Inhibition of Nuclear Protein Import by a Monoclonal Antibody against a Novel Class of Nuclear Pore Proteins

Nuclear import of proteins is mediated by the nuclear pore complexes in the nuclear envelope and requires the presence of a nuclear localization signal (NLS) on the karyophilic protein. In this paper, we describe studies with a monoclonal antibody, Mab E2, which recognizes a class of nuclear pore proteins of 60-76 kDa with a common phosphorylated epitope on rat nuclear envelopes. The Mab E2-reactive proteins fractionated with the relatively insoluble pore complex-containing component of the envelope and gave a finely punctate pattern of nuclear staining in immunofluorescence assays. The antibody did not bind to any cytosolic proteins. Mab E2 inhibited the interaction of a simian virus 40 large T antigen NLS peptide with a specific 60-kDa NLS-binding protein from rat nuclear envelopes in photoaffinity labeling experiments. The antibody blocked the nuclear import of NLS--albumin conjugates in an in vitro nuclear transport assay with digitonin-permeabilized cells, but did not affect passive diffusion of a small non-nuclear protein, lysozyme, across the pore. Mab E2 may inhibit protein transport by directly interacting with the 60-kDa NLS-binding protein, thereby blocking signal-mediated nuclear import across the nuclear pore complex.     

Two Isoforms of Npap60 (Nup50) Differentially Regulate Nuclear Protein Import

Npap60 (Nup50) is a nucleoporin that binds directly to importin α. In humans, there are two Npap60 isoforms: the long (Npap60L) and short (Npap60S) forms. In this study, we provide both in vitro and in vivo evidence that Npap60L and Npap60S function differently in nuclear protein import. In vitro binding assays revealed that Npap60S stabilizes the binding of importin α to classical NLS-cargo, whereas Npap60L promotes the release of NLS-cargo from importin α. In vivo time-lapse experiments showed that when the Npap60 protein level is controlled, allowing CAS to efficiently promote the dissociation of the Npap60/importin α complex, Npap60S and Npap60L suppress and accelerate the nuclear import of NLS-cargo, respectively. These results demonstrate that Npap60L and Npap60S have opposing functions and suggest that Npap60L and Npap60S levels must be carefully controlled for efficient nuclear import of classical NLS-cargo in humans. This study provides novel evidence that nucleoporin expression levels regulate nuclear import efficiency.     

RNA binding protein Musashi1 is expressed in sertoli cells in the rat testis from fetal life to adulthood.

The Musashi1 (Msi1) gene identified in mouse is a member of a subfamily of RNA binding proteins that are highly conserved across species. Msi1 expression is highly enriched in proliferative cells within the developing central nervous system. Within the testis, proliferation and differentiation of germ cells takes place within the seminiferous epithelium, where these cells are supported physically and functionally by Sertoli cells that do not themselves proliferate following the onset of puberty. RNA binding proteins expressed in testicular germ cells are essential for normal fertility. Preliminary data suggested the mRNA for Msi1 was present in ovary; therefore, we used an Msi1-specific cRNA and monoclonal antibody to investigate whether Msi1 was expressed in the testis. Msi1 mRNA was expressed in rat testis from birth until adulthood; in situ hybridization revealed silver grains within the seminiferous epithelium. Immunohistochemical studies demonstrated that at all ages examined (from Fetal Day 14.5 until adulthood) Msi1 protein was expressed in Sertoli cells. In fetal and adult rat ovaries, Msi1 was detected in granulosa cells and their precursors. In Sertoli cells, protein was detected in both cytoplasmic and nuclear compartments; in adult testes, the immunointensity of the nuclear staining was stage dependent, with highest levels of expression in Sertoli cells at stages I-VI. In rat gonads, the RNA binding protein Msi1 is expressed in both proliferating and nonproliferating Sertoli and granulosa cells.     

