Báo cáo khoa học: NARG2 encodes a novel nuclear protein with (S/T)PXX motifs that is expressed during development docx

NARG2 localizes to the nucleus in transfected cells, and deletion of a canonical basic nuclear localization signal suggests that this and other sequences in the protein cooperate for nuc

NARG2 encodes a novel nuclear protein with (S/T)PXX motifs

that is expressed during development

Naoaki Sugiura*, Vladimir Dadashev† and Roderick A Corriveau

Department of Cell Biology and Anatomy, LSU Health Sciences Center, New Orleans, LA, USA

We previously identified a partial expressed sequence tag

clone corresponding to NARG2 in a screen for genes that

are expressed in developing neurons and misexpressed in

transgenic mice that lack functional N-methyl-D-aspartate

receptors Here we report the first characterization of the

mouse and human NARG2 genes, cDNAs and the proteins

that they encode Mouse and human NARG2 consist of 988

and 982 amino acids, respectively, and share 74% identity

NARG2 does not display significant homology to other

known genes, and lower organisms such as Saccharomyces

cerevisiae, Drosophila melanogaster and Fugu rubripes

appear to lack NARG2 orthologs In vitro translation of the

mouse cDNA yields a 150 kDa protein NARG2 localizes to

the nucleus in transfected cells, and deletion of a canonical

basic nuclear localization signal suggests that this and other

sequences in the protein cooperate for nuclear targeting

NARG2consists of 16 exons in both mice and humans, 11 of which are identical in length, and alternative splicing is evi-dent in both species Exon 10 is the largest, and exhibits a much higher rate of nonsynonymous nucleotide substitution than the others In addition, NARG2 contains (S/T)PXX motifs (11 in mouse NARG2, six in human NARG2) Northern blot analysis and RNase protection demonstrated that NARG2 is expressed at relatively high levels in dividing and immature cells, and that it is down-regulated upon ter-minal differentiation The results indicate that NARG2 encodes a novel (S/T)PXX motif-containing nuclear protein, and suggest that NARG2 may play an important role in the early development of a number of different cell types Keywords: human; mouse P19 embryonic carcinoma cells; cDNA; nuclear protein; SPXX

The N-methyl-D-aspartate (NMDA) receptor, a

glutamate-gated ion channel that is permeable to Ca2+, plays an

important role in brain development by regulating neuronal

survival [1,2], migration [3], proliferation [4] and the

formation of precise neural circuits [5–7] Programs of gene

expression are also critical for brain development [8–10] In

an earlier study we used cDNA microarray analysis of mice

that lack NMDAR1, the obligatory subunit for NMDA

receptor function, to screen for genes that are abnormally

expressed in the developing brain in the absence of NMDA

receptors A group of three genes was identified (termed

NMDA receptor-regulated genes): NARG1, NARG2 and

NARG3[11] These genes lack homology with one another,

but all three are expressed at the highest levels in the

neonatal brain and fail to be appropriately down-regulated

in NMDAR1 knockout animals NARG1 (now termed

mNAT1) combines with its evolutionarily conserved cosubunit, mARD1, to form a functional acetyltransferase that may facilitate entry into the G0phase of the cell cycle [12,13] in higher animals, as it does in yeast The significance

of NARG3 is unknown, as NARG3 cDNAs corresponding

to the longest NARG3 transcript on Northern blots lack an open reading frame (N Sugiura and R Corriveau, unpub-lished observations) Here we report the cDNA sequence and exon–intron structure of the mouse and human NARG2 genes, and provide evidence that NARG2 encodes a nuclear protein that is expressed early in the development of a number of different cell types Moreover, NARG2 contains repeats of (S/T)PXX, a putative DNA-binding motif that is found in many gene regulatory proteins including Kruppel, Hunchback and Antennapedia [14] The results suggest that NARG2 is a regulatory protein that is present in the nucleus

of dividing cells and then down-regulated as progenitors exit the cell cycle and begin to differentiate

Experimental procedures

cDNA library screening Isolation of mouse NARG2 cDNA by cDNA microarray analysis originally identified NARG2, an EST (AA472833) that is expressed at higher than normal levels in the developing brain of NMDAR1 knockout mice [11] PCR primers were designed based on the sequence of this EST Because embryonic mouse brain cDNA libraries are not readily available, and because the testis expresses significant levels of NARG2 relative to other adult tissues [11], we used

Correspondence to R A Corriveau, Department of Cell Biology and

Anatomy, LSU Health Sciences Center, 1901 Perdido Street,

New Orleans, LA 70112, USA Fax: +1 504 568 4392,

Tel.: +1 504 568 2011, E-mail: rcorri@lsuhsc.edu

Abbreviations: NMDA, N-methyl- D -aspartate; EST, expressed

sequence tag; NLS, nuclear localization signal; h, human; m, mouse.

Present addresses: *Department of Biochemistry, St Marianna

University, Sugao, Miyamae-ku Kawasaki, Kanagawa, Japan;

Department of Neurological Surgery, Emory University School of

Medicine, Atlanta, GA, USA.

