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

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 NA RG2 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 a nd human NARG2 gen es, cDNAs a nd the p roteins
that they encode. M ouse and human NARG2 consist o f 988
and 982 amino acids, respectively, and share 74% identity.
NARG2 does not display s ign ificant 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 localizatio n signal suggests that t his and other
sequences in the protein cooperate for nuclear targeting.
NARG2 consists of 16 exons in both mice and humans, 11 of
which are ide ntical i n length, and alternative splicing is evi-
dent in both species. Exon 10 i s 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 m ouse NARG2, six in human NARG2).
Northern blot analysis and RNase protection d emonstrated
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 NAR G2 may play an important role in the
early d evelopment of a number of different cell types.
Keywords: human; mouse P19 embryonic carcinoma c ells;
cDNA; nuclear protein; SPXX.
The N-methyl-
D
-aspartate (NMDA) receptor, a glutamate-
gated ion channel that is p ermeable to Ca
2+
, plays an
important role in brain d evelopment by regulating n euronal
survival [1,2], migration [ 3], p roliferation [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 cDN A microarray analysis of m ice
that lack NMDAR1, the obligatory subunit for NMDA
receptor function, to screen for genes that are abnormally
expressed in the developing brain i n 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 w ith one another,
but all three are e xpressed at t he highest levels i n the
neonatal brain and fail to b e 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 e ntry into the G

0
phase of the cell cycle
[12,13] in higher animals, as it does in yeast. T he significance
of NARG3 is unknown, as NARG3 cDNAs corresponding