Development of a Nuclear Export Signal Trapping Method for Isolating Genes with HIV Rev Activity

We have developed a method for nuclear export signal trapping (NEST) to isolate functional Rev clones from various types of libraries such as libraries of Rev mutants. The expression libraries are cotransfected into COS cells together with a novel Rev-dependent immunoselectable CD28 expression plasmid, pCMV128-CD28. CD28-positive cells are recovered by FACS or by immune precipitation with magnetic beads, and the low-molecular-weight extra chromosomal DNA is recovered, amplified for Rev-containing DNA by PCR and recloned into expression plasmids. The resulting clones are enriched for functional Rev clones. These can be recovered efficiently after several repetitive NEST cycles. This technique may be usefully applied to study various regions of Rev, such as the RNA binding domain and the nuclear export signal, or effector domain and potentially to the isolation of cellular factors with nuclear export capabilities.     

Cytosolic RNA:DNA hybrids activate the cGAS–STING axis

Intracellular recognition of non‐self and also self‐nucleic acids can result in the initiation of potent pro‐inflammatory and antiviral cytokine responses. Most recently, cGAS was shown to be critical for the recognition of cytoplasmic dsDNA. Binding of dsDNA to cGAS results in the synthesis of cGAMP(2′–5′), which then binds to the endoplasmic reticulum resident protein STING. This initiates a signaling cascade that triggers the induction of an antiviral immune response. While most studies on intracellular nucleic acids have focused on dsRNA or dsDNA, it has remained unexplored whether cytosolic RNA:DNA hybrids are also sensed by the innate immune system. Studying synthetic RNA:DNA hybrids, we indeed observed a strong type I interferon response upon cytosolic delivery of this class of molecule. Studies in THP‐1 knockout cells revealed that the recognition of RNA:DNA hybrids is completely attributable to the cGAS–STING pathway. Moreover, in vitro studies showed that recombinant cGAS produced cGAMP upon RNA:DNA hybrid recognition. Altogether, our results introduce RNA:DNA hybrids as a novel class of intracellular PAMP molecules and describe an alternative cGAS ligand next to dsDNA.     

Phosphoinositide Interactions Position cGAS at the Plasma Membrane to Ensure Efficient Distinction between Self- and Viral DNA

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Nuclear Export of NBN Is Required for Normal Cellular Responses to Radiation

Nijmegen breakage syndrome arises from hypomorphic mutations in the NBN gene encoding nibrin, a component of the MRE11/RAD50/nibrin (MRN) complex. In mammalian cells, the MRN complex localizes to the nucleus, where it plays multiple roles in the cellular response to DNA double-strand breaks. In the current study, sequences in mouse nibrin required to direct the nuclear localization of the MRN complex were identified by site-specific mutagenesis. Unexpectedly, nibrin was found to contain both nuclear localizing signal (NLS) sequences and a nuclear export signal (NES) sequence whose functions were confirmed by mutagenesis. Both nuclear import and export sequences were active in vivo. Disruption of either the NLS or NES sequences of nibrin significantly altered the cellular distribution of nibrin and Mre11 and impaired survival after exposure to ionizing radiation. Mutation of the NES sequence in nibrin slowed the turnover of phosphorylated nibrin after irradiation, indicating that nuclear export of nibrin may function, in part, to downregulate posttranslationally modified MRN complex components after DNA damage responses are complete.Exposure to ionizing radiation (IR) results in a spectrum of damage to cells that includes the induction of DNA double-strand breaks (DSBs). In mammalian cells, sensing of DNA DSBs is extremely rapid, occurring within seconds of exposure to IR, and very sensitive, responding to as little as a single DSB in a cell. The sensitivity and speed of this response require immediate access to genomic DNA and raise the possibility that nuclear localization of key components of the damage-sensing or signaling cascade could play an important regulatory role in the process.The earliest measurable cellular response to DNA DSBs is phosphorylation of the protein kinase ATM on serine 1981. ATM exists normally in cells as an inactive dimer which, upon the induction of DNA DSBs, undergoes a transphosphorylation reaction and dissociates into active monomers (1). ATM is recruited to the sites of DNA DSBs via an interaction with the C-terminal end of the nibrin protein, amino acids 735 to 754 (9, 23), and subsequently phosphorylates nibrin (7, 10, 17, 21, 24) and other substrates. Phosphorylated nibrin then plays two key roles, one as a transducer of signals necessary to activate the S-phase checkpoint and the other as a scaffold for the recruitment and phosphorylation of other ATM substrates.The MRE11/RAD50/nibrin (MRN) complex, of which nibrin is a component, has well-defined DNA repair functions, including DNA binding and nuclease activity. Consistent with these functions, hypomorphic mutations in nibrin and MRE11 result in radiation sensitivity disorders, Nijmegen breakage syndrome (NBS) and ataxia telangiectasia-like disorder, respectively. MRE11 interacts with a conserved binding site at the C-terminal end of nibrin, adjacent to the binding site for ATM (6, 9, 23). In NBS cells, where full-length nibrin is absent, MRE11 and RAD50 lose their nuclear localization and are distributed randomly throughout the cell, indicating a requirement for nibrin to maintain the correct subcellular localization of the MRN complex (3). Similar effects are observed in ataxia telangiectasia-like disorder cells, which have mutations in MRE11 that impair its binding to nibrin (20). Nibrin mutants lacking the C-terminal 100 amino acids that include the MRE11 binding site localize to the nucleus when expressed in NBS cells but fail to relocalize either MRE11 or RAD50 or to complement the cellular radiosensitivity associated with NBS (6, 15). These results suggest that sequences mediating nuclear localization of nibrin are located 5′ of the C-terminal 100 amino acids.Given the critical role that nuclear localization plays in the function of the MRN complex, and hence the mammalian DNA DSB response, in the current study we used in vitro mutagenesis to map and identify sequences in mouse nibrin that affect the nuclear localization of the MRN complex. We demonstrate that the nuclear localization of nibrin and MRE11 represents an equilibrium state in a dynamic process of active import and export mediated by specific sequences in nibrin. Maintenance of this equilibrium by nibrin-mediated shuttling between the cytoplasm and the nucleus is required for normal cellular responses to DNA DSBs and may play a role in downregulating responses after damage.     

