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Genetics. Aug 2006; 173(4): 1835–1850.
PMCID: PMC1569712

Long-Term Evolution and Functional Diversification in the Members of the Nucleophosmin/Nucleoplasmin Family of Nuclear Chaperones

Abstract

The proper assembly of basic proteins with nucleic acids is a reaction that must be facilitated to prevent protein aggregation and formation of nonspecific nucleoprotein complexes. The proteins that mediate this orderly protein assembly are generally termed molecular (or nuclear) chaperones. The nucleophosmin/nucleoplasmin (NPM) family of molecular chaperones encompasses members ubiquitously expressed in many somatic tissues (NPM1 and -3) or specific to oocytes and eggs (NPM2). The study of this family of molecular chaperones has experienced a renewed interest in the past few years. However, there is a lack of information regarding the molecular evolution of these proteins. This work represents the first attempt to characterize the long-term evolution followed by the members of this family. Our analysis shows that there is extensive silent divergence at the nucleotide level suggesting that this family has been subject to strong purifying selection at the protein level. In contrast to NPM1 and NPM-like proteins in invertebrates, NPM2 and NPM3 have a polyphyletic origin. Furthermore, the presence of selection for high frequencies of acidic residues as well as the existence of higher levels of codon bias was detected at the C-terminal ends, which can be ascribed to the critical role played by these residues in constituting the acidic tracts and to the preferred codon usage for phosphorylatable amino acids at these regions.

NUCLEOSOMES and ribosomes are highly organized nucleoprotein complexes. Orderly assembly of the protein components onto the nucleic acid component is facilitated in part by assembly proteins that are called molecular (or nuclear) chaperones (Laskey et al. 1978; Dingwall and Laskey 1990; Philpott et al. 2000). Among the different nuclear chaperones present in metazoan organisms, the nucleophosmin/nucleoplasmin (NPM) family comprises three major functional types: NPM1, NPM2, and NPM3.

NPM1 is also known as nucleophosmin, B23, numatrin in mammals, and NO38 in amphibians. The N-terminal domain of NO38 forms an eight-stranded β-barrel and a set of five monomers fit together to form a stable pentamer (Namboodiri et al. 2004). Members of this type are among the most abundant phosphorylated proteins in the nucleolus of somatic cells and have been implicated in ribosome assembly. They have been shown to be involved in nucleic acid binding (Dumbar et al. 1989), ribonuclease activity (Herrera et al. 1995), and association with maturing preribosomal ribonucleoprotein particles (Olson et al. 1986). Their role during cell proliferation was originally believed to be restricted to ribosome maturation (Feuerstein et al. 1988; Chan et al. 1989). However, NPM1 is a multifunctional protein that can also bind pRb synergistically stimulating DNA polymerase α (Takemura et al. 1999). NPM1 also appears to be intimately involved in centrosome duplication, as it associates specifically with unduplicated centrosomes, and phosphorylation of NPM1 by CDK-2/cyclin E is required for duplication to occur (Okuda et al. 2000). Furthermore, NPM1 members are involved in several transcription processes through their interaction with transcription factors and histone chaperone activity (Kondo et al. 1997; Okuwaki et al. 2001; Swaminathan et al. 2005; Weng and Yung 2005). NPM1 is induced after genotoxic stress, protecting cells against DNA damage by either increasing their DNA repair capacity or decreasing the apoptotic signal (Wu et al. 2002; Maiguel et al. 2004). In addition, members of the NPM1 family also may be involved in the transport of ribosomal or other nuclear proteins across the nuclear membrane, as these proteins are known to shuttle between the cytoplasm and nucleus and to stimulate the nuclear importation of proteins (Borer et al. 1989).

The NPM2 type (or nucleoplasmin) was first isolated from Xenopus laevis egg extracts and this protein represents an archetypal molecular chaperone (Laskey et al. 1978; Earnshaw et al. 1980; Dingwall and Laskey 1990), which is found natively bound to histones H2A and H2B facilitating their storage in oocytes and eggs (Dingwall and Laskey 1990; Philpott et al. 2000; Burns et al. 2003). NPM2 proteins are acidic, thermostable, and form oligomeric complexes consisting of five identical subunits of ~22 kDa that can both bind histones and promote chromatin assembly in vitro (Laskey et al. 1978; Dingwall et al. 1987; Ohsumi and Katagiri 1991b). Nucleoplasmin is the most abundant protein in Xenopus oocytes and participates in the decondensation of sperm chromatin after fertilization by removing the sperm nuclear basic proteins (SNBPs) (Ohsumi and Katagiri 1991a; Philpott et al. 1991). In concert with the unrelated protein N1/N2, which binds histones H3 and H4, it participates in the assembly of nucleosomes in the male pronucleus (Dingwall and Laskey 1990; Philpott et al. 2000). The chromatin remodeling ability of Xenopus NPM2 at fertilization has been shown to be dependent on its extent of phosphorylation. This post-translational modification enhances the H2A/H2B exchange activity during the decondensation of sperm chromatin, and it increases its ability to promote nucleosome assembly in vitro (Leno et al. 1996).

