Mouse Npm3 is located approximately 5 kb upstream of the Fgf8 gene in the same transcriptional orientation 2021. To determine if human NPM3 is similarly located and to identify genomic clones of NPM3, we analyzed FGF8-containing human genomic lambda clones 22 by Southern blotting using a mouse Npm3 cDNA probe. A 4.3-kb Hind III-Xho I fragment that hybridized to Npm3 was identified, subcloned and used to screen a human liver cDNA library. Several partial cDNAs with strong sequence homology to mouse Npm3 were isolated. The 5' coding region was subsequently isolated using a reverse transcriptase-PCR approach and was fused to one of the partial cDNAs at a common restriction site to produce a cDNA with full coding potential. Sequencing of subcloned genomic fragments and comparison with the cDNA sequence allowed us to construct an exon map of the NPM3 gene (Figure 1A). NPM3 has the same exon structure as the mouse ortholog and is located approximately 5.5 kb upstream of FGF8, in the same transcriptional orientation (Figure 1B). The NPM3 exon/intron boundaries and organization information is presented in Table 1. Based on its close linkage to FGF8, NPM3 maps to chromosome 10q24-26 2324.

The amino acid sequence deduced from the human NPM3 cDNA sequence was 87% identical and 95% similar to mouse Npm3 (Figure 2). The human sequence is three amino acids longer than that of the mouse; these additional residues lie in the acidic domain in the C-terminal portion of the protein. At least eight potential serine and threonine phosphorylation sites are present in the human protein involving such kinases as casein kinase I and II, protein kinase A and C, glycogen synthase kinase 3 and calmodulin-dependent protein kinase II (Figure 2). All of these consensus sites are also found in mouse Npm3 with the exception of the casein kinase I site at residue 16. A cluster of amino acids at the C-terminus that is rich in basic residues and glycines forms a potential nuclear localization signal 2526 in the human as well as the mouse protein (Figure 2). Multiple phosphorylation sites and nuclear localization signals, or the ability to bind proteins with such signals, are also characteristics of nucleophosmin and nucleoplasmin 16272829. When the functions of NPM3 are elucidated, it will be of interest to determine the contribution of phosphorylation to these activities, since the functions of both nucleoplasmin and nucleophosmin are regulated by phosphorylation, which is extensive in both proteins 717).

To enable an amino acid comparison between NPM3 with other members of the nucleophosmin/nucleoplasmin family, we searched the nonredundant GenBank database using human NPM3, Xenopus nucleoplasmin and human nucleophosmin as BLASTP queries, and 11 additional full-length proteins were retrieved (Table 2). An amino acid comparison of NPM3 with these other members of the nucleophosmin/nucleoplasmin family from various metazoans reveals extensive sequence identities and similarities throughout all of NPM3 except the C-terminal 16 residues (Figure 3). All members of this family have a core region of relatively close similarity in the N-terminal half of the proteins. C-terminal to this region, all have one or more acidic domains consisting of a total of 17 to more than 100 aspartic acid and glutamic acid residues per molecule. These two residues can comprise more than 25% of the total amino acids in some proteins of this family; in NPM3 they make up approximately 18% of the residues. The N-terminal core region of high similarity between family members correlates to a region in nucleophosmin/B23 that has been shown to be involved in oligomerization as well as chaperone activity 44. A central portion between nucleophosmin's two acidic domains is required for ribonuclease activity 44; this region does not have a corresponding domain in either NPM3 or nucleoplasmin, suggesting that these proteins would lack such activity. A nucleic acid binding domain in the C-terminus of nucleophosmin is also lacking in NPM3 and nucleoplasmin, but the known functions of nucleoplasmin in nucleosome assembly and sperm decondensation suggest that this protein, and possibly NPM3, can accomplish intermolecular reactions involving nucleic acids in other ways, perhaps by associating with other proteins that have this binding activity. Indeed, NO29, a Xenopus protein with significant si milarity to NPM3, is found to associate with NO38, the Xenopus ortholog of mammalian nucleophosmin 43.

