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.     

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.     

利用酵母双杂交技术筛选与AtbZIP1相互作用的蛋白质

碱性亮氨酸拉链bZIP类转录因子在植物的生长发育、光形态建成、光信号传导及非生物胁迫反应中发挥重要的作用.为研究AtbZIP1基因的作用机理,本研究首先验证了该基因的自激活转录活性,通过缺失突变确定了该转录因子的转录激活结构域;以AtbZIP1缺失突变体AtbZ3为诱饵蛋白,采用Matchmaker Gold Yeast Two-Hybrid System(Clonetch),共筛选获得5个与诱饵蛋白相互作用的蛋白质;并通过AbA(Aureobasidin A)抗生素标记基因,His营养缺陷和LacZ蓝白斑检测验证了阳性克隆.亚细胞定位分析发现,AtbZIP1蛋白除了定位于细胞核外,还定位于叶绿体细胞.通过分析这些靶蛋白的已知功能,为研究AtbZIP1蛋白的未知生物学功能提供重要信息.     

蛋白质的入核运送和它在基因表达中的调节作用

蛋白质进入细胞核是由蛋白质分子内部的核定位信号（nuclear localization signal, NLS）引导的.NLS蛋白首先与NLS受体结合,然后在多种胞浆因子及核孔复合物蛋白的作用下穿过核孔、转位入核.蛋白质上存在NLS并不一定总能够引导蛋白质入核.当NLS被修饰或遮掩时,它们便不能被核转运装置所识别.因而,NLS的遮掩被解除之前,蛋白质一直被扣留在胞浆中.以调节转录因子的入核运送来控制转录因子的活性是基因表达调节的一个新概念,也是细胞生长和分化的另一水平的调节.     

Import Pathway of Nuclear-Encoded Cytochrome c Oxidase Subunit 2 Using Yeast as a Model

Abstract: Subunit 2 of cytochrome c oxidase (Cox2) is a mitochondrial-encoded protein in most organisms. In soybean Glycine max a second Cox2 gene was identified in the nucleus which is functional, whereas the mitochondrial-encoded cox2 gene is silent. For import and sorting of the nuclear-encoded soybean Cox2 protein ( Gm Cox2p) into mitochondria, the protein has acquired an N-terminal extension of 136 amino acid residues that is cleaved off in three steps during import. To study the function and processing of the Gm Cox2p leader peptide, we used yeast as a model system. Using different leader peptide-GFP constructs, we were able to show that the i₁ intermediate is generated in the mitochondrial matrix and the mature protein is generated in the inner membrane space. Mitochondrial processing peptidase (MPP) is involved in processing the first part of the leader peptide, processing of the last part is catalysed by the inner membrane peptidase (IMP). Oxa1p is necessary for insertion of the protein into the inner mitochondrial membrane. Gm Cox2p therefore utilises many of the same components as its mitochondrial-encoded predecessor, for sorting and maturation, following its import into the mitochondria.     

Functional Analysis of Tpr: Identification of Nuclear Pore Complex Association and Nuclear Localization Domains and a Role in mRNA Export

Tpr is a 270-kD coiled-coil protein localized to intranuclear filaments of the nuclear pore complex (NPC). The mechanism by which Tpr contributes to the structure and function of the nuclear pore is currently unknown. To gain insight into Tpr function, we expressed the full-length protein and several subdomains in mammalian cell lines and examined their effects on nuclear pore function. Through this analysis, we identified an NH₂-terminal domain that was sufficient for association with the nucleoplasmic aspect of the NPC. In addition, we unexpectedly found that the acidic COOH terminus was efficiently transported into the nuclear interior, an event that was apparently mediated by a putative nuclear localization sequence. Ectopic expression of the full-length Tpr caused a dramatic accumulation of poly(A)⁺ RNA within the nucleus. Similar results were observed with domains that localized to the NPC and the nuclear interior. In contrast, expression of these proteins did not appear to affect nuclear import. These data are consistent with a model in which Tpr is tethered to intranuclear filaments of the NPC by its coiled coil domain leaving the acidic COOH terminus free to interact with soluble transport factors and mediate export of macromolecules from the nucleus.     