(Received 10 August 2004, revised 29 September 2004,

accepted 4 October 2004)

these primers to screen a mouse testis cDNA library

(Origene, Rockville, MO) for full-length NARG2 cDNA

The screen yielded two clones, clone 3H (construct

NARG2–3H) and clone 2C (construct

pCMV6-NARG2–2C) The coding region of 3H was sequenced on

both strands and the cDNA sequence was registered in the

GenBank database (accession number AY244558)

pT7-NARG2, the construct used for in vitro translation,

was generated by replacing the luciferase cDNA in

Lucif-erase T7 control plasmid DNA (Promega, Pittsburgh, PA)

with an EcoRI(blunted)/Bpu10I(blunted) NARG2 fragment

from pCMV6-NARG2–3H An epitope-tagged NARG2

construct, pCS2+MT-NARG2, was generated by

ligat-ing an EcoRI/Bpu10I(blunted) NARG2 fragment from

pCMV6-NARG2–3H into the EcoRI and SnaBI sites

of pCS2+MT [15,16] The nuclear localization signal

(NLS)-deleted NARG2 mutant construct,

pCS2+MT-NARG2DNLS, was prepared as described below A

326 bp NARG2 fragment was generated by PCR

using pCMV6-NARG2–3H as a template and the

follow-ing primers: 5¢-GCTTTTAAAACCAGTTTCCAGG-3¢,

and 5¢-GAAATTGTCTTCGCGTGGTCTCGTTTCTAC

CCT-3¢ The latter primer consists of a fusion of the

sequences from both adjacent sides of the 21 bp cDNA

encoding the NLS and therefore the resulting PCR fragment

lacks the NLS sequence This fragment was cloned into

pBluescript II SK and the sequence was verified The 200 bp

fragment containing the mutated site was excised by MscI/

BbsI digestion and used to replace the corresponding

wild-type sequence of pBS-NARG2-Pst930, which contains the

930 bp PstI fragment of pCMV6-NARG2–3H

Subse-quently, the 519 bp mutant EcoNI/SanDI fragment was

excised from the resulting plasmid and used to replace

the corresponding fragment of pCS2+MT-NARG2 to

generate pCS2+MT-NARG2DNLS

In vitrotranslation of NARG2 was performed in the

pre-sence of Redivue [35S]methionine (Amersham, Pitscataway,

NJ) using a TNT T7 Quick coupled transcription/translation

system (Promega) and pT7-NARG2 Post-translational

modifications take place in this reticulocyte-lysate based

system, including acetylation [17], isoprenylation [18],

myristolation [19,20], O-linked glycosylation [21] and

phos-phorylation [22] The translation product was resolved on by

7% SDS/PAGE and analyzed by autoradiography

Analysis of genomic and cDNA sequences

For genomic analysis we used the mouse and human

genomic database at NCBI/NIH (http://www.ncbi.nlm

nih.gov) EST and open reading frame analyses, as well as

Saccharomyces cerevisiae and Drosophila melanogaster

genomic analyses, were also carried out using the NCBI/

NIH website The Caenorhabditis elegans, Fugu rubripes

(pufferfish) and zebrafish genomic databases are available

at http://www.sanger.ac.uk, genome.jgi-psf.org, and

zfBl-astA.tch.harvard.edu, respectively Other programs used for

data analysis include nucleotide alignment, CLUSTALW

(http://www.clustalw.genome.jp); amino acid

align-ment, CLUSTALW and MULTIPLE ALIGN SHOW (http://

www.ualberta.ca/
stothard/javascript/); protein sequence

analysis,PREDICTPROTEIN (http://www.embl-heidelberg.de/

Predictprotein/); NLS analysis, (http://cubic

bioc.columbia.edu/predictnls/) Exon–intron boundaries were determined by genomic DNA and cDNA sequence comparisons, coupled with the identification of conserved GT:AG nucleotides of intron splice sites

Synonymous and nonsynonymous substitution rates between the human and mouse NARG2 cDNAs, as well

as insertions and deletions, were calculated based on the method of Nei and Gojobori [23] using the synonymous/ nonsynonymous analysis program (SNAP; http://www.hiv lanl.gov/content/hiv-db/SNAP/WEBSNAP/SNAP.html) Proline usage in Mus musculus proteins was found at http:// bioinformatics.weizmann.ac.il/blocks/help/CODEHOP/ codon.html

Cellular localization of NARG2 Rat NRK fibroblast cells (ATCC, CRL-6509) were main-tained in Dulbecco’s modified Eagle’s medium containing 5% (v/v) fetal bovine serum, and 3· 105cells were replated

on a 35 mm dish 24 h before transfection One microgram of pCS2 + MT-NARG2 DNA was transiently transfected into the cells using FuGENE6 transfection reagent (Roche, Florence, SC) The cells were fixed with 3.7% (v/v) formal-dehyde/NaCl/Pifor 10 min and then blocked and permea-bilized in 0.1% (v/v) Triton X-100 and 10% (v/v) goat serum

in NaCl/Pifor 10 min For immunostaining, 9E10 mouse anti-(c-Myc) monoclonal ascites fluid (Sigma, St Louis, MO) was used at a dilution of 1 : 1000, followed by Alexa 488-conjugated goat anti-(mouse IgG) IgG at a dilution of