The nucleoplasmin monomer folds into two different domains: an N-terminal core domain, which consists of an eight-stranded β-barrel that fits snugly within a stable pentamer, and a solvent-exposed C-terminal acidic domain, which is rich in turn structures (Hierro et al. 2001; Namboodiri et al. 2004). In addition to the presence of two acidic tracts, the C-terminal region contains a bipartite nuclear localization sequence. Within amphibians, nucleoplasmin is a highly conserved protein (Prado et al. 2004; Frehlick et al. 2006) that has been shown to bind equally well to the three major types of SNBPs: protamines (Rice et al. 1995; Prieto et al. 2002), protamine-like type (Rice et al. 1995; Prado et al. 2004; Ramos et al. 2005), and histone type (Ramos et al. 2005), both in vivo and in vitro.

The lack of specificity with which amphibian nucleoplasmin binds to SNBPs contrasts with the higher specificity with which it binds to core histones (Arnan et al. 2003), especially to H2A-H2B (Dutta et al. 2001).

An intriguing aspect of other NPM2 members in mammals is that their absence has no effect on meiosis, sperm DNA decondensation, or the first S-phase after fertilization. It is possible in this instance that NPM1 and NPM3, which are present in oocytes (such as in mice), could possibly provide a mechanism of compensation (Burns et al. 2003).

NPM3 (also known as NO29 in Xenopus) includes a group of less well-characterized proteins. In the case of Xenopus, NO29 migrates in SDS–PAGE gels as a 29-kDa protein and shares many physical characteristics with NPM1 and NPM2 at the structural level, including an acidic domain, multiple potential phosphorylation sites, and a putative nuclear localization signal (Zirwes et al. 1997). Proteins of the NPM3 type include a group of molecular chaperones that are ubiquitously expressed in many tissues, showing the highest levels in pancreas and testis and the lowest in lung, in the case of humans (Shackleford et al. 2001). Like NPM1, NPM3 is mainly localized to the nucleolus in interphase cells and active rRNA transcription appears to be required for this localization (Huang et al. 2005). NPM3 forms a complex with NPM1 and has been implicated in regulation of NPM1 function in ribosome biogenesis (Huang et al. 2005). In addition, inhibition of NPM3 expression in mammalian oocytes prohibits paternal chromatin decondensation (McLay and Clarke 2003), suggesting this family member may also serve multiple functions.

The diversification of NPM proteins during animal evolution must have been determined by the presence of different structural and functional constraints acting on these proteins. In this article we take advantage of the molecular data currently available for NPM proteins of different taxonomic groups to analyze their long-term evolution. Special attention is paid to the relative importance of the functional and structural constraints acting at the protein and nucleotide level.

MATERIALS AND METHODS

A total of 106 nucleotide coding sequences belonging to 25 different species of metazoans have been used in our analyses (see supplemental Table 1 at http://www.genetics.org/supplemental/). These include 104 NPM family sequences (54 NPM1, 27 NPM2, 13 NPM3, 2 NPM3 pseudogenes, 8 NPM-like from invertebrates) and 2 outgroup sequences (N1/N2 nuclear histone binding protein from X. laevis and nuclear autoantigenic sperm protein, NASP, from human). Sequences retrieved from databases were subsequently corrected for errors in accession numbers and nomenclature, and they were aligned on the basis of their translated amino acid sequences using the CLUSTAL_X (Thompson et al. 1997) and BIOEDIT programs (Hall 1999) with the default parameters. The alignment of the complete set of sequences consisted of 3873 nucleotide positions (excluding the start and stop codons) corresponding to 1293 amino acid sites. The independent alignments for each of the four NPM lineages are shown in supplemental alignments 1–4 (http://www.genetics.org/supplemental/) and in all cases were checked for errors by visual inspection. The distinction between the N-terminal and the acidic C-terminal regions of NPM proteins was established in this work on the basis of the information available for tertiary protein structure of the core regions: NPM1 [N terminus, nucleotide position (nt pos.) 1–363; C terminus, nt pos. 364–927] (Namboodiri et al. 2003, 2004) and NPM2 (N terminus, nt pos. 1–369; C terminus, nt pos. 370–684) (Dutta et al. 2001). For the cases of NPM3 (N terminus, nt pos. 1–435; C terminus, nt pos. 436–630) and NPM-like from invertebrates (N terminus, nt pos. 1–363; C terminus, nt pos. 364–1287) this was deduced from the alignment of these sequences with those of NPM1 and NPM2.

All molecular evolutionary analyses in this work were carried out using the program MEGA v. 3.1 (Kumar et al. 2004). The extent of nucleotide and amino acid divergence between sequences was estimated by means of the uncorrected differences (p-distance) as this distance is known to give better results than more complicated methods when the number of sequences is large and the number of positions used is relatively small, because of its smaller variance (Nei and Kumar 2000). The numbers of synonymous (pS) and nonsynonymous (pN) nucleotide differences per site were computed using the modified Nei–Gojobori method (Zhang et al. 1998), providing in both cases the transition/transversion ratio (R). Distances were estimated using the complete-deletion option (except for the case of the protein and nucleotide phylogenetic tree reconstructions, where the pairwise-option deletion was used) and standard errors were calculated by the bootstrap method with 1000 replicates. The presence and nature of selection was tested in NPM genes by using the codon-based Z-test for selection, establishing the alternative hypothesis as H1: pN < pS and the null hypothesis as H0: pN = pS (Nei and Kumar 2000). The Z-statistic and the probability that the null hypothesis is rejected were obtained, indicating the significance level as **P (P < 0.001) and *P (P < 0.05).

