- Research article
- Open Access
Cloning, expression and nuclear localization of human NPM3, a member of the nucleophosmin/nucleoplasmin family of nuclear chaperones
© Shackleford et al; licensee BioMed Central Ltd. 2001
- Received: 7 August 2001
- Accepted: 6 November 2001
- Published: 6 November 2001
Studies suggest that the related proteins nucleoplasmin and nucleophosmin (also called B23, NO38 or numatrin) are nuclear chaperones that mediate the assembly of nucleosomes and ribosomes, respectively, and that these activities are accomplished through the binding of basic proteins via their acidic domains. Recently discovered and less well characterized members of this family of acidic phosphoproteins include mouse nucleophosmin/nucleoplasmin 3 (Npm3) and Xenopus NO29. Here we report the cloning and initial characterization of the human ortholog of Npm3.
Human genomic and cDNA clones of NPM3 were isolated and sequenced. NPM3 lies 5.5 kb upstream of FGF8 and thus maps to chromosome 10q24-26. In addition to amino acid similarities, NPM3 shares many physical characteristics with the nucleophosmin/nucleoplasmin family, including an acidic domain, multiple potential phosphorylation sites and a putative nuclear localization signal. Comparative analyses of 14 members of this family from various metazoans suggest that Xenopus NO29 is a candidate ortholog of human and mouse NPM3, and they further group both proteins closer with the nucleoplasmins than with the nucleophosmins. Northern blot analysis revealed that NPM3 was strongly expressed in all 16 human tissues examined, with especially robust expression in pancreas and testis; lung displayed the lowest level of expression. An analysis of subcellular fractions of NIH3T3 cells expressing epitope-tagged NPM3 revealed that NPM3 protein was localized solely in the nucleus.
Human NPM3 is an abundant and widely expressed protein with primarily nuclear localization. These biological activities, together with its physical relationship to the chaparones nucleoplasmin and nucleophosmin, are consistent with the proposed function of NPM3 as a molecular chaperone functioning in the nucleus.
- Anaplastic Lymphoma Kinase
- Acidic Domain
- Ribosome Assembly
- Centrosome Duplication
- Nuclear Autoantigenic Sperm Protein
The proper assembly of basic proteins with nucleic acids, such as occurs in the packaging of histones with DNA to produce chromatin and in the packaging of ribosomal proteins with rRNA to form ribosomes, is a reaction that must be facilitated so as to prevent the aggregation of these oppositely charged groups of molecules. Proteins that mediate these reactions, generally termed molecular (or nuclear) chaperones, have been identified biochemically. Two well studied proteins that participate in the processes of chromatin and ribosome assembly are nucleoplasmin and nucleophosmin, respectively, two related proteins whose characteristic acidic domains have been shown to bind the basic proteins involved in these processes and present them to the nucleic acid.
Nucleoplasmin is the most abundant protein in the Xenopus oocyte nucleus and is the protein for which the term molecular chaperone was coined due to its multiple roles in the assembly of nucleosomes during early frog development . Nucleoplasmin forms a pentamer and its stretches of acidic residues bind to histone H2A and H2B. In concert with the unrelated acidic protein N1/N2, which binds histones H3 and H4, they act together and with other factors to assemble nucleosomes [2, 3]. In addition to assembly, nucleoplasmin and other proteins are involved in the chromatin remodeling and nucleosome disassembly that occurs, for example, during transcription to allow the access of transcription factors to nucleosomal DNA . Another major function of nucleoplasmin is the decondensation of sperm chromatin at fertilization. In this case, nucleoplasmin acts to exchange the sperm specific basic proteins, which allow the dense packing of DNA in sperm, with the histones H2A and H2B, thus effecting chromatin decondensation [5, 6]. Phosphorylation of nucleoplasmin appears to be important in regulating this activity, as heavily phosphorylated nucleoplasmin is significantly more active .