1 : 1000 (Molecular Probes, Eugene, OR) Samples were examined using a Nikon Eclipse TE2000-S microscope, and acquired images were subject to analysis of average pixel intensity in the cytoplasm and the nucleus usingMETAMORPH software (Universal Imaging Corporation, Marlow, Buck-inghamshire, UK) For each cell (11 for wild-type NARG2, and 19 for NARG2 lacking the NLS), a ratio of average pixel intensity in the cytoplasm divided by the average pixel intensity in the nucleus was calculated, and statistical comparison was carried out using a two-tailed t-test The background average signal intensities were negligible com-pared to the signal in transfected cells (<1%), were not included in the calculations, and do not significantly impact upon the numbers reported Our staining protocol did not detect endogenous c-Myc protein in untransfected cells P19 cell culture and RNase protection

Mouse P19 embryonic carcinoma cell culture was carried out as described previously [13,24] Briefly, monodispersed P19 cells were seeded in bacteriological grade culture dishes (Asahi Techno Glass Corp., Funabashi, Japan) at 1· 105 cellsÆmL)1 in the presence of 1 lM retinoic acid These aggregate cultures were maintained for 4 days, trypsinized, and then replated on tissue culture dishes in the absence of retinoic acid Two days after replating the medium was replaced with fresh medium containing 5 lgÆmL)1cytosine arabinoside (Sigma); cultures were then maintained for up

to another six days for a total of eight days after retinoic acid treatment Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA) RNase protection was carried out as described previously using a NARG2 antisense probe corresponding to nucleotides 102–365 of AA472833 [11,25]

Northern blot analysis

A human poly(A)+RNA blot (OriGene HB-2010)

con-taining RNA from 12 adult tissues, and a Fetal Multiple

Human Tissue Blot (Clontech 7756–1, Palo Alto, CA), each

using 2 lg poly(A)+ RNA per lane, were processed in

parallel The intactness of the RNA samples and

equival-ence in amounts from lane to lane were verified by

denaturing gel electrophoresis with ethidium bromide

staining, and by Northern blot analysis for human b-actin

controls performed by OriGene and Clontech

Hybridiza-tion was carried out with 1· 106cpmÆmL)1of32P-labeled

cDNA probe in ULTRAhyb buffer (Ambion, Austin, TX)

at 42C for 18 h The NARG2 probe used here corresponds

to a 531 bp PstI fragment from the human NARG2 EST

AL549015 The highest stringency wash was in 0.1· NaCl/

Cit and 0.1% SDS at 42C for 15 min

Results

NARG2 cDNA and protein

EST AA472833 was originally identified by cDNA

micro-array analysis as one of a group of three independent genes

that are expressed at higher than normal levels in the

developing brain of NMDAR1 knockout mice [11] We

termed the corresponding gene mNARG2 To investigate

the significance of mNARG2 further, we screened a mouse

cDNA library and obtained two full-length clones, 2C and

3H Sequence analysis of clone 3H indicated that this clone

contains an open reading frame of 2964 bp encoding 988

amino acids with a predicted molecular mass of 109 880

(Fig 1A) The open reading frame of clone 2C lacks a

130 bp sequence that is present in 3H In vitro translation

performed using the 3H cDNA yielded a 150 kDa protein,

as determined by SDS/PAGE and autoradiography This

migration is slower than predicted, but may be explained by

post-translational modification (see Experimental

proce-dures) or the relatively high proline content of NARG2

(8.1% vs the Mus musculus average of 6.0%) High proline

levels in other proteins, for example in as1-casein B (8.6%),

have been reported to slow their migration in SDS/PAGE,

limiting the accuracy of this method for size determination

of such proteins [26]

Genomic organization of NARG2

Analysis of EST, cDNA and genomic sequences available

in public databases was performed to investigate both the

origin of the difference in sequence between mouse clones

2C and 3H, as well as to begin to evaluate the evolutionary

significance of NARG2 Human NARG2 (hNARG2)

cDNA was identified in the GenBank database

(AL832046, AK055752), and hNARG2 shares 74% amino

acid identity with mouse NARG2 (mNARG2) (Fig 1A)

However, neither mNARG2 nor hNARG2 have significant

similarity with other known genes Only one gene encoding

NARG2 was identified in both human and mouse:

mNARG2 is present on chromosome 9D, and hNARG2 is

present on chromosome 15q21.3 The NARG2 gene is

present on chromosomal regions that conserve human–

mouse synteny Three putative pseudogenes with significant

homology to NARG2 were identified in human: two are tandem repeats on chromosome 4 that correspond to 2 kb

of the 3¢ end of the cDNA, and a third is present on chromosome 3 that corresponds to the full-length cDNA All three putative pseudogenes display high nucleotide homology to NARG2 cDNA, lack open reading frames long enough to encode NARG2 (< 250 amino acids), and lack intervening sequences that would correspond to introns No NARG2 ortholog was identified in lower organisms such as S cerevisiae, C elegans, D melanogas-ter, pufferfish and zebrafish