Nanos3 of the frog Rana rugosa: Molecular cloning and characterization

Nanos is expressed in the primordial germ cells (PGCs) and also the germ cells of a variety of organisms as diverse as Drosophila, medaka fish, Xenopus and mouse. In Nanos3‐deficient mice, PGCs fail to incorporate into the gonad and the size of the testis and ovary is thereby dramatically reduced. To elucidate the role of Nanos in an amphibian species, we cloned Nanos3 cDNA from the testis of the R. rugosa frog. RT‐PCR analysis showed strong expression of Nanos3 mRNA in the testis of adult R. rugosa frogs, but expression was not sexually dimorphic during gonadal differentiation. In Nanos3‐knockdown tadpoles produced by the CRISPR/Cas9 system, the number of germ cells decreased dramatically in the gonads of both male and female tadpoles before sex determination and thereafter. This was confirmed by three dimensional imaging of wild‐type and Nanos3 knockdown gonads using serial sections immunostained for Vasa, a marker specific to germ cells. Taken together, these results suggest that Nanos3 protein function is conserved between R. rugosa and mouse.     

Npap60/Nup50 is a tri-stable switch that stimulates importin-alpha:beta-mediated nuclear protein import

Many nuclear-targeted proteins are transported through the nuclear pore complex (NPC) by the importin-alpha:beta receptor. We now show that Npap60 (also called Nup50), a protein previously believed to be a structural component of the NPC, is a Ran binding protein and a cofactor for importin-alpha:beta-mediated import. Npap60 is a tri-stable switch that alternates between binding modes. The C terminus binds importin-beta through RanGTP. The N terminus binds the C terminus of importin-alpha, while a central domain binds importin-beta. Npap60:importin-alpha:beta binds cargo and can stimulate nuclear import. Endogenous Npap60 can shuttle and is accessible from the cytoplasmic side of the nuclear envelope. These results identify Npap60 as a cofactor for importin-alpha:beta nuclear import and as a previously unidentified subunit of the importin complex.     

Expression of 5α-reductase in bacteria as a trp E fusion protein and its use in the production of antibodies for immunocytochemical localization of 5α-reductase

A cDNA encoding a full-length rat 5α-reductase was isolated using female rat liver mRNA and the polymerase chain reaction, and fused to the Escherichia coli trp E gene in a pATH expression vector. The trp E-5α-reductase fusion protein expressed in bacteria and a synthetic oligopeptide corresponding to the C-terminus of rat 5α-reductase were used as antigens to produce rabbit polyclonal antibodies to 5α-reductase. Antibodies to the 5α-reductase portion of the fusion protein and to the peptide were purified by affinity chromatography. Antibodies against the 5α-reductase fusion protein reacted with a single component of rat liver microsomes with M_r 26,000 on Western blots, consistent with the size of 5α-reductase predicted from its cDNA, and with a M_r 23,000 component on Western blots of detergent extracts of rat ventral prostate nuclei; other rat ventral prostate cellular fractions (mitochondrial, microsomal, cytosol) bound little or no antibody. Antibody against the synthetic peptide reacted with a M_r 26,000 component of rat liver microsomes as well as with several components in various cellular fractions of rat ventral prostate. With anti-5α-reductase fusion protein antibodies, specific immunocytochemical staining was observed in the epithelial cell nuclei of the rat ventral prostate, seminal vesicle, epididymis and other accessory sex glands. This nuclear staining was specific, since antibodies from non-immunized rabbits did not give nuclear staining and preincubation of the anti-5α-reductase fusion protein antibodies with the trp E-5α-reductase fusion protein eliminated nuclear staining. Incubation of antibodies with trp E (without the 5α-reductase fusion) had no effect on nuclear staining. Specific staining was not detected in the cytoplasm of these epithelial cells. Little or no specific staining was observed in stromal cells in these rat tissuess. Human prostate was also immunocytochemically stained with this antibody. Specific staining was found in both epithelial and stromal cell nuclei.     