Nucleophosmin (also called B23 , NO38  or numatrin ), a protein related to nucleoplasmin, is implicated in ribosome assembly due to its abundance, localization in the nucleolus and its multiple activities that are consistent with such a function. Some of these activities include nucleic acid binding , ribonuclease activity (for processing preribosomal RNA)  and association with maturing preribosomal ribonucleoprotein particles [13, 14]. It may also be involved in the transport of ribosomal or other nucleosomal proteins across the nuclear membrane, as it is known to shuttle between the cytoplasm and nucleus and to stimulate the nuclear importation of proteins [15, 16]. Nucleophosmin also appears to be intimately involved in centrosome duplication. It associates specifically with unduplicated centrosomes, and its phosphorylation by CDK-2/cyclin E, the trigger for centrosome duplication, is required for duplication to occur . The nucleophosmin gene is also known for its fusion with the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase in cases of this disease with (2;5)(p23;q35) translocations . The nucleophosmin portion contributes to transformation by providing a dimerization domain, which allows activation of the fused kinase and signal transduction .
We previously discovered and initially characterized a novel member of this family in the mouse, namely nucleophosmin/nucleoplasmin 3 (Npm3). To identify the human ortholog of Npm3 and begin its characterization, we have cloned a human NPM3 cDNA, determined its genomic structure and relationship to other family members and show its expression in multiple tissues and subcellular localization in the nucleus.
NPM3 cDNA cloning and genomic structure
Exon/intron organization of the NPM3 gene
Exon size (bp)
5' Splice site*
Intron size (bp)
3' Splice site*
Amino acid at splice**
NPM3 sequence analysis
Accession numbers of amino acid sequences
Nucleophosmin; NO38; B23; Numatrin
Nucleophosmin; NO38; B23; Numatrin
Nucleophosmin; NO38; B23; Numatrin
Nucleophosmin; NO38; B23; Numatrin
Nucleophosmin; NO38; B23; Numatrin
Nucleic acid-associated protein 36; ANO39
Mitotic apparatus protein p62
CG7911 gene product
Nuclear histone-binding protein N1/N2
Nuclear autoantigenic sperm protein
NPM3 expression in human tissues
NPM3 protein localizes to the nucleus
The striking similarities in amino acid sequence and domain structure between NPM3 and its chaperone relatives, nucleophosmin and nucleoplasmin, together with abundant expression levels and nuclear localization, strongly suggests that NPM3 shares fundamental nuclear chaperone functions with these proteins. It will be important in future work to refine these results and test, for example, whether NPM3 may have chromatin or ribosome assembly functions that are similar or complementary to nucleoplasmin or nucleophosmin, or whether it may have completely independent functions.
Isolation of NPM3 genomic probes
We digested previously isolated lambda genomic clones of the human FGF8 region  with restriction endonucleases (Promega, Madison, WI) and analyzed them by Southern blotting using a mouse Npm3 cDNA  probe, as previously described . An 8.4-kb Xho I fragment from these clones that hybridized to the probe was subcloned into pBluescript (Stratagene) for analysis. Deletion of non-Npm3 sequences from this clone resulted in a 4.3-kb Hin d III-Xho I fragment, which was then isolated and used as a probe for the isolation of a human NPM3 cDNA.
Isolation of NPM3 cDNA
The 4.3-kb Hin d III-Xho I human genomic fragment described above was used as a probe to screen a λgt11 human liver cDNA library for the NPM3 cDNA. Three positive clones were obtained from 250,000 plaques. Following isolation of pure plaques, the insert DNAs were prepared by PCR methods with the following conditions: 100 μl reactions containing 1X Pfu buffer (20 mM Tris-Cl, pH 8.75, 10 mM KCl, 2 mM MgSO4, 10 mM (NH4)2SO4, 0.1% v/v Triton X-100, 100 μg/ml bovine serum albumin), 1 μM λgt11 forward and reverse primers (Promega, Madison, WI), 0.2 mM deoxyribonucleotides, and 5 Units of recombinant Pfu DNA Polymerase (Stratagene, La Jolla, CA). The thermocycling conditions were as follows: 95°C for 3 minutes, then 30 cycles of 95°C for 45 seconds, 50°C for 30 seconds and 75°C for 60 seconds, then 75°C for 10 minutes, using a PTC-100 Thermocycler (M.J. Research, Watertown, MA). The resulting cDNA inserts were purified by agarose gel electrophoresis, digested with Eco R I and cloned into pBluescript KS- (Stratagene, La Jolla, CA). The cDNA inserts were thermocycle-sequenced using the fmol kit (Promega, Madison, WI) and found to be identical and to lack a portion of the 5' coding region.