The human and mouse NARG2 genes have highly conserved exon–intron structures including 16 exons, 11 of which are identical in size in the two species (Fig 1B, Table 1, Table 2) A comparison of cDNA and genomic sequences revealed that the 130 bp sequence present in mouse clone 3H but absent in clone 2C corresponds to exon

12, and suggests that this exon can be eliminated by alternative splicing In addition to the alternative splicing that yields the different mouse mRNAs described above, the human NARG2 gene generates an alternatively spliced mRNA that lacks both exon 4 and exon 5 (e.g BE814990) Exon 10 is considerably larger than the other exons, and most of the differences between the mouse and human gene products are localized to this exon (Fig 1A) To quantify the differences between mouse and human NARG2, non-synonymous (amino acid altering), non-synonymous (silent), and insertion/deletion nucleotide differences per codon [23] were plotted along the amino acid sequence (Fig 2) Based

on the slope, it is clear that nonsynonymous substitutions have accumulated faster in exon 10 than in the other coding exons The ratio dn/ds, a measure of the relative pressure of evolutionary selection based on the rate of nonsynonymous substitutions (dn) divided by the rate of synonymous substitutions (ds) [23,27], was calculated for exon 10 and the rest of the coding region outside of exon 10 The overall dn/ds ratio for NARG2 is < 1, indicating that, on balance, this protein is under evolutionary pressure to resist amino acid substitutions, and that it is likely to have a functional importance in multiple species However, the dn/ds ratio is higher for exon 10 (0.40) than for the other exons (0.13) As the ds value is similar for exon 10 (0.62) and the other exons (0.56), this difference in ratio is predominantly attributable

to dn, i.e nonsynonymous substitutions (exon 10, 0.25; other exons, 0.07), confirming the observation that nonsynonymous substitutions are more frequent in exon

10, and raising the possibility that parts of exon 10 are under positive selection (see Discussion)

Further examination of exon 10 revealed the presence of

a number of repeats of (S/T)PXX, a putative DNA-binding domain that is present in many transcription factors including Kruppel, Hunchback and Antennapedia [14], as well as other DNA-binding proteins [28,29] There are 11 repeats of (S/T)PXX in mNARG2 (seven in exon 10), and six in hNARG2 (four in exon 10)

Cellular localization of NARG2 hNARG2 and mNARG2 both contain a possible nuclear localization signal (NLS) that consists of a canonical stretch

of basic amino acids near the C-terminus of the protein (KKRKKIRR, amino acids 764–771 of the mouse sequence

in Fig 1A; [30]) A search of the PredictNLS database

did not reveal any other known NLS (http://cubic

bioc.columbia.edu/predictnls [31]) To determine whether

NARG2 localizes to the nucleus, and the role of the putative

NLS, we examined NRK fibroblast cells transfected with

Myc epitope-tagged NARG2 (pCS2+MT-NARG2), and

pCS2+MT-NARG2DNLS, in which the NLS has been

deleted by mutagenesis Results were visualized by

immu-nofluorescent staining using a primary antibody against

c-Myc, followed by an Alexa 488-conjugated secondary

antibody

Wild-type NARG2 localized almost exclusively to the

nucleus in most cells (Fig 3B) A few cells expressed NARG2

at abnormally high levels, in which case it was localized to the cytoplasm and excluded from the nucleus (< 20%; data not shown) This latter result may be an artefact resulting from excessive and pathological accumulation of the protein, as moderate expression of a short NLS–MT fusion protein results in nuclear localization while very high levels of expression result in exclusion from the nucleus (data not shown) Alternatively, it is possible that under biological conditions NARG2 localization may be regulated by its concentration Finally, NARG2 that lacks the NLS displays nuclear localization similar to that of wild-type, although significantly more than the typical wild-type levels of cytoplasmic NARG2 are observed (Fig 3C; ratios of

Fig 1 Human and mouse NARG2 (A) Alignment of deduced amino acid sequences of hNARG2 and mNARG2 Identities and conserved amino acid substitutions are in black and grey shaded backgrounds, respectively The most divergent region of the protein, encoded by exon 10, is indicated between arrowheads The canonical NLS is underlined (S/T)PXX repeats are indicated by asterisks (present in both mNARG2 and hNARG2), filled circles (mouse-specific), and open circles (human-specific) (B) Exon–intron structure of hNARG2 and mNARG2 Exon numbers and sizes (bp) are indicated Coding regions are indicated by filled boxes (above) or vertical bars (below).

cytoplasmic to nuclear signals were 0.37 ± 0.07 and

0.49 ± 0.10 for wild-type NARG2 and for NARG2 lacking

the NLS, respectively, P < 0.005) The results indicate that

NARG2 is usually localized to the nucleus, that the canonical

NLS plays a supporting role in nuclear localization, and that

there is probably an NLS in NARG2 that is not currently

represented in the PredictNLS database [31]