The presence of selection in the three main NPM lineages (1–3) was further studied by testing for deviations from neutrality. The GC content at fourfold degenerate sites was assumed to represent the genomic GC content and in addition was considered as an approximation to the neutral expectation. The influence of selection on certain amino acids was analyzed by determining the correlation between the genomic GC content and the proportion of GC-rich (GAPW) and GC-poor (FYMINK) residues. Under the neutral model, GC-rich and GC-poor amino acids will be positively and negatively correlated with genomic GC content, respectively (Kimura 1983). If the frequency of these amino acids is influenced by selection, no correlation between genomic GC content and amino acid frequency would be expected (Rooney 2003). Correlations were computed for complete sequences and for discriminating between the N-terminal and C-terminal segments by using the Spearman rank correlation coefficient. The statistical significance was assessed through regression analysis. Given that all changes at second codon positions are nonsynonymous, whereas changes at fourfold degenerate sites are synonymous, the effects of selection and mutation bias will be apparent by comparing these two types of sites. Under the neutral model, nucleotide frequencies at second codon positions should not be significantly different from the frequencies at fourfold degenerate sites (Rooney et al. 2000). If selection is biasing the frequency of concrete amino acids, frequencies of nucleotides at second codon positions will be higher than expected under the neutral model, represented by fourfold degenerate sites. Comparisons were also made for NPM lineages by discriminating between N-terminal and C-terminal domains and the statistical significance was determined by t-tests once the homogeneity of standard deviations was confirmed.

The neighbor-joining tree-building method (Saitou and Nei 1987) was used to reconstruct the phylogenetic trees. To assess that our results are not dependent on this choice, phylogenetic inference analyses were completed by the reconstruction of a maximum-parsimony tree (Rzhetsky and Nei 1992) using the close-neighbor-interchange (CNI) search method with search level 1 and with 10 replications for the random addition trees option. We decided to combine the bootstrap (Felsestein 1985) and the interior-branch test methods (Rzhetsky and Nei 1992; Sitnikova 1996) to test the reliability of the obtained topologies, producing the bootstrap probability (BP) and the confidence probability (CP) values for each internal branch, assuming BP > 80% and CP ≥ 95% as statistically significant (Sitnikova et al. 1995). Human NASP (Welch et al. 1990) and the nuclear histone binding N1/N2 protein from X. laevis (Kleinschmidt et al. 1986) were used as outgroups in the reconstruction, given that they are also histone-binding proteins but unrelated to NPM proteins (Shackleford et al. 2001).

The analysis of the nucleotide variation across different NPM coding regions was performed using a sliding-window approach, by estimating the total (π) and the synonymous (πS) nucleotide diversity (average number of nucleotide differences per site between two sequences) with a window length of 20 bp and a step size of 5 bp (for π) and a window length of 10 bp and a step size of 5 bp (for πS). The codon usage bias in NPM genes was estimated as the effective number of codons (ENC) (Wright 1990), where the highest value (61) indicates that all synonymous codons are used equally (no bias) and the lowest (20) that only a preferred codon is used in each synonymous class (extreme bias). Preferences in the usage of different synonymous triplets were tested by multiple range tests, discriminating among means using the Fisher's least significant difference (LSD) procedure. Both analyses were conducted with the program DnaSP v. 4.10 (Rozas et al. 2003).

The three-dimensional structure of X. laevis NO29 (NPM3) core monomer was modeled using the coordinates determined for the crystal of the Xenopus NO38-core monomer (Namboodiri et al. 2004) as a reference using the SWISS-MODEL server (Schwede et al. 2003). Post-translational phosphorylation sites at serines, threonines, and tyrosines in the NPM protein sequences were predicted by using the NetPhos v. 2.0 server (Blom et al. 1999), selecting only those positions scoring >0.5 in phosphorylation potential.

RESULTS

Evolution of the NPM protein family:

The protein phylogeny was reconstructed from 106 NPM sequences (including 52 nonredundant sequences) of 25 species belonging to different metazoan phyla (Figure 1; see supplemental Table 1 at http://www.genetics.org/supplemental/). The three NPM types and the invertebrate NPM-like type are well defined by the topology and by the confidence levels calculated for each internal node. The different taxonomic groups are also well differentiated with respect to each of the NPM types. Given that many NPM sequences are not yet annotated or are wrongly assigned to their corresponding subtype in the databases, the present topology represents a powerful tool that allowed 17 previously unannotated sequences to be unequivocally ascribed to a given NPM type. Seven of these sequences were identified in silico from the draft genomes of rhesus monkey, orangutan, zebrafish, the fish Tetraodon nigroviridis, the yellow fever mosquito, and the African malaria mosquito.

Figure 1.
Phylogenetic relationships among NPM proteins. The reconstruction was carried out by calculating the evolutionary amino acid p-distances from the NPM sequences of all the organisms analyzed (see supplemental Table 1 at http://www.genetics.org/supplemental/ ...