To obtain the 5' end of the NPM3 cDNA, we performed 5' Rapid Amplification of cDNA Ends (RACE), using a Marathon-Ready™ human testis cDNA library (Clontech, Palo Alto, CA) and the Marathon™ cDNA amplification kit (Clontech, Palo Alto, CA). The RACE conditions were as follows: 50 μl reactions containing 1X KlenTaq buffer and Advantage KlenTaq Polymerase Mix (Clontech, Palo Alto, CA), 0.2 mM deoxyribonucleotides, 0.2 μM NPM3 GSP1 (5'-CGG TGA GGC AGA GCA TGG TTA GTG C-3'), and 0.2 μM API primer (Clontech, Palo Alto, CA). Touchdown PCR conditions were employed as follows: 95°C for 5 minutes, then 5 cycles of 95°C for 10 seconds, 72°C for 2 minutes, then 5 cycles of 95°C for 10 seconds, 70°C for 2 minutes, then 25 cycles of 95°C for 10 seconds, 68°C for 2 minutes. The resulting band was purified by agarose gel electrophoresis and subcloned into pCR II (Invitrogen, Carlsbad, CA). Multiple clones were thermocycle sequenced to confirm the 5' sequence of human NPM3 cDNA. Finally, to produce a full-length human NPM3 cDNA, we spliced together the 5' RACE NPM3 cDNA with the original partial cDNA obtained from the lambda library at a unique Sac I site present in both fragments. The final cDNA sequence was submitted to GenBank (accession number AY049737).
Determination of exon structure
Fragments of the 4.3-kb Hin d III-Xho I NPM3 genomic DNA isolated above that hybridized to a mouse Npm3 cDNA probe were further subcloned into pBS and sequenced using an automated ABI 377 sequencer. Comparison of these compiled sequences with the human NPM3 cDNA sequence allowed the localization of exon sequences within the genomic DNA. The genomic sequence and exon placement was later confirmed with human genome sequences that subsequently appeared in GenBank (accession number AC010789).
Amino acid sequences were aligned using CLUSTAL W (v. 1.81) with default parameters on a European Molecular Biology Laboratory web server http://www.ebi.ac.uk. An alignment output from this source was used to create a dendrogram using TreeTop  with PHYLIP output (default parameters) at a node of the European Molecular Biology Network http://www.genebee.msu.su. Potential phosphorylation sites were identified using NetPhos 2.0 http://www.cbs.dtu.dk/services/NetPhos. After alignment with CLUSTAL W, identical and similar amino acids in Figure 3 were identified using MacBoxshade 2.15 http://www.isrec.isb-sib.ch/ftp-server/boxshade/MacBoxshade.
Northern blot analysis
Human tissue northern blots of poly(A)+ RNA were obtained from Clontech (Palo Alto, CA) and sequentially hybridized with radiolabeled NPM3 cDNA and beta-actin probes as previously described .
A cDNA that encodes a hemagglutinin-tagged NPM3 protein was produced by PCR under the following conditions: 100 μl reactions with 1X Pfu buffer, 0.2 mM deoxyribonucleotides, 1 μM HA-F primer (5'-AAA GAA TTC AGC ATG TAC CCA TAC GAC GTC CCA GAC TAC GCC GCC GCC GGT ACT GCA GCT GCC-3'), 1 μM NPM3-R2 primer (5'-AAA GAA TTC CTA GGG CCT GCC CCC CTG CTT TTT GGC AGG AAG GAT GGG-3'), 1 ng of human NPM3 cDNA insert, and 2.5 Units of recombinant Pfu DNA Polymerase. The thermocycling conditions were as follows: 95°C for 3 minutes, then 30 cycles of 95°C for 45 seconds, 75°C for 60 seconds, then 75°C for 10 minutes, using a PTC-100 Thermocycler (M.J. Research, Watertown, MA). The resulting DNA fragment was purified by agarose gel electrophoresis, cleaved with Eco R I, and subcloned into pBluescript KS-. The resulting plasmids were thermocycle sequenced, and an insert with the correct sequence was identified and subcloned into pMIRB . The orientations of the resulting inserts were determined by restriction digests with Sac I.