Expression of NARG2 in human tissues

Previous studies in mice demonstrated that, in the brain,

NARG2is expressed at the highest levels in neonates, and is

subsequently down-regulated [11] In the adult mouse, NARG2 is expressed at very low levels in all tissues examined, with the most appreciable levels of expression observed in the kidney, testes, liver and brain [11] Northern blot analyses demonstrate a similar expression pattern for NARG2in humans (Fig 4) Significant NARG2 expression was detected in fetal kidney, liver, lung and brain (Fig 4A) but little or no expression was observed in adult kidney, liver, brain or a number of other tissues (Fig 4B) A small amount of expression was detected in adult lung, and, as previously reported in mouse [11], significant expression was present in adult testes (Fig 4B) Taken together, these

Table 1 Exon–intron boundary sequences of the mouse NARG2 Exon sequences are shown in uppercase, introns in lowercase Exons of the same size in human and mouse are underlined.

Boundary sequences

Intron size (kb)

Table 2 Exon–intron boundary sequences of the human NARG2 Exon sequences are shown in uppercase, introns in lowercase Exons of the same size in human and mouse are underlined.

Boundary sequences

Intron size (kb)

a

Some ESTs (e.g accession AL549015) lack part of exon 4; a few ESTs lack both exon 4 and exon 5 (e.g accession BE814990).

results suggest a trend of relatively abundant NARG2 expression during development followed by low expression

in the adult that is conserved among mammals

Down-regulation of NARG2 in P19 cells undergoing neuronal differentiation

Mouse P19 embryonic carcinoma cells are multipotential cells that can be induced to exit the cell cycle and attain a neuron-like phenotype in vitro [24] Following treatment with retinoic acid under specific culture conditions, P19 cells start to express neuronal markers including glutamic acid decarboxylase, neural cell adhesion molecule, NMDA receptors and metabotropic glutamate receptors [13,32– 35] We extracted RNA from P19 cells at various stages of differentiation and used RNase protection to determine whether, as is the case in vivo (Fig 4; [11]), NARG2 is down-regulated during cellular differentiation in vitro

NARG2 is expressed at the highest levels in P19 cells before the addition of retinoic acid, and is progressively down-regulated as the cells differentiate and acquire a neuron-like phenotype (Fig 5) This pattern of expression is opposite to that found for neuronal markers For example, RNase protection analyses performed on aliquots of the P19 RNA samples used here demonstrated strong up-regulation of NMDAR1 following treatment with retinoic acid [13] Increased NMDAR1 expression with NARG2down-regulation in differentiating P19 cells concurs with the pattern of regulation of these genes during neuronal

Fig 2 Comparison of mouse and human NARG2 coding sequences

reveals a high rate of nonsynonymous nucleotide substitutions as well as

insertions and deletions in exon 10 Cumulative indexes of

synony-mous nucleotide substitutions and nonsynonysynony-mous substitutions per

codon, and insertions and deletions are plotted vs the NARG2

amino acid sequence, starting at the N-terminus of the protein The

stretch of amino acids derived from exon 10 is indicated between two

broken vertical lines The rate of synonymous substitutions remains

relatively constant in all exons, including in exon 10 Figure

gener-ated by SNAP

Fig 3 Localization of NARG2 (A) Autoradiogram of in vitro

trans-lated mouse NARG2 (lane 2), and vector without insert (negative

control) (lane 1) (B,C) Cellular localization of myc-tagged wild-type

(B, WT) and mutant (C, DNLS) mNARG2 in rat NRK fibroblast

cells Mutant mNARG2 lacks the putative nuclear localization

sequence Cells were transiently transfected with NARG2 constructs

and the proteins were visualized by immunofluorescence using a

primary antibody (anti-Myc) followed by an Alexa 488-conjugated

secondary antibody.

Fig 4 Autoradiograms of Northern blot analyses of NARG2 expres-sion in human fetal and adult tissues (A) Fetal tissues, by lane: 1, 19–23 week kidney; 2, 18–24 week liver; 3, 22–23 week lung; 4, 19–22 week brain (B) Adult tissues, by lane: 1, brain; 2, colon; 3, heart;

4, kidney; 5, liver; 6, lung; 7, muscle; 8, placenta; 9, small intestine; 10, spleen; 11, stomach; 12, testes.

development in vivo, and is consistent with NMDA receptor

function playing a role in the down-regulation of NARG2

[11] The finding that some NARG2 expression remains after

12 days of differentiation is probably due to the presence of

significant numbers of cells that do not differentiate upon

treatment with retinoic acid [24] These results provide

evidence that, as is the case in vivo [11], NARG2 expression

is inversely related to the degree of differentiation in

cell culture

Discussion

In a previous study we identified NARG2 as one of a group

of three genes, NARG1 (now referred to as mNAT1),

NARG2and NARG3, which are expressed at higher than

normal levels in the brain of NMDAR1 knockout mice [11]