While the tree topology shows the presence of a monophyletic origin for NPM1 proteins, the polyphyletic origin observed for NPM2 and NPM3 is the result of differences between mammals and amphibians, giving rise to independent groups in the phylogeny. In NPM2, two differentiation events (nodes 1 and 1′ in the tree) occurred that led first to the nucleoplasmin lineage from mammals and subsequently to the differentiation of the nucleoplasmin lineage from amphibians and fish. It is important to note that, as with NPM2, amphibian and fish NPM3 proteins (closer to NPM1) also differentiate from mammalian NPM3 proteins. This pattern of differentiation, which was also corroborated by reconstructing a maximum-parsimony phylogenetic tree, may bear important functional implications and will be further discussed below.

The lineages corresponding to NPM3 and NPM1 differentiated later than NPM2 (nodes 2 and 3 in the tree, respectively). The relationship between types 1 and 3 (p = 0.300 ± 0.033 substitutions per site) was the closest of all, followed by types 2 and 3 (p = 0.411 ± 0.071), and types 1 and 2 (p = 0.453 ± 0.035). This is most likely the result of their different polyphyletic origin as well as the longer time elapsed since the differentiation of NPM2 (and thus, more accumulated changes). This observation is supported by the protein variation observed within lineages (Table 1), which is higher for NPM2 (p = 0.401 ± 0.024 substitutions per site), followed by NPM3 and NPM1 (p = 0.371 ± 0.022 and p = 0.218 ± 0.014, respectively), in agreement with the temporal differentiation frame of the NPM types. The group corresponding to NPM-like sequences from invertebrates also shows a monophyletic origin and shares the closest common ancestor with the NPM1 group of proteins.

TABLE 1
Average numbers of amino acid (pAA) and nucleotide (pNT) differences per site, and average synonymous (pS) and nonsynonymous (pN) differences per site in the four NPM lineages defined in Figure 1, discriminating among complete coding regions, N-terminal ...

After the differentiation of the three vertebrate NPM lineages, the early diversification of NPM2, which apparently took place at the same point in mammals and amphibians/fishes (node A in the tree), was followed by that of NPM-like from invertebrates (node B) and by NPM1 (node C) and NPM3 (node D). Interestingly, the differentiation process leading to the appearance of the three vertebrate NPM types appears to be absent in invertebrates. Nevertheless, some diversification is also present in the invertebrate NPM-like proteins, as is evidenced from their presence in protostomes (such as arthropods) and deuterostomes (such as echinoderms) (see Figure 1).

Nucleotide variation among NPM genes:

To further examine the evolutionary relationships among NPM family members, an additional phylogenetic tree was reconstructed (Figure 2) from the genomic nucleotide regions coding for the different NPM proteins analyzed in Figure 1. The two mouse NPM3 pseudogenes were also included in this analysis (MacArthur and Shackleford 1997). However, because any NPM comparison of sequences within and between species was close to or had even reached the saturation level, the nucleotide-based tree was of low reliability and in what follows we mainly focus our discussion on the protein phylogeny (Figure 1). In the tree that is based on the synonymous nucleotide differences per site, shown in Figure 2, different NPM types intersperse extensively with each other. This, together with the long branch lengths observed by this analysis, reveals that the nature of the nucleotide variation in the different NPM lineages is essentially synonymous. The level of silent variation was very similar for each of the three vertebrate lineages (NPM1, pS = 0.508 ± 0.018; NPM2, pS = 0.604 ± 0.018; NPM3 pS = 0.573 ± 0.019, Table 1) and slightly higher in the invertebrate NPM-like genes (pS = 0.682 ± 0.020). When comparing these values with the nonsynonymous differences, we found that pS was always significantly greater than pN [**P < 0.001, Z-test of selection, (Table 1)].

Figure 2.
Phylogenetic neighbor-joining tree of NPM complete nucleotide-coding sequences. The reconstruction was performed by using the number of synonymous nucleotide differences per site (pS) estimated by the modified Nei–Gojobori method (p-distance). ...

Although the nucleotide coding sequences of these proteins have diverged extensively through silent substitutions, different NPMs from the same species do not necessarily cluster together in the phylogenies on the basis of their protein sequences (Figure 1) and synonymous nucleotide substitutions (Figure 2). In general, the amount of silent variation was very high between NPM coding regions and the range of pS values was nearly the same when these regions were compared both within and between related species (supplemental Table 2 at http://www.genetics.org/supplemental/). From comparison of NPMs of different types from different vertebrate species we observed that genes from a species are not more closely related to each other than they are to NPM genes belonging to very different species of vertebrates (supplemental Table 3 at http://www.genetics.org/supplemental/). For instance, the average synonymous divergence between human NPM1 and NPM2 genes is ~0.855 ± 0.048 substitutions/site, which is significantly higher than that observed between human NPM1 and any of the other types in either human or any other vertebrates.

Importantly, the relative divergence of mouse NPM3 pseudogenes from functional NPM3 genes was estimated as 0.222 ± 0.012 nucleotide substitutions/site, which is not significantly different from the variation presented within functional NPM3 genes (0.287 ± 0.011 substitutions/site). This observation, together with the short branch lengths exhibited by pseudogenes in the nucleotide phylogeny and their clustering with mouse functional NPM3 genes, suggests that the pseudogenization events are not very ancestral, as there was not enough time to accumulate a high level of divergence.