Molecular characterization and expression pattern of dmrt1 in the immature Chinese sturgeon Acipenser sinensis

In this study, the cDNA of dmrt1 gene from the Chinese sturgeon Acipenser sinensis was isolated and its expression pattern was characterized in different tissues of immature A. sinensis. By real‐time quantitative PCR (qrtPCR) analysis, the A. sinensis dmrt1 mRNA was detected mainly in gonad and with a higher level in the testis than the ovary, especially in 3 and 4 year‐old samples. This indicated that the dmrt1 expression exhibited gradual testis specificity with development. The subcellular localization analysis indicated that the Dmrt1 protein exists only in germ cells and not in somatic cells. These results suggest that A. sinensis dmrt1 might be a highly specific sex differentiation gene for testis development and spermatogenesis.     

Cloning and characterization of the gene encoding the bovine BOULE protein

The Deleted in Azoospermia (DAZ) genes encode potential RNA-binding proteins that are expressed exclusively in the germ-line. The bovine Deleted in Azoospermia-like gene is a strong candidate for male cattle-yak infertility. In this work, with the goe goal to further reveal the genetic cause of male cattle-yak sterility, another bovine DAZ family gene, b-boule, was isolated and characterized. The b-boule gene is predicted to encode a polypeptide of 295 amino acids with an RNP-type RNA recognition domain. Tertiary structure analysis shows that b-boule binds specifically to polypyrimidine RNAs and might act as a nuclear ribonucleoprotein particle auxiliary factor during germ cell formation and morphological changes of germ cells. RT-PCR assays revealed that b-boule was expressed specifically in the adult testis. However, an extremely low level of expression was detected in the testis of sterile male cattle-yaks. Microstructure of the testes from sterile males showed that type A spermatogonia were the only germ cells present and that few germ cells developed further than the stage of pachytene spermatocytes. These results suggest that b-boule may function in bovine spermatogenesis, and that low levels of b-boule expression might lead to male sterility in cattle-yaks.     

Molecular cloning and characterization of the germ cell‐related nuclear orphan receptor in chickens

Nuclear receptor subfamily 6, group A, member 1 (NR6A1), also known as germ cell nuclear factor/retinoid receptor‐related testis‐associated receptor and neuronal cell nuclear factor, is a member of the nuclear orphan receptor superfamily. NR6A1 has been cloned in various species including humans and mice, but it has been scarcely investigated in avian species. In the present study, we cloned the chicken NR6A1 (cNR6A1) from a testis cDNA library. The cloned cNR6A1 sequence was mapped to chromosome 17 and contained an open reading frame of 1.4 kb encoding 445 amino acids. Multiple alignment analysis of the cNR6A1 protein‐coding sequence with NR6A1s from humans, mice, boars, rats, zebrafish, and Xenopus showed high degrees of homology, 89%, 90%, 89%, 88%, 83%, and 87%, respectively. Using RNA interference, changes in the expression of pluripotency‐, germ cell‐, and differentiation‐related key genes by silencing of cNR6A1 were validated in chicken blastoderm‐derived embryonic stem cells. Among those genes, the relative expression levels of POU5F1, CRIPTO, DAZL, DDX4, BMP15, GSC, and SOX7 changed significantly compared to the control group. We also confirmed that the activity of alkaline phosphatase, known as a pluripotency marker, was maintained by cNR6A1 gene silencing in chicken blastodermal cells. Collectively, our data suggest that cNR6A1 may play an important role during chicken embryonic development and differentiation. Mol. Reprod. Dev. 77: 273–284, 2010. © 2009 Wiley‐Liss, Inc.     

Developmental expression of heat shock protein 60 (HSP60) in the rat testis and ovary