Permeable FGF-1 Nuclear Localization Signal Peptide Stimulates DNA Synthesis in Various Cell Types but Is Cell-Density Sensitive and Unable to Support Cell Proliferation

An earlier report indicated that a 26-amino-acid peptide (SA), comprised of the nuclear localization signal (NLS) of fibroblast growth factor-1 (FGF-1) and a membrane-permeable peptide, was able to stimulate DNA synthesis after it was taken up by NIH3T3 fibroblasts. Here, we report that SA, but not a mutant with the NLS motif destroyed, induced DNA synthesis in BALB/c3T3 murine fibroblasts, human vascular endothelial (HUVE) cells, and primary cultured hepatocytes, although the activity was weaker than that of FGF-1. The kinetics of SA-induced DNA synthesis and G₁cyclin expression were similar to those elicited by FGF-1, indicating that SA induces cell cycle progression. Kinetic analysis also suggested that SA stimulates only a fraction of the DNA replication in BALB/c3T3 cells. At high cell densities, SA-induced G₁cyclin expression and DNA synthesis were more strongly inhibited than those induced by FGF-1. SA did not induce cell division in HUVE and BALB/c3T3 cells and did not interfere with FGF-1-stimulated proliferation of HUVE cells. These results indicate that SA is able to partially induce cell cycle progression through a contact-inhibition sensitive signaling pathway, but it is insufficient to support cell mitosis. We also suggest that signaling by SA does not interfere with that of FGF-1.     

The N-Terminal Domain of cGAS Determines Preferential Association with Centromeric DNA and Innate Immune Activation in the Nucleus

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Nuclear localization signal of SV40 T antigen directs import of plasmid DNA into sea urchin male pronuclei in vitro

Nuclear import of plasmid DNA mediated by a nuclear localization signal (NLS) derived from SV40 T antigen was investigated in a cell-free extract. In vitro assembled sea urchin male pronuclei were incubated in a 100,000g supernatant of a zebrafish fertilized egg lysate, together with fluorescently labeled plasmid DNA bound to NLS or nuclear import deficient reverse NLS (revNLS) peptides. After 3 hr, DNA-NLS, but not DNA-revNLS, complexes were bound around the nuclear periphery. We demonstrate that nuclear import of DNA-NLS complexes is a two-step process involving binding to, and translocation across, the nuclear envelope. Binding is ATP-independent, occurs at 0°C and is Ca²⁺-independent. By contrast, translocation requires ATP hydrolysis, Ca²⁺, is temperature dependent and is blocked by the lectin wheat germ agglutinin. Both binding and translocation are competitively inhibited by albumin-NLS conjugates, require heat-labile cytosolic factors, and are inhibited by N-ethylmaleimide treatment of the cytosol. Binding and translocation are differentially affected by cytosol dilutions, suggesting that at least two distinct soluble fractions are required for nuclear import. The requirements for NLS-mediated nuclear import of plasmid DNA are similar to those for nuclear import of protein-NLS conjugates in permeabilized cells. © 1996 Wiley-Liss, Inc.     