By discriminating between the N-terminal core region and the disordered C terminus, it is possible to ascribe a significantly lower amino acid variation to the former when compared with the latter (Table 1). Conversely, the nucleotide variation is roughly the same in terms of silent variation in both domains of the molecule and always significantly greater than the nonsilent variation (**P < 0.001, Z-test of selection). Nevertheless, the nonsilent variation is significantly lower at N-terminal domains of the proteins. This suggests the presence of the strongest functional constraints in this region, which in turn is the main target of the purifying selection acting on NPM proteins. The NPM-like proteins from invertebrates appear to depart from this observation, as similar levels of protein, nucleotide, and silent and nonsilent variation are observed when comparing the complete sequence with that of the different domains of the protein (Table 1).

The nature of the nucleotide variation exhibited by sequences among different species was further analyzed by calculating the nucleotide diversity (π) and the synonymous nucleotide diversity (πS) across NPM sequences using a sliding-window approach, as shown in Figure 3. The relative contribution of πS to π is evident, as in most cases the overall amount of nucleotide variation is the result of the underlying synonymous variation. While on average the amount of πS ranges between 0.55 and 0.70 substitutions per site along the four different types of NPM sequences, a slight increase in the value of π in the case of NPM1 and NPM3 can be observed at C-terminal regions. This is most likely due to a relaxation of the structural and functional constraints in these regions of the molecules. In contrast, the inverse peaks of nucleotide diversity observed at C-terminal regions of the different NPM genes are determined by the presence of acidic tracts exclusively composed by either glutamic or aspartic acid residues, which are both encoded by twofold degenerate codons. The values of π and πS appear also to be constrained by the presence of the nuclear localization signal (NLS) in the C-terminal region of NPM2, resulting in a reduced nucleotide variation in the segment composing this element. In the case of NPM-like genes from invertebrates, the massive divergence results in a great number of indels when comparing the different sequences, which makes it very difficult to discern between different patterns of variation.

Figure 3.
Total (π) and synonymous (πS) nucleotide diversity (expressed as the average number of nucleotide difference per site) across the coding regions of NPM1 (A), NPM2 (B), NPM3 (C), and NPM-like from invertebrates (D). The diversity values ...

Amino acid frequency and nucleotide composition of NPMs:

The different members of the NPM family are characterized by glutamic and aspartic acid-rich acidic tracts located at the C-terminal regions of the molecule, which are involved in interactions with other proteins (Figure 4). The presence of selection for certain biased amino acids in the NPM lineages was first analyzed by determining the correlation coefficients between GC content and the frequency of GC-rich and GC-poor amino acids, schematized in Figure 5. In the case of the NPM1 lineage, neither the frequency of GC-rich nor that of GC-poor amino acids is correlated with the GC content in the complete molecule or discriminating in the N-terminal and the C-terminal domains (Table 2). In the case of NPM2, a significant negative correlation was observed between the GC content and the frequency of GC-rich and GC-poor amino acids when analyzing the complete molecule, as well as in the concrete case of the C-terminal domain, where a significant negative correlation is observed between the GC content and the frequency of GC-rich residues. In the case of NPM3, significant correlations were observed only in the case of the complete molecule, positive between GC content and GC-rich amino acids and negative with respect to GC-poor residues. Thus, the only case in which a significant correlation agrees with the predictions of the neutral model is the negative correlation observed between GC content and the frequency of GC-poor residues in the case of the complete NPM2 molecules.

Figure 4.
Amino acid alignments corresponding to the C-terminal segment of NPM1, NPM2, and NPM3 in different representative species showing the characteristic acidic tracts (solid boxes).
Figure 5.
Relationship between GC content and the frequencies of GC-rich (GAPW) and GC-poor (FYMINK) amino acid classes in NPM1 (A), NPM2 (B), and NPM3 (C) discriminating between the complete proteins, the N-terminal domains, and the C-terminal domains.
TABLE 2
Correlations between genomic GC content and the frequency of GC-rich (GAPW) and GC-poor (FYMINK) amino acids in complete NPM proteins and discriminating between N-terminal and C-terminal domains

While in the case of GC-rich amino acids there are no representatives found with significantly higher frequencies than the others, lysine shows high frequencies in all the NPM lineages for the case of GC-poor amino acids. Codons for lysine possess adenine at the second positions, as do the codons for glutamic acid and aspartic acid, which are the major components of the acidic tracts present in the C-terminal domains of NPM lineages. To further assess the effect of selection on these amino acids, the nucleotide frequencies for adenine at the second codon positions were compared with those presented by fourfold degenerate positions. The adenine content at second codon positions was significantly higher than the observed frequency for this nucleotide at fourfold degenerated positions (Table 3). The differences were most evident in the case of the C-terminal domain of NPMs, where the acidic tracts (rich in glutamic and aspartic acids) are present. These results suggest that selection influences nucleotide composition, especially at the C-terminal regions, maintaining high frequencies of acidic residues.