We identified these genes by cDNA microarray analysis,

and all three were previously uncharacterized in vertebrates

Moreover, they share regulatory properties including high

levels of expression in the neonatal brain, dramatic

down-regulation during early postnatal development, and high

levels of expression in proliferating cells We now report that

NARG2is a novel gene that encodes a nuclear protein that is

conserved in mammals, but appears to be absent in lower

organisms NARG2 contains repeats of (S/T)PXX, a motif

present in many transcription factors as well as other

regulatory proteins that bind to DNA such as histones and

RNA polymerase II [14,28,29]

The classic monopartite NLS present near the C-ter-minus of NARG2 appears to cooperate with other regions of the protein for nuclear localization However, sequence analyses did not reveal evidence of a second typical NLS In this context, functional cloning has demonstrated a higher frequency of atypical amino acid sequences that target proteins to the nucleus than was previously appreciated [30] To evaluate fully the signifi-cance of the monopartite NLS, it will be necessary to identify other amino acids in NARG2 that participate in nuclear localization Examples of proteins that have two characterized nuclear targeting signals that contribute to nuclear localization in different ways and to different degrees include E1a [36], hnRNP K [37], and USF2 [38] These proteins all contain at least one atypical NLS, as appears to be the case for NARG2

Although NARG2 shares regulatory features with NARG1/mNAT1 and NARG3, these three genes do not share sequence homology and probably have very different functions For example, while NARG2 is a nuclear protein, NARG1/mNAT1 encodes a critical subunit of an N-terminal acetyltransferase that is localized to the cyto-plasm [13] These findings illustrate that, in contrast to cDNA screens based on sequence homology, cDNA microarray screens are driven by similarities in the regula-tion of gene expression that are not necessarily reflected by similarities in structure or function Nevertheless, groups of coregulated genes may play diverse roles in determining a specific phenotype It will be interesting to determine whether, in the absence of NMDAR1, the increased levels

of NARG1, NARG2 and NARG3 each contribute in different ways to, for example, maintaining the cell in an undifferentiated state

NARG2 as a whole is well-conserved between human and mouse, with 74% overall identity, suggesting that this protein has functional significance Of particular interest is exon 10, the largest and most divergent of the 16 exons When the amino acids from exon 10 are excluded the identity between mouse and human NARG2 rises to 86%, indicating that most of NARG2 may already be fixed for function across mammals However, the dn/ds ratio for exon 10 (0.40) is higher than that for the other exons, which have a dn/ds value (0.13) that is similar to the average of that for mouse–human 1 : 1 orthologs (0.115; [39]) In other words, a dn/ds ratio of about 0.115 is expected for mouse– human orthologs when functional domains are conserved and nonessential domains experience random drift in their amino acid composition The dn/ds ratio of 0.40 for exon 10 indicates an accumulation of nonsynonymous changes faster than in the rest of the coding sequence, and faster than would be expected by random drift This suggests that nonsynonymous substitutions are subject to positive selec-tion in exon 10 Although dn/ds for exon 10 is not high enough to be formally defined as being subject to positive selection (dn/ds > 1; [39]), we suggest that a subset of amino acids encoded by exon 10 may be under positive selection for substitutions as a result of a need for diversity

in this domain, possibly for a species-specific function The contribution to dn/ds by positively selected sites in exon 10 may be offset by other amino acids in this exon that are not under positive selection, i.e by amino acids that are randomly drifting or being actively conserved [40] A classic

Fig 5 Down-regulation of NARG2 during neuronal differentiation of

mouse P19 embryonic carcinoma cells Neuronal differentiation of P19

cells was induced with 1 l M retinoic acid The cells were harvested at

the indicated times and total RNA was extracted Single-stranded

antisense 32 P-labeled riboprobe complementary to mNARG2 was

synthesized, isolated, and hybridized with 5 lg of the indicated total

RNA sample, digested with RNases, and fractionated by gel

electro-phoresis An autoradiogram of the gel is shown, with the sizes (bp) of

the undigested probe (arrow) and the protected species (arrowhead)

indicated As a control, 2 lg aliquots of the samples used for RNase

protection were run in parallel on an ethidium bromide-stained

denaturing agarose gel That each lane contains similar amounts of

good quality total RNA is indicated by the relative signals and intact

28S and 18S ribosomal bands (inset) The t ¼ 0 sample was not treated

with retinoic acid (–RA) For additional details see Experimental

procedures Probe, undigested probe; tRNA, negative control; d, day.

example of positive selection is the class I major

histocom-patibility family of proteins, which have specific domains

that require diversity for effective immune function [27]

Olfactory receptor molecules are also under positive

selec-tion [41]