Heat shock protein 60 (HSP60), a member of the chaperonin family, has an essential role in mediating correct folding of nuclear encoded proteins imported to mitochondria. We have investigated immunocytochemical expression of HSP60 in developing fetal, newborn, postnatal, and pubertal testis and ovary, and in the adult ovary of the rat. In the fetal gonads, HSP60 was expressed in the germ cells organized into sex cords and in the developing Leydig cells of the testis. In the pubertal testis, Leydig cells were strongly, spermatogonia and premeiotic spermatocytes moderately labeled, spermatids unlabeled. In the postnatal ovary, oocytes at all stages of folliculogenesis were positive for HSP60. In the pubertal ovary, glandular theca cells, and in the mature ovary, also the cells of the corpora lutea exhibited intense cytoplasmic labeling. At the electron microscopic level, immunogold particles were localized in the mitochondrial matrix, and in the Western blot analysis the antibody detected one single band of 60 kDa. Anti-HSP60 labeling in male and female sex steroid producing cells and their progenitors seems to be coordinated with the functional differentiation of these endocrine cells of the gonad. In the oocytes, a key element required for proper folding of imported mitochondrial proteins seems to be constitutively expressed throughout folliculogenesis. However, the data suggest that in the male germ cells mitochondrial chaperonin HSP60 is either not needed during the haploid phase of spermatogenesis or its level becomes extensively reduced and therefore undetectable by the methods used in the study.     

Npap60: a new player in nuclear protein import

Until very recently, the vertebrate protein Npap60/Nup50 was thought merely to be a component of the nuclear pore complex (NPC). This conclusion was based on the observations that Npap60/Nup50 localizes at the NPC by immunofluorescence and electron microscopy and also contains FG (Phe-Gly) repeats, a motif commonly found in nucleoporins but not in proteins located elsewhere. However, far from being a fixed structural component of the NPC, it now appears as though Npap60 can shuttle from one side of the NPC to the other. Most significantly, a recent paper shows that Npap60 enhances the nuclear import of a cargo possessing a basic nuclear localization sequence by associating directly with the import cargo-carrier complex and (presumably) moving through the NPC with it. Several NPC proteins have now been shown to be mobile in the NPC, and this new report might indicate that these 'mobile' nucleoporins play a more active role in the nuclear transport of cargo than was previously appreciated.     

Primary biliary cirrhosis sera recognize not only gp210 but also proteins of the p62 complex bearing N-acetylglucosamine residues from rat liver nuclear envelope

We have recently observed reactivity of primary biliary cirrhosis (PBC) sera with several proteins bearing N-acetylglucosamine residues from rat liver nuclear envelopes. The aim of this study was to characterize the reactive antigens. Sera from 31 patients with PBC, 30 with rheumatoid arthritis (RA) and 30 with Sjögren's syndrome (SS) were examined. Rim-like immunofluorescence staining was observed in 15 of 31 (48%) sera from patients with PBC, in 1 of 30 with RA and in 1 of 30 with SS. Upon immunoblotting using preparations of whole rat liver nuclear envelopes and their Triton X 100-KCl extract as antigen souces, a 200 kDa protein band was observed in 9 of sera with PBC. Furthermore, upon immunoblotting using the wheat germ aggulutinin-bound fraction of rat liver envelope as antigen, 62, 60 and 54 kDa protein bands corresponding to components of the p62 complex in the nuclear pore complex (Kita et al. Biochem. 113, 377–382) were observed in 7, 5 and 6 samples respectively, of the 31 PBC sera. Our data suggest that PBC sera recognize not only the 210 kDa protein but also the p62 complex proteins.Abbreviations ANA antinuclear antibody - AMA anti-mitochondrial antibodies - IF immunofluorescence - LAP2 lamina-associated polypeptide 2 - LBR lamin B receptor - anti-NBP 60 anti-nuclear localization signal binding protein 60 - NE nuclear envelope - NPC nuclear pore complex - PBC primary biliary cirrhosis - RA rheumatoid arthritis - SLE systemic lupus erythematosus - SS Sjögren's syndrome - WGA wheat germ agglutinin     

Product of the oncogene-activating gene Tpr is a phosphorylated protein of the nuclear pore complex