The N-terminus of porcine circovirus type 2 replication protein is required for nuclear localization and ori binding activities

Porcine circovirus type 2 possesses a circular, single-stranded DNA genome that requires the replication protein (Rep) for virus replication. To characterize the DNA binding potential and the significant region that confers the nuclear localization of the Rep protein, the defined coding regions of rep gene were cloned and expressed. All of the recombinant proteins except for the N-terminal 110 residues deletion mutant could bind to the double-stranded minimal binding site of replication origin (ori). In addition, the N-terminal deletion mutant lacking 110 residues exhibited mainly cytoplasmic staining in the transfected cells in contrast to the others, which localized dominantly in the nucleus, suggesting that this N-terminal domain is essential for nuclear localization. Furthermore, a series of green fluorescence proteins (GFP) containing potential nuclear localization signal (NLS) sequences were tested for their cellular distribution. The ability of the utmost 20 residues of the N-terminal region to target the GFP to the nucleus confirmed its role as a functional NLS.     

核定位信号分析示踪载体——pGST-EGFP的构建及功能鉴定

目的 构建谷胱甘肽转硫酶（GST）与EGFP相融合的新型蛋白质示踪载体--pGST-EGFP,以用于蛋白质细胞亚定位信号序列的深入分析.方法 以质粒pEGFP-N1为骨架,融合从pGEX-2TK载体中扩增的GST编码序列,构建成pGST-EGFP融合表达质粒;再插入人工合成的已知核定位蛋白SV40的核定位序列（NLS）,构建成pGST-EGFP-SV40 NLS作为阳性对照;另外,构建小分子量蛋白TNNI2在pGST-EGFP的融合表达质粒.将对照pEGFP-N1和各重组质粒分别用脂质体介导,瞬时转染HeLa细胞,荧光显微镜下观察蛋白的核定位情况.结果 单独表达的EGFP呈全细胞分布,而GST-EGFP融合蛋白只存在于细胞浆;SV40 NLS能将GST-EGFP融合蛋白带进细胞核.虽然TNNI2-EGFP融合蛋白的细胞亚定位呈现核内丰度更高的特点,但TNNI2-GST-EGFP融合蛋白仅限定于胞浆分布,提示TNNI2不能主动定位到细胞核中.结论 成功构建了蛋白质细胞亚定位示踪载体--pGST-EGFP.作为核定位信号分析系统,其对小分子蛋白细胞亚定位的示踪效果优于传统的pEGFP载体,更适用于科研工作中小分子量蛋白质核定位信号序列的研究.     

A Nuclear Localization Signal at the SAM-SAM Domain Interface of AIDA-1 Suggests a Requirement for Domain Uncoupling Prior to Nuclear Import

The neuronal scaffolding protein AIDA-1 is believed to act as a convener of signals arising at postsynaptic densities. Among the readily identifiable domains in AIDA-1, two closely juxtaposed sterile alpha motif (SAM) domains and a phosphotyrosine binding domain are located within the C-terminus of the longest splice variant and exclusively in four shorter splice variants. As a first step towards understanding the possible emergent properties arising from this assembly of ligand binding domains, we have used NMR methods to solve the first structure of a SAM domain tandem. Separated by a 15-aa linker, the two SAM domains are fused in a head-to-tail orientation that has been observed in other hetero- and homotypic SAM domain structures. The basic nuclear import signal for AIDA-1 is buried at the interface between the two SAM domains. An observed disparity between the thermal stabilities of the two SAM domains suggests a mechanism whereby the second SAM domain decouples from the first SAM domain to facilitate translocation of AIDA-1 to the nucleus.     