Mediators of Nuclear Protein Import Target Karyophilic Proteins to Pore Complexes of Cytoplasmic Annulate Lamellae

Nuclear pore complexes are constitutive structures of the nuclear envelope in eukaryotic cells and represent the sites where transport of molecules between nucleus and cytoplasm takes place. However, pore complexes of similar structure, but with largely unknown functional properties, are long known to occur also in certain cytoplasmic cisternae that have been termed annulate lamellae (AL). To analyze the capability of the AL pore complex to interact with the soluble mediators of nuclear protein import and their karyophilic protein substrates, we have performed a microinjection study in stage VI oocytes ofXenopus laevis.In these cells AL are especially abundant and can easily be identified by light and electron microscopy. Following injection into the cytoplasm, fluorochrome-labeled mediators of two different nuclear import pathways, importin β and transportin, not only associate with the nuclear envelope but also with AL. Likewise, nuclear localization signals (NLS) of the basic and M9 type, but not nuclear export signals, confer targeting and transient binding of fluorochrome-labeled proteins to cytoplasmic AL. Mutation or deletion of the NLS signals prevents these interactions. Furthermore, binding to AL is abolished by dominant negative inhibitors of nuclear protein import. Microinjections of gold-coupled NLS-bearing proteins reveal specific gold decoration at distinct sites within the AL pore complex. These include such at the peripheral pore complex-attached fibrils and at the central “transporter” and closely resemble those of “transport intermediates” found in electron microscopic studies of the nuclear pore complex (NPC). These data demonstrate that AL can represent distinct sites within the cytoplasm of transient accumulation of nuclear proteins and that the AL pore complex shares functional binding properties with the NPC.     

A Novel In Vivo Assay Reveals Inhibition of Ribosomal Nuclear Export in Ran-Cycle and Nucleoporin Mutants

To identify components involved in the nuclear export of ribosomes in yeast, we developed an in vivo assay exploiting a green fluorescent protein (GFP)-tagged version of ribosomal protein L25. After its import into the nucleolus, L25-GFP assembles with 60S ribosomal subunits that are subsequently exported into the cytoplasm. In wild-type cells, GFP-labeled ribosomes are only detected by fluorescence in the cytoplasm. However, thermosensitive rna1-1 (Ran-GAP), prp20-1 (Ran-GEF), and nucleoporin nup49 and nsp1 mutants are impaired in ribosomal export as revealed by nuclear accumulation of L25-GFP. Furthermore, overexpression of dominant-negative RanGTP (Gsp1-G21V) and the tRNA exportin Los1p inhibits ribosomal export. The pattern of subnuclear accumulation of L25-GFP observed in different mutants is not identical, suggesting that transport can be blocked at different steps. Thus, nuclear export of ribosomes requires the nuclear/cytoplasmic Ran-cycle and distinct nucleoporins. This assay can be used to identify soluble transport factors required for nuclear exit of ribosomes.     

体内转录的多聚腺苷酸加速外源基因mRNA的出核转运

目的:探索体内转录的短多聚腺苷酸[poly(A)]对外源基因mRNA的表达及出核转运的影响。方法:构建依赖于H1启动子体内转录的poly(A)载体,用脂质体转染方法将其与外源绿色荧光蛋白(GFP)基因表达质粒导入MCF-7细胞,采用qPCR和Western印迹分别检测GFP在mRNA和蛋白水平的表达情况,并通过体外实验检测体内转录的poly(A)对GFP mRNA的加尾影响;采用qPCR法考察16种内源基因的mRNA水平;将其与p53表达质粒共转染MCF-7细胞后,采用MTT法检测细胞增殖情况。结果:在MCF-7细胞中,依赖于H1启动子转录的poly(A)能够加速外源GFP mRNA的poly(A)加尾,促进其从细胞核向细胞质输出,从而提高12 h内GFP的表达量,对内源基因mRNA水平没有影响;它还能加速外源p53基因的表达。结论:建立了通过体内转录poly(A)从而加速外源基因表达的策略,可能对基因治疗或快速研究某个特异基因的功能具有重要的应用价值。     

The ADP-ribosyltransferase PARP10/ARTD10 Interacts with Proliferating Cell Nuclear Antigen (PCNA) and Is Required for DNA Damage Tolerance

All cells rely on genomic stability mechanisms to protect against DNA alterations. PCNA is a master regulator of DNA replication and S-phase-coupled repair. PCNA post-translational modifications by ubiquitination and SUMOylation dictate how cells stabilize and re-start replication forks stalled at sites of damaged DNA. PCNA mono-ubiquitination recruits low fidelity DNA polymerases to promote error-prone replication across DNA lesions. Here, we identify the mono-ADP-ribosyltransferase PARP10/ARTD10 as a novel PCNA binding partner. PARP10 knockdown results in genomic instability and DNA damage hypersensitivity. Importantly, we show that PARP10 binding to PCNA is required for translesion DNA synthesis. Our work identifies a novel PCNA-linked mechanism for genome protection, centered on post-translational modification by mono-ADP-ribosylation.     

