cells that stained positive for glial fibrillary acidic protein (GFAP+) expressed gastrin de novo through a mechanism that required PKA. Gastrin-induced nuclear export of menin via cholecystokinin B receptor (CCKBR)-mediated activation of PKA. Once exported from the nucleus, menin was ubiquitinated and degraded by the proteasome. GFAP and other markers of enteric glial cells (eg, p75 and S100B), colocalized with gastrin in human duodenal gastrinomas. MEN1-associated gastrinomas, which develop in the submucosa, might arise fromenteric glial cells through hormone-dependent PKA signaling. This pathway disrupts nuclear menin function, leading to hypergastrinemia and associated sequelae.

Dual function of the nuclear export signal of the Borna disease virus nucleoprotein in nuclear export activity and binding to viral phosphoprotein

BackgroundBorna disease virus (BoDV), which has a negative-sense, single-stranded RNA genome, causes persistent infection in the cell nucleus. The nuclear export signal (NES) of the viral nucleoprotein (N) consisting of leucine at positions 128 and 131 and isoleucine at positions 133 and 136 overlaps with one of two predicted binding sites for the viral phosphoprotein (P). A previous study demonstrated that higher expression of BoDV-P inhibits nuclear export of N; however, the function of N NES in the interaction with P remains unclear. We examined the subcellular localization, viral polymerase activity, and P-binding ability of BoDV-N NES mutants. We also characterized a recombinant BoDV (rBoDV) harboring an NES mutation of N.ResultsBoDV-N with four alanine-substitutions in the leucine and isoleucine residues of the NES impaired its cytoplasmic localization and abolished polymerase activity and P-binding ability. Although an alanine-substitution at position 131 markedly enhanced viral polymerase activity as determined by a minigenome assay, rBoDV harboring this mutation showed expression of viral RNAs and proteins relative to that of wild-type rBoDV.ConclusionsOur results demonstrate that BoDV-N NES has a dual function in BoDV replication, i.e., nuclear export of N and an interaction with P, affecting viral polymerase activity in the nucleus.

Deregulation of kinase signaling and lymphoid development in EBF1-PDGFRB ALL leukemogenesis.

The chimeric fusion oncogene EBF1-PDGFRB is a recurrent lesion observed in Ph-like B-ALL and is associated with particularly poor prognosis. While it is understood that this fusion activates tyrosine kinase signaling, the mechanisms of transformation and importance of perturbation of EBF1 activity remain unknown. EBF1 is a nuclear transcription factor required for normal B-lineage specification, commitment, and development. Conversely, PDGFRB is a receptor tyrosine kinase that is normally repressed in lymphocytes, yet PDGFRB remains a common fusion partner in leukemias. Here, we demonstrate that the EBF1-PDGFRB fusion results in loss of EBF1 function, multimerization and autophosphorylation of the fusion protein, activation of STAT5 signaling, and gain of IL-7-independent cell proliferation. Deregulation and loss of EBF1 function is critically dependent on the nuclear export activity of the TM domain of PDGFRB. Deletion of the TM domain partially rescues EBF1 function and restores IL-7 dependence, without requiring kinase inhibition. Moreover, we demonstrate that EBF1-PDGFRB synergizes with loss of IKAROS function in a fully penetrant B-ALL in vivo. Thus, we establish that EBF1-PDGFRB is sufficient to drive leukemogenesis through TM-dependent loss of transcription factor function, increased proliferation and synergy with additional genetic insults including loss of IKAROS function.

Dnase1L3 Regulates Inflammasome-Dependent Cytokine Secretion.

Pediatric-onset systemic lupus erythematosus arises in humans and mice lacking the endonuclease Dnase1L3. When Dnase1L3 is absent, DNA from circulating apoptotic bodies is not cleared, leading to anti-DNA antibody production. Compared to early anti-DNA and anti-chromatin responses, other autoantibody responses and general immune activation in Dnase1L3−/− mice are greatly delayed. We investigated the possibility that immune activation, specifically inflammasome activation, is regulated by Dnase1L3. Here, we report that Dnase1L3 inhibition blocked both NLR family, pyrin domain containing 3 (NLRP3) and NLRC4 inflammasome-mediated release of high-mobility group box 1 protein and IL-1β. In contrast to IL-1β release, Dnase1L3 inhibition only mildly impaired NLRP3-dependent pyroptosis, as measured by propidium iodide uptake or LDH release. Mechanistically, we found that Dnase1L3 was needed to promote apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) nuclear export and speck formation. Our results demonstrate that Dnase1L3 inhibition separates cytokine secretion from pyroptosis by targeting ASC. These findings suggest that Dnase1L3 is necessary for cytokine secretion following inflammasome activation.