We have identified a component of the human nuclear pore complex and have shown that it is the product of a gene involved in oncogenic activation. A monoclonal antibody raised against purified nuclear matrix proteins recognizes a single protein with an electrophoretic mobility of approximately 300 kDa and stains the nuclear envelope in a punctate pattern typical of nuclear pores. The antibody was used to screen λgt11 human cDNA libraries, and the resulting clones were sequenced and compared to sequences in the Genbank database. An exact match was found with the human tpr (for translocated promoter region) gene, a gene shown previously to be involved in the oncogenic activation of several protein kinases. Double-label immunofluorescent microscopy with the anti-Tpr antibody and an antibody to the previously characterized nuclear pore complex protein nup153 confirms that Tpr is localized to the nuclear pore complex. Tpr is located on the cytoplasmic face of the nucleus, as demonstrated by immunofluorescent staining of cells permeabilized with digitonin. Tpr is a 2,349-amino acid protein with extensive coiled-coil domains and an acidic globular C-terminus. The protein contains 10 leucine zipper motifs and numerous sites for phosphorylation by a variety of protein kinases. Immunoprecipitation of Tpr from ³²P-orthophosphate-labeled cells shows that it is a phosphoprotein. Potential functions for Tpr and possible mechanisms for the transforming activity of Tpr fusion proteins are discussed. © 1996 Wiley-Liss, Inc.     

Cloning and mapping of Np95 gene which encodes a novel nuclear protein associated with cell proliferation

We previously obtained a monoclonal antibody (Th-10a mAb) that recognizes a single 95-kDa mouse nuclear protein (NP95). Immunostaining analyses revealed that the NP95 was specifically stained in the S phase of normal mouse thymocytes. In contrast, mouse T cell lymphoma cells exhibited a constantly high level of NP95 accumulation irrespective of cell stages during the cell cycle. In the present study, we isolated the cDNA encoding the NP95 from a λgt-11 cDNA expression library, using the Th-10a mAb. Sequencing of the whole 3.5-kb cDNA revealed that NP95 is a novel nuclear protein with an open reading frame (ORF) consisting of 782 amino acids. The ORF contains a zinc finger motif, a potential ATP/GTP binding site, a putative cyclin A/E-cdk2 phosphorylation site, and the retinoblastoma protein (RB)-binding motif ``IXCXE'. The chromosomal location of Np95 gene was determined by fluorescence in situ hybridization. Np95 gene locates on mouse Chromosome (Chr) 17DE1.1. and rat Chr 9q11.2–q12.1. Np95 was strongly expressed in the testis, spleen, thymus, and lung tissues, but not in the brain, liver, or skeletal muscles. These results collectively implicate this novel nuclear protein in cell cycle progression and/or DNA replication. Received: 24 June 1998 / Accepted: 12 August 1998     

Identification of a soluble precursor complex essential for nuclear pore assembly in vitro

We analysed the soluble form in which the nuclear pore complex protein p68 is stored in Xenopus laevis eggs and its involvement in pore complex assembly processes. We have shown previously that p68, which is the major wheat germ agglutinin (WGA)-binding glycoprotein of nuclear pore complexes from Xenopus oocytes, is located in the pore channel and participates in mediated transport of karyophilic proteins. Using a monoclonal antibody directed against p68 (PI1) we removed this protein from Xenopus egg extract by immunoadsorption. On addition of lambda DNA the immunodepleted extract supported reconstitution of nuclei which were surrounded by a continuous double-membrane envelope but lacked pore complexes and were unable to import karyophilic proteins such as nucleoplasmin or lamin L_III. Essentially identical results were obtained with extract depleted of WGA-binding proteins. Our finding that both the anti-p68 antibody and WGA efficiently removed components from the extract necessary for pore complex assembly but did not interfere with nuclear membrane formation demonstrates that these processes are independent of each other. Analysis of the immunoprecipitate on silver-stained SDS-polyacrylamide gels indicated that the antibody adsorbed other proteins besides p68, notably two high molecular weight components. By sucrose gradient centrifugation and gel filtration we showed that p68 together with associated protein(s) forms a stable, approximately globular complex plex with an M_r of 254,000, a Stokes radius of 5.2 nm and a sedimentation coefficient of 11.3 S. Our finding that p68 occurs in the form of larger macromolecular assemblies offers an explanation for the distinctly punctate immunofluorescence pattern observed in the cytoplasm of mitotic cells after staining with antibodies to p68.W. Hennig