A CRM1-Mediated Nuclear Export Signal Is Essential for Cytoplasmic Localization of Neurogenin 3 in Neurons

Neurogenin 3 (Ngn3), a proneural gene, regulates dendritogenesis and synaptogenesis in mouse hippocampal neurons. Ngn3 is transiently exported from the cell nucleus to the cytoplasm when neuronal polarity is initiated, suggesting that the nucleo-cytoplasmic transport of the protein is important for its action on neuronal development. In this study, we identified for the first time a functional nuclear export sequence (NES2; ¹³¹YIWALTQTLRIA¹⁴²) in Ngn3. The green fluorescent protein (EGFP)-NES2 fusion protein was localized in the cytoplasm and its nucleo-cytoplasmic shuttling was blocked by the CRM1 specific export inhibitor leptomycin B. Mutation of a leucine residue to alanine (L135A) in the NES2 motif resulted in both cytoplasmic and nuclear localization of the EGFP-NES2 fusion protein and in the nuclear accumulation of ectopic full-length myc-Ngn3. In addition, point mutation of the leucine 135 counteracted the effects of Ngn3 on neuronal morphology and synaptic inputs indicating that the cytoplasmic localization of Ngn3 is important for neuronal development. Pharmacological perturbation of the cytoskeleton revealed that cytoplasmic Ngn3 is associated with microtubules.     

利用核定位信号筛选系统初步筛选小鼠胚胎核定位蛋白基因

在已建立的核定位信号 (nuclearlocalizationsignal,NLS)筛选系统的基础上 ,对这一系统进行了改进并对改进的系统进行了验证。将小鼠 1 1天胚胎cDNA文库插入改进后的筛选载体的多克隆位点 ,转化酵母宿主菌。然后将约 1 0 4 个酵母克隆接种于选择性平板上进行筛选 ,得到了 2 2个可在选择性培养基上生长的克隆。分析了其中 1 8个克隆的DNA序列 ,见到 1 3个克隆含有以正确读框融合的编码NLS的基因片段。取其中 3个克隆的插入片段与绿色荧光蛋白基因融合后在哺乳类细胞内表达 ,证明了其在哺乳类细胞中的核定位功能。研究证明 ,构建的核定位信号筛选系统 ,能够有效地从cDNA文库中筛选核定位蛋白的基因     

Rac1 Protein Signaling Is Required for DNA Damage Response Stimulated by Topoisomerase II Poisons

To investigate the potency of the topoisomerase II (topo II) poisons doxorubicin and etoposide to stimulate the DNA damage response (DDR), S139 phosphorylation of histone H2AX (γH2AX) was analyzed using rat cardiomyoblast cells (H9c2). Etoposide caused a dose-dependent increase in the γH2AX level as shown by Western blotting. By contrast, the doxorubicin response was bell-shaped with high doses failing to increase H2AX phosphorylation. Identical results were obtained by immunohistochemical analysis of γH2AX focus formation, comet assay-based DNA strand break analysis, and measuring the formation of the topo II-DNA cleavable complex. At low dose, doxorubicin activated ataxia telangiectasia mutated (ATM) but not ATM and Rad3-related (ATR). Both the lipid-lowering drug lovastatin and the Rac1-specific inhibitor NSC23766 attenuated doxorubicin- and etoposide-stimulated H2AX phosphorylation, induction of DNA strand breaks, and topo II-DNA complex formation. Lovastatin and NSC23766 acted in an additive manner. They did not attenuate doxorubicin-induced increase in p-ATM and p-Chk2 levels. DDR stimulated by topo II poisons was partially blocked by inhibition of type I p21-associated kinases. DDR evoked by the topoisomerase I poison topotecan remained unaffected by lovastatin. The data show that the mechanisms involved in DDR stimulated by topo II poisons are agent-specific with anthracyclines lacking DDR-stimulating activity at high doses. Pharmacological inhibition of Rac1 signaling counteracts doxorubicin- and etoposide-stimulated DDR by disabling the formation of the topo II-DNA cleavable complex. Based on the data we suggest that Rac1-regulated mechanisms are required for DNA damage induction and subsequent activation of the DDR following treatment with topo II but not topo I poisons.     

CK2 Inhibits TIMELESS Nuclear Export and Modulates CLOCK Transcriptional Activity to Regulate Circadian Rhythms

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