In addition to the relatively high value of dn/ds for exon

10, it contains most of the (S/T)PXX repeats found in both

mNARG2 (seven of 11) and hNARG2 (four of six) The

prevalence of this motif in NARG2 lends support to

the conclusion that it is a nuclear protein, and raises the

possibility that NARG2 is involved in regulating gene

expression [14] Future studies will address the specific role

of the (S/T)PXX motifs, and whether NARG2 is a

regulatory protein that binds to DNA

Numerous eukaryotic proteins are conserved from single

cell organisms to higher mammals However, about 22% of

vertebrate proteins do not have obvious homologues in

lower organisms [39,42] Genes that are involved in immune

and nervous system function are particularly enriched in this

group [42] Here we propose that one such gene, NARG2,

plays a role in development Examples of vertebrate genes

not represented in lower organisms that regulate cell growth

and differentiation include dkk and krm These genes

encode proteins that functionally cooperate to block Wnt/

b-catenin signaling [43] From an evolutionary perspective it

may be significant that dkk and krm are not essential for

signaling, but rather modulate the transmission of signals

through the Wnt/b-catenin pathway by regulating proteins

that are critical for signaling We hypothesize that NARG2,

similar to the proteins encoded by dkk and krm, interacts

with and modulates the function of evolutionarily conserved

proteins The findings reported here provide the primary

characterization required for studies that will test this

hypothesis and determine the function of this novel nuclear

protein

Acknowledgements

This work was supported by a Louisiana Board of Regents Research

Competitiveness Subprogram Award to R.A.C We thank Rajan Patel

for expert technical assistance, and Dr Oliver Wessely for his careful

reading of the manuscript and helpful suggestions We are also grateful

to Dr David Turner and Stacy DeRuiter for vectors, cells, protocols

and advice, and to Dr Jeffrey Loeb, Dr Qunfang Li and Dr Kunio

Takishima for assistance with preliminary experiments.

References

1 Ikonomidou, C., Bosch, F., Miksa, M., Bittigau, P., Vockler, J.,

Dikranian, K., Tenkova, T.I., Stefovska, V., Turski, L & Olney,

J.W (1999) Blockade of NMDA receptors and apoptotic

neuro-degeneration in the developing brain Science 283, 70–74.

2 Adams, S.M., de Rivero Vaccari, J.C & Corriveau, R.A (2004)

Pronounced cell death in the absence of NMDA receptors in the

developing somatosensory thalamus J Neurosci 24, 9441–9450.

3 Komuro, H & Rakic, P (1993) Modulation of neuronal

migra-tion by NMDA receptors Science 260, 95–97.

4 Gould, E., Cameron, H.A & McEwen, B.S (1994) Blockade of

NMDA receptors increases cell death and birth in the developing

rat dentate gyrus J Comp Neurol 340, 551–565.

5 Goodman, C.S & Shatz, C.J (1993) Developmental mechanisms

that generate precise patterns of neuronal connectivity Cell 72

(Suppl.), 77–98.

6 Constantine-Paton, M & Cline, H.T (1998) LTP and activity-dependent synaptogenesis: the more alike they are, the more dif-ferent they become Curr Opin Neurobiol 8, 139–148.

7 Corriveau, R.A (1999) Electrical activity and gene expression in the development of neural circuits J Neurobiol 41, 148–157.

8 Krumlauf, R., Marshall, H., Studer, M., Nonchev, S., Sham, M.H & Lumsden, A (1993) Hox homeobox genes and regionalization of the nervous system J Neurobiol 24, 1328–1340.

9 Acampora, D & Simeone, A (1999) The TINS lecture: Under-standing the roles of Otx1 and Otx2 in the control of brain mor-phogenesis Trends Neurosci 22, 116–122.

10 Puelles, L & Rubenstein, J.L (2003) Forebrain gene expression domains and the evolving prosomeric model Trends Neurosci 26, 469–476.

11 Sugiura, N., Patel, R.G & Corriveau, R.A (2001) NMDA receptors regulate a group of transiently expressed genes in the developing brain J Biol Chem 276, 14257–14263.

12 Mullen, J.R., Kayne, P.S., Moerschell, R.P., Tsunasawa, S., Gri-bskov, M., Colavito-Shepanski, M., Grunstein, M., Sherman, F.

& Sternglanz, R (1989) Identification and characterization of genes and mutants for an N-terminal acetyltransferase from yeast EMBO J 8, 2067–2075.

13 Sugiura, N., Adams, S.M & Corriveau, R.A (2003) An evolu-tionarily conserved N-terminal acetyltransferase associated with neuronal development J Biol Chem 278, 40113–40120.

14 Suzuki, M (1989) SPXX, a frequent sequence motif in gene reg-ulatory proteins J Mol Biol 207, 61–84.

15 Turner, D.L & Weintraub, H (1994) Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate Genes Dev 8, 1434–1447.

16 Rupp, R.A., Snider, L & Weintraub, H (1994) Xenopus embryos regulate the nuclear localization of XMyoD Genes Dev 8, 1311– 1323.

17 Palmiter, R.D., Gagnon, J & Walsh, K.A (1978) Ovalbumin: a secreted protein without a transient hydrophobic leader sequence Proc Natl Acad Sci USA 75, 94–98.

18 Sanford, J., Codina, J & Birnbaumer, L (1991) c-Subunits of

G proteins, but not their a- or b-subunits, are

polyisoprenylat-ed Studies on post-translational modifications using in vitro translation with rabbit reticulocyte lysates J Biol Chem 266, 9570–9579.

19 Heuckeroth, R.O., Towler, D.A., Adams, S.P., Glaser, L & Gordon, J.I (1988) 11-(Ethylthio) undecanoic acid A myristic acid analogue of altered hydrophobicity which is functional for peptide N-myristoylation with wheat germ and yeast acyl-transferase J Biol Chem 263, 2127–2133.