Cellular localization of HA-NPM3 by cell fractionation and immunoblotting
The resulting pMIRB-HA-NPM3 plasmids (both sense and antisense orientations) were transiently transfected into NIH 3T3 cells, using transfection conditions with Lipofectamine and OptiMEM serum-free medium (Gibco-BRL, Bethesda, MD) as described . Following a six-hour incubation of the DNA-Lipofectamine complexes in OptiMEM, the cells were washed and incubated in 10-cm dishes with their usual growth media (DMEM with 10% v/v fetal calf serum, 2 mM L-glutamine, 100 Units/ml of Penicillin G and 100 μg/ml Streptomycin) for 48–72 hours at 37°C in humidified 5% CO2 incubators.
Following the 48–72 hour incubation, the media was collected and placed on ice with the following protease inhibitors added: 1 mM DTT (Sigma, St. Louis, MO), 0.5 mM PMSF (Sigma, St. Louis, MO), 5 μg/ml Pepstatin (Sigma, St. Louis, MO), 3 μg/ml Leupeptin (Sigma, St. Louis, MO) and 5 μg/ml Aprotinin (Sigma, St. Louis, MO). The cells were washed 3X in cold PBS and collected by cell scrapers (Nunc) in 1 ml of cold PBS. The cells were transferred to microfuge tubes, pelleted (5 seconds at 14,000 rpm, Eppendorf 5415C, 4°C) and resuspended in hypotonic Buffer A (10 mM K-HEPES pH 7.9, 1.5 mM MgCl2, and 10 mM KCl, with inhibitors added as for the media above) for 15 minutes on ice. The suspension was vortexed vigorously for 10 seconds to lyse the cells, and then microcentrifuged (1 minute at 14,000 rpm, Eppendorf 5415C, 4°C). The resulting supernatant was called "cytoplasm" but actually contained cytoplasmic membranes as well. The resulting nuclear pellet was resuspended in 20 μl of Buffer C (20 mM K-HEPES pH 7.9, 1.5 mM MgCl2, 0.2 mM EDTA, 20% v/v glycerol, and 0.42 M NaCl, with protease inhibitors as above), incubated on ice for 20 minutes, then microcentrifuged (5 minutes at 14,000 rpm, Eppendorf 5415C, 4°C). This final supernatant was the nuclear extract.
The protein solutions were quantified by Bradford Assay (Biorad, Hercules, CA), and 50 mg of each sample were subjected to SDS-PAGE (15% acrylamide). Following electrophoresis, the gel contents were electrophoretically-transferred to nitrocellulose (500 mA, 60–90 V, 1 hour, 4°C, Transfer Buffer II from Harlow and Lane ). The membrane was blocked with 5% w/v nonfat dried milk in PBS-0.1% v/v Tween 20 (PBS-T) overnight at 4°C, then subjected to immunoblotting and ECL (Amersham, Arlington, IL) detection. The primary antibody was monoclonal mouse anti-hemagglutinin (dilution 1:1000 in PBS-T, gift of Guojon Bu, Washington University, St. Louis), and the secondary antisera was horseradish peroxidase-conjugated sheep anti-mouse IgG (dilution 1:1000 in PBS-T, Amersham, Arlington, IL).
This work was supported by grants to G.M.S. from the Department of Defense Breast Cancer Research Program (DAMD 17-96-1-6039) and the T.J. Martell Foundation for Leukemia, Cancer and AIDS, and by grants to C.A.M. from the National Institutes of Health (CA70106), Edward G. Mallinckrodt, Jr., Foundation, and the Elsa U. Pardee Foundation.
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