编码新型转录因子样蛋白的小鼠及人类同源cDNA的克隆(英文)

 通过检索GenBank的表达序列标签 (EST)数据库并结合cDNA末端快速扩增法 (RACE) ,从小鼠胸腺克隆到一个新的cDNA序列 ,并从人类肝癌组织中克隆出了其同源cDNA .根据读码框架分析 ,这两个cDNA分别编码 541和 555个氨基酸的蛋白质 两个蛋白质之间氨基酸序列一致率为77% ,和已知蛋白无显著同源性 .分子生物学软件和网上分析表明 ,两个蛋白质所含功能序列与STAT家族成员极为相似 ,均含有包括酪氨酸蛋白激酶在内的多种蛋白激酶的磷酸化位点和核定位信号 (NLS) ,可能是一种新型转录因子 .RT PCR分析显示 ,两个基因在正常组织中选择性表达 ,其分布相似 ,而且都具有一定程度的与分化或增殖相关的趋势 .     

The chaperone function of cyclophilin 40 maps to a cleft between the prolyl isomerase and tetratricopeptide repeat domains

Cyclophilin 40 (CyP40), an immunophilin cochaperone present in steroid receptor-Hsp90 complexes, contains an N-terminal peptidylprolyl isomerase (PPIase) domain separated from a C-terminal Hsp90-binding tetratricopeptide repeat (TPR) domain by a 30-residue linker. To map CyP40 chaperone function, CyP40 deletion mutants were prepared and analysed for chaperone activity. CyP40 fragments containing the PPIase domain plus linker or the linker region and the adjoining TPR domain retained chaperone activity, whilst individually, the catalytic and TPR domains were devoid of chaperoning ability. CyP40 chaperone function then, is localized within the linker that forms a binding cleft with potential to accommodate non-native substrates.     

Investigating nucleo-cytoplasmic shuttling of the human DEAD-box helicase DDX3

The human DEAD-box helicase DDX3 is a multi-functional protein involved in the regulation of gene expression and additional non-conventional roles as signalling adaptor molecule that are independent of its enzymatic RNA remodeling activity. It is a nucleo-cytoplasmic shuttling protein and it has previously been suggested that dysregulation of its subcellular localization could contribute to tumourigenesis. Indeed, both tumour suppressor and oncogenic functions have been attributed to DDX3. In this study, we investigated the regulation of DDX3’s nucleocytoplasmic shuttling. We confirmed that an N-terminal conserved Nuclear Export Signal (NES) is required for export of human DDX3 from the nucleus, and identified three regions within DDX3 that can independently facilitate its nuclear import. We also aimed to identify conditions that alter DDX3’s subcellular localisation. Viral infection, cytokine treatment and DNA damage only induced minor changes in DDX3’s subcellular distribution as determined by High Content Analysis. However, DDX3’s nuclear localization increased in early mitotic cells (during prophase) concomitant with an increase in DDX3 expression levels. Our results are likely to have implications for the proposed use of (nuclear) DDX3 as a prognostic biomarker in cancer.     

The C-Terminal Domain of SRA1p Has a Fold More Similar to PRP18 than to an RRM and Does Not Directly Bind to the SRA1 RNA STR7 Region

Steroid receptor activator RNA protein (SRA1p) is the translation product of the bi-functional long non-coding RNA steroid receptor activator RNA 1 (SRA1) that is part of the steroid receptor coactivator-1 acetyltransferase complex and is indicated to be an epigenetic regulatory component. Previously, the SRA1p protein was suggested to contain an RNA recognition motif (RRM) domain. We have determined the solution structure of the C-terminal domain of human SRA1p by NMR spectroscopy. Our structure along with sequence comparisons among SRA1p orthologs and against authentic RRM proteins indicates that it is not an RRM domain but rather an all-helical protein with a fold more similar to the PRP18 splicing factor. NMR spectroscopy on the full SRA1p protein suggests that this structure is relevant to the native full-length context. Furthermore, molecular modeling indicates that this fold is well conserved among vertebrates. Amino acid variations in this protein seen across sequenced human genomes, including those in tumor cells, indicate that mutations that disrupt the fold occur vary rarely and highlight that its function is well conserved. SRA1p had previously been suggested to bind to the SRA1 RNA, but NMR spectra of SRA1p in the presence of its 80-nt RNA target suggest otherwise and indicate that this protein must be part of a multi-protein complex in order to recognize its proposed RNA recognition element.