To gain further insight into the relationship of NPM3 with the other known members of this family, we used CLUSTAL W and TreeTop 45 to produce a dendrogram of these relationships (Figure 4). In this analysis, we included the 14 proteins from Figure 3 together with two other histone-binding proteins that are unrelated to this family. These two proteins, Xenopus N1/N2 36 and human NASP (nuclear autoantigenic sperm protein) 41, are closely related to each other and may be orthologs 46. This analysis shows that NPM3 is more closely related to NO29 and the nucleoplasmins from Xenopus than to the nucleophosmins from multiple species. No nucleoplasmin ortholog in humans has been reported to date, and our screening of GenBank has not detected such a sequence. The relationship between N1/N2 and NASP can be seen in this dendrogram and appears similar in closeness to that of NPM3 and NO29. The kinship of NPM3 and NO29 is supported by recent GenBank screens with the NPM3 amino acid sequence as a TBLASTN query against the entire nonredundant GenBank database, which resulted in NO29 sequences as the best non-NPM3 match, and vice versa. Although other genomic and functional studies would be required to prove an orthologous relationship between NPM3 and NO29, they appear by several analyses to share a relatively close evolutionary history and as such could be considered candidate orthologs.

We examined the expression of NPM3 in 16 human tissues by Northern blot analysis (Figure 5). Abundant and relatively equal expression of NPM3 was found in all tissues with the highest levels, relative to actin, in pancreas and testis and the lowest in lung. Previous analysis of Npm3 expression in mouse tissues also showed generally equal and strong expression in the tissues tested with especially strong expression in testis 21. However, in the mouse, the brain, rather than the lung, displayed the lowest Npm3 RNA levels. Nevertheless, the widespread and relatively strong expression of the gene in both species suggests that the protein has a fundamental function(s) in cells of many, if not all, tissues. The apparently ubiquitous expression and abundant levels of NPM3 RNA are consistent with other members of this gene family and with a proposed role as a molecular chaperone.

All of the nucleophosmin and nucleoplasmin homologs that have been studied to date are localized in the nucleus. To allow a determination of the subcellular localization of the NPM3 protein, we tagged the protein at its N-terminus with a hemagglutinin (HA) epitope. We then expressed this protein in NIH3T3 cells, fractionated the cells into nuclear, cytoplasmic and extracellular (culture medium) fractions and analyzed these by immunoblotting using an anti-HA antibody. This analysis revealed that NPM3 was localized only in the nuclear fraction (Figure 6). The NPM3 protein migrated in SDS-PAGE gels as a doublet with apparent molecular masses of 25 and 27 kDa, which are larger than the 20.5 kDa mass predicted by the deduced amino acid sequence including the HA tag. Similarly, other members of the nucleophosmin/nucleoplasmin family also migrate more slowly than expected: Xenopus NO29 migrates at 29 kDa versus the expected 20 kDa 44, Xenopus NO38 migrates at 38 kDa versus the expected 33.5 kDa 9, and Xenopus nucleoplasmin migrates at 38 kDa rather than 33.5 kDa 9. The slower migration is likely primarily due to an electrophoretic anomaly reflecting the amino acid composition, although phosphorylation or other modifications could conceivably contribute. Supporting this notion, the in vitro transcription/translation of a plasmid encoding the closely related NO29 protein produces a product, presumably unmodified, with slow mobility that is similar to the mobility of NO29 extracted from cells 43. The smaller-sized polypeptide of the NPM3 doublet is probably a degradation product, as is observed in analyses of the NO29 protein 43. This subcellular localization of NPM3, together with the other results here, suggests that this protein is a molecu lar chaperone with functions in the nucleus.

We digested previously isolated lambda genomic clones of the human FGF8 region 22 with restriction endonucleases (Promega, Madison, WI) and analyzed them by Southern blotting using a mouse Npm3 cDNA 21 probe, as previously described 20. 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 Hind III-Xho I fragment, which was then isolated and used as a probe for the isolation of a human NPM3 cDNA.

The 4.3-kb Hind 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 EcoR 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).

Fragments of the 4.3-kb Hind 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 45 with PHYLIP output (default parameters) at a node of the European Molecular Biology Network http://www.genebee.msu.su47. Potential phosphorylation sites were identified using NetPhos 2.0 http://www.cbs.dtu.dk/services/NetPhos48. 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.

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 20.

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 EcoR 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 49. The orientations of the resulting inserts were determined by restriction digests with Sac I.

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 49. 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 50). 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).