20 Deichaite, I., Casson, L.P., Ling, H.P & Resh, M.D (1988)

In vitro synthesis of pp60 v–src : myristylation in a cell-free system Mol Cell Biol 8, 4295–4301.

21 Starr, C.M & Hanover, J.A (1990) Glycosylation of nuclear pore protein p62 Reticulocyte lysate catalyzes O-linked N-acetylglucosamine addition in vitro J Biol Chem 265, 6868– 6873.

22 Joshi, B., Cai, A.L., Keiper, B.D., Minich, W.B., Mendez, R., Beach, C.M., Stepinski, J., Stolarski, R., Darzynkiewicz, E & Rhoads, R.E (1995) Phosphorylation of eukaryotic protein synthesis initiation factor 4E at Ser-209 J Biol Chem 270, 14597– 14603.

23 Nei, M & Gojobori, T (1986) Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide sub-stitutions Mol Biol Evol 3, 418–426.

24 Rudnicki, M.A & McBurney, M.W (1987) Cell culture methods and induction of differentiation of embryonal carcinoma cell lines In Teratocarcinomas and Embryonic Stem Cells:

a Practical Approach (Robertson, E.J., ed.), pp 19–49 IRL Press, Oxford.

25 Corriveau, R.A & Berg, D.K (1993) Coexpression of multiple

acetylcholine receptor genes in neurons: quantification of

tran-scripts during development J Neurosci 13, 2662–2671.

26 Noelken, M.E., Wisdom, B.J Jr & Hudson, B.G (1981)

Estima-tion of the size of collagenous polypeptides by sodium dodecyl

sulfate-polyacrylamide gel electrophoresis Anal Biochem 110,

131–136.

27 Hughes, A.L & Nei, M (1988) Pattern of substitution at major

histocompatibility complex class I loci reveals overdominant

selection Nature 335, 167–170.

28 Suzuki, M (1990) The heptad repeat in the largest subunit of

RNA polymerase II binds by intercalating into DNA Nature 344,

562–565.

29 Lindsey, G.G & Thompson, P (1992) S (T) PXX motifs promote

the interaction between the extended N-terminal tails of histone

H2B with linker DNA J Biol Chem 267, 14622–14628.

30 Christophe, D., Christophe-Hobertus, C & Pichon, B (2000)

Nuclear targeting of proteins, how many different signals? Cell.

Signal 12, 337–341.

31 Cokol, M., Nair, R & Rost, B (2000) Finding nuclear localization

signals EMBO Report 1, 411–415.

32 Bain, G., Ramkumar, T.P., Cheng, J.M & Gottlieb, D.I (1993)

Expression of the genes coding for glutamic acid decarboxylase in

pluripotent cell lines Brain Res Mol Brain Res 17, 23–30.

33 Husmann, M., Gorgen, I., Weisgerber, C & Bitter-Suermann, D.

(1989) Up-regulation of embryonic NCAM in an EC cell line by

retinoic acid Dev Biol 136, 194–200.

34 Ray, W.J & Gottlieb, D.I (1993) Expression of ionotropic

glu-tamate receptor genes by P19 embryonal carcinoma cells.

Biochem Biophys Res Commun 197, 1475–1482.

35 MacPherson, P.A., Jones, S., Pawson, P.A., Marshall, K.C & McBurney, M.W (1997) P19 cells differentiate into glutamatergic and glutamate-responsive neurons in vitro Neuroscience 80, 487–499.

36 Standiford, D.M & Richter, J.D (1992) Analysis of a devel-opmentally regulated nuclear localization signal in Xenopus.

J Cell Biol 118, 991–1002.

37 Michael, W.M., Eder, P.S & Dreyfuss, G (1997) The K nuclear shuttling domain: a novel signal for nuclear import and nuclear export in the hnRNP K protein EMBO J 16, 3587–3598.

38 Luo, X & Sawadogo, M (1996) Functional domains of the transcription factor USF2: atypical nuclear localization signals and context-dependent transcriptional activation domains Mol Cell Biol 16, 1367–1375.

39 Mouse Genome Sequencing Consortium (2002) Initial sequencing and comparative analysis of the mouse genome Nature 420, 520– 562.

40 Sharp, P.M (1997) In search of molecular darwinism Nature 385, 111–112.

41 Gilad, Y., Segre, D., Skorecki, K., Nachman, M.W., Lancet, D & Sharon, D (2000) Dichotomy of single-nucleotide polymorphism haplotypes in olfactory receptor genes and pseudogenes Nat Genet 26, 221–224.

42 International Human Genome Sequencing Consortium (2001) Initial sequencing and analysis of the human genome Nature 409, 860–921.

43 Mao, B., Wu, W., Davidson, G., Marhold, J., Li, M., Mechler, B.M., Delius, H., Hoppe, D., Stannek, C.W., Glinka, A & Niehrs,

C (2002) Kremen proteins are Dickkopf receptors that regulate Wnt/b-catenin signaling Nature 417, 664–667.