TABLE 3
Differences between the frequencies of the nucleotide adenine at second codon positions vs. the frequencies at fourfold degenerate positions in the functional domains of NPM genes

NPM codon usage bias and phosphorylation:

The presence of functional constraints at the protein level allows for a large extent of silent variation in the nucleotide sequences, resulting in a subsequent decrease in the codon bias exhibited by NPM genes. As shown in Figure 6, the overall ENC for NPM genes ranges from 49.415 ± 9.708 (for NPM-like genes of invertebrates) to 54.200 ± 4.222 (for NPM2). When discriminating between the different protein domains, the C-terminal region displays a trend that is slightly more biased than the N-terminal core, with the exception of NPM-like proteins of invertebrates. This quite unexpected observation may be related to the presence of more phosphorylatable residues at the C-terminal segments of NPM proteins, which have been shown to be critical for their correct structure and interaction with histones (Dutta et al. 2001; Arnan et al. 2003; Prado et al. 2004). The predicted phosphorylation sites and the codon usage for each residue of NPM proteins are detailed in Figure 7. Most of the potential phosphorylation targets occur at the N- or C-terminal segments, in agreement with the proposed role for this post-translational modification in unfolding this highly negatively charged segment of the molecule (Bañuelos et al. 2003; Prado et al. 2004).

Figure 6.
Codon usage bias in NPM genes. The effective number of codons (ENC) was calculated for complete NPM genes (solid circles) and discriminating between the N-terminal (open boxes) and the C-terminal (open circles) structural domains. Values are given as ...
Figure 7.
Predicted phosphorylation sites in NPM proteins. The potential phosphorylation sites at serine (S), threonine (T), and tyrosine (Y) residues (threshold >0.5) were predicted and the corresponding codon usage at these positions for NPM proteins ...

Few residues are predicted to be phosphorylated across all taxa at the same position but many positions are conserved potential targets of phosphorylation when the groups of mammals, birds, and amphibians are individually considered (see supplementary alignments 1–4 at http://www.genetics.org/supplemental/). With NPM1, all predicted phosphorylatable tyrosines are encoded by the codon TAT, and codons TCT and ACA are also used to encode most of the serines and threonines amenable to phosphorylation. The analysis of the codon usage for these three residues in NPM1 genes show that they are significantly preferred over the remaining synonymous triplets (*P < 0.05, Fisher's LSD; see supplemental Table 4 at http://www.genetics.org/supplemental/).

In the case of NPM2 and NPM3, the differences in codon usage observed between amphibians and mammals extend beyond their amino acid primary structure as shown by the protein phylogeny, resulting in the usage of different preferred codons. In mammals, AGC is the preferred codon of most of the putative phosphorylatable serines of NPM2 and NPM3, whereas TCA and TCT codons are the preferred ones in the case of amphibians. By contrast, the phosphorylatable threonines are primarily encoded by ACC in amphibian NPM2 and NPM3 proteins, while the codons ACC and ACA are preferentially used in mammals. Finally, AGC and ACC are also the preferred codons for serine and threonine, respectively, for the NPM-like proteins from invertebrates (Figure 7; supplemental Tables 5–7 at http://www.genetics.org/supplemental/).

DISCUSSION

Despite the critical functional roles of the members of the nucleophosmin/nucleoplasmin family in cell metabolism and early development of metazoan organisms, there is a lack of information regarding the mechanisms involved in the evolution of these proteins. This is mainly due to the limited amount of sequence data available until very recently. The work presented here represents a first attempt to characterize the mechanisms behind the evolutionary change of the NPM genes.

An evolutionary overview of the NPM family:

The protein phylogeny shown in Figure 1 reveals that NPM protein evolution is subject to strong functional and structural constraints leading to the diversification of the family members to accomplish a broad variety of functions in the cell nucleus, as evidenced from the clustering by type of different NPM proteins from different taxa. These types of analyses have proven to be very useful in assessing the paralogous relationship that exists between amphibian NO29 and NO38 proteins and mammalian NPM3 and NPM1 members, respectively, in agreement with what had been previously suggested by Shackleford et al. (2001). Furthermore, as a consequence of the functional diversification exhibited by these proteins along their evolution, the protein phylogenies have allowed us to clearly determine the type (NPM1, -2, or -3) of several previously unassigned sequences.

As shown by the phylogeny in Figure 1, NPM types 1 and 3 are the most closely related. Both of these types, in contrast to NPM2, are ubiquitously expressed in many tissues (Shackleford et al. 2001) and localize to the nucleolus (Huang et al. 2005). In addition, NPM1 and NPM3 form a complex in the cell and through this interaction NPM3 may help regulate NPM1's role in ribosome biogenesis (Huang et al. 2005). This suggests the potential for shared functional constraints between NPM1 and NPM3.

The phylogeny indicates that the NPM1 lineage was the one to most recently appear in the evolution of NPMs. In contrast, NPM2 seems to have been the earliest lineage, a conclusion that is further supported by the higher amino acid variation among taxa within this type. While the role played by NPM2 in histone storage in the oocyte is common for most organisms, the sperm chromatin remodeling function played after fertilization is not as obvious, mainly in the case of amphibians lacking protamines in their sperm (Frehlick et al. 2006). A possibility would involve the acquisition of this sperm chromatin remodeling function following the differentiation of specific sperm nuclear basic proteins. Although further studies are needed to clearly trace this process, the functional diversification presented by NPM2 along vertebrate evolution is in good agreement with the notion of its early origin. In contrast to the widespread expression of NPM1 and -3 across different tissue types, NPM2 in both mammals and amphibians is only expressed in oocytes and eggs. Thus, NPM2 may have differentiated early to fulfill the critical role of storing histones in oocytes and eggs.

Invertebrate NPM-like proteins seem to deviate from the differentiation/diversification process observed in vertebrate NPMs as they still retain a monophyletic origin, exhibiting some extent of similarities with the NPM1 lineage. This suggests that they are following a different evolutionary path that is also functionally constrained.

An intriguing feature of the phylogenetic protein trees is the polyphyletic origin of NPM2 and NPM3 proteins, where mammals apparently constitute a well-differentiated group. The polyphyletic origin for NPM2 and NPM3 is further supported by the maximum-parsimony reconstruction, discarding a possible dependency on the neighbor-joining method in obtaining this result. Furthermore, the codon usage shows different trends between mammals and the rest of organisms in the case of the polyphyletic lineages, which is mainly determined by the differentiation observed between mammals and amphibians. This phenomenon raises very important questions regarding the evolution of NPM2 and NPM3 that, as referred in Frehlick et al (2006), seems to play different roles in mammals compared to that played in amphibians. More specifically, the sperm chromatin remodeling function of NPM2 is not necessarily critical in mammals, as NPM2-null mice still show normal progression of the paternal chromatin remodeling immediately after fertilization (Burns et al. 2003). Otherwise, in amphibians such as X. laevis, NPM2 is required for this initial step. In addition, the downregulation of NPM3 expression in mammalian oocytes prohibits paternal chromatin condensation, suggesting that NPM3 and not NPM2 may be the family member required for decondensation of the paternal chromatin in mammals (McLay and Clarke 2003). These observations hint at a potential difference between the roles fulfilled by mammalian and amphibian NPM2 and NPM3 proteins, at least during early embryonic development, which may account for the observed polyphyly. Further studies on NPM2 and NPM3 structure and function in mammals and other organisms will be needed to better understand the reason behind the different observed functions and separate polyphyletic origins.

Although less time appears to have elapsed since the appearance of the NPM3 lineage, it shows a similar vertebrate class-dependent differentiation to that observed for NPM2. NPM3 may have differentiated following the loss of the nucleic acid binding domain found in the C terminus of NPM1, which may allow NPM3 to regulate ribosome biogenesis by regulating the binding of NPM1 to RNA (Huang et al. 2005).

Functional constraints and NPM structure:

NPM genes diverge extensively and significantly through silent substitutions (Table 1; supplemental Tables 2 and 3 at http://www.genetics.org/supplemental/), indicating that their long-term evolution is subject to a strong purifying selection process that operates at the protein level. Such a mode of evolution is also common for somatic histones and SNBPs, with the exception of protamines, which experienced a shift toward an adaptive selection process (Eirín-López et al. 2004; Nei and Rooney 2005; Eirín-López et al. 2006a,b). The NPM N-terminal core region represents the most conserved domain of the protein and contributes importantly to the structural identity of each NPM type. While the synonymous variation in the N terminus of the molecule is as high as in the less-constrained C-terminal region, the nonsynonymous variation is reduced in the N-terminal regions. The higher extent of nonsynonymous variation at the C-terminal end is probably due to the low selectivity for the occurrence of either aspartic or glutamic acid in the acidic tracts at C-terminal regions, as either residue will equally maintain the overall negative charge of the molecule, which is required for the function of this domain. The selective pressure on the maintenance of a correct protein structure can be further ascertained from the highly conserved pentameric organization of NPM1 and NPM2, which is the structural conformation required for binding to core histones (Dutta et al. 2001; Arnan et al. 2003; Namboodiri et al. 2003, 2004; Prado et al. 2004). Such quaternary structural arrangement is ultimately determined by the tertiary organization (folding) of residues constituting the N-terminal part of the NPM monomers, which contrasts with the intrinsically disordered (Hansen et al. 2005) C-terminal region (Hierro et al. 2001; Prieto et al. 2002).

While the NPM2 protein N-terminal folded regions are subject to critical structural constraints, the post-translational phosphorylation of serine, threonine, and tyrosine residues at the N-terminal exposed regions of the molecule and at the C-terminal tail are critical for the interaction of this protein with other chromosomal proteins and for chromatin remodeling (Dutta et al. 2001; Arnan et al. 2003; Prado et al. 2004). The predicted phosphorylation sites shown in Figure 7 and in the supplemental alignments 1–4 (http://www.genetics.org/supplemental/) reveal that only a few residues are potentially phosphorylated in all species at a given position for all NPM types. It appears that the phosphorylation sites in each of the three NPM lineages are specific either for mammals or for all the other vertebrate species, a fact that reflects the divergence of these molecules during the transition to mammals. The functional constraint imposed by the phosphorylation of these residues is evidenced not only by the conservation of the positions they occupy at the protein level, but also by the usage of preferred triplets, which ultimately resulted in the observed codon bias. Interestingly, the preferred codons used by mammals are also different from those preferentially used in other vertebrate classes. Hence, the nucleotide combination used to encode preferentially phosphorylatable residues appears to be taxa specific, which contrasts with the functional clustering of NPM types independent of the species to which they belong.

Furthermore, there is a codon usage heterogeneity within amphibians in organisms with different types of SNBPs. In amphibians, the nature of NPM2 appears to be independent of the SNBP type, as amphibians with SNBPs of the protamine (P) type (Bufo), protamine-like (PL) type (Xenopus), and histone (H) type (Rana) all have NPM2 proteins with similar primary structures (Frehlick et al. 2006). However, at the nucleotide level each of these amphibians use different codons in most of their potentially phosphorylatable NPM2 residues. An important exception is that of tyrosine 128 (see supplemental alignment 2 at http://www.genetics.org/supplemental/), which is encoded in all four amphibian sequences by a TAC codon. The biased codon usage conservation at this position must underlie a critically important functional constraint in the NPM2 molecule. Indeed, dephosphorylation of this residue has been shown to occur during apoptosis in Xenopus egg extracts and has been shown to regulate chromatin condensation mediated by changes in the interaction of NPM2 with chromatin and the loss of its chromatin decondensation activity (Lu et al. 2005).

While the massive synonymous nucleotide variation among NPM genes may result in a decrease of the codon usage bias, the overall effect of a preferred codon usage by phosphorylatable amino acids results in the higher codon bias that is observed at the C-terminal regions (where most of this post-translational modification takes place). Although this explanation is valid for the case of NPM1 and NPM-like lineages, further analyses are necessary in the case of NPM2 and NPM3, since the number of predicted phosphorylation sites is similar in both (N-terminal and C-terminal) protein regions. In addition, the presence of acidic tracts composed of either aspartic acid or glutamic acid residues within these regions could contribute to enhance the codon bias displayed by NPM1 and NPM3, where these two amino acids are encoded by preferred codons.

Selection for biased amino acid frequency at C-terminal regions of NPM proteins:

Under the neutral model, a protein's amino acid content is influenced by the nucleotide composition of its corresponding gene (Kimura 1983). However, the effect of selection at the protein level could alter nucleotide composition bias if it was strong enough. This was shown to occur in the case of mammalian protamine 1, in which the maintenance of arginine (essential for the DNA-binding function) at high levels can be related to the primary function of the protein, attesting to its importance (Rooney et al. 2000). A similar effect of selection for high frequencies of alanine and lysine has been described in the tolA gene from proteobacteria, in which a bias in the C + A nucleotide composition of the gene has resulted due to selection for high levels of alanine and lysine (Rooney 2003).

In the case of NPM proteins, comparisons between genomic GC content and the frequency of GC-rich and GC-poor amino acids reveal a deviation from neutrality, as no significant correlations are detected in most cases (Figure 5, Table 2). As can be ascertained from the compositional analysis of NPM proteins, it seems that high levels of lysine and, in addition, glutamic and aspartic acid, are required for the correct structure and function of NPM members. Given that codons for lysine, glutamic acid, and aspartic acid invariably show the presence of an adenine at second positions (Table 3), the frequency of this base was compared between second codon positions and fourfold degenerated positions. Our results reveal significantly higher frequencies in the case of the second codon positions, which run counter to the expectations of the neutral model (Rooney et al. 2000). The greatest differences were detected in the case of C-terminal regions, suggesting the presence of selection maintaining acidic residues at high frequencies at these domains, which are critical for the interactions of NPM proteins with other molecules through their acidic tracts. In contrast to protamines, where the overall arginine content is maintained at high levels regardless of position in the proteins (Rooney et al. 2000), the acidic tracts of NPMs are located at specific positions in C-terminal regions, indicating that in this case the underlying selection mechanisms operate under constraints determined by both the length and the position of these regions in the NPM molecules.

Long-term evolution of the NPM family:

The long-term evolution of the different members of the NPM family follows a process involving a strong purifying selection acting at the protein level, evidenced from the functional diversification of the different NPM proteins along their evolution and from the extensive silent divergence observed at the nucleotide level. The functional and structural constraints exhibited by the N-terminal core region of these proteins are determined by a primary structure that contributes to the structural identity of each NPM type. In the case of the C-terminal domains of these proteins, the constraints transcend from the protein level to the nucleotide level, as revealed by the codon usage bias observed for the acidic residues and potentially phosphorylatable residues of these regions and by the high level of adenine at second codon positions determined by the selection for high frequencies of acidic amino acids. Thus, the amino acid code involved in the post-translational modifications (phosphorylation) of NPMs represents one of the main selective constraints in these proteins, maintained not only at the protein level but also by a preferred codon usage at these positions.

Notably, the long-term evolution of NPM proteins and histones (for which at least NPM1 and NPM2 operate as chaperones) exhibit some common trends. Both groups of proteins are under a strong purifying selection at the protein primary structure level, with an extensive silent variation at the nucleotide level and the presence of a code in the post-translational modifications (yet to be established in NPMs) referred to as histone code in the case of histones (Strahl and Allis 2000). A very exciting possibility would involve a process of parallel evolution between these two groups of interacting proteins resulting in the functional diversification in some of the NPM lineages. However, more functional and structural data are still needed before this hypothesis can be tested.

Acknowledgments

We thank two anonymous reviewers and Shozo Yokoyama for useful comments on the early version of this manuscript. This work was supported by grants from Natural Sciences and Engineering Research Council of Canada (NSERC), grant number OGP 0046399 (to J.A.), by a Postdoctoral Marie Curie International Fellowship within the 6th European Community Framework Programme (to J.M.E.-L.), and by NSERC PGS-D fellowship (to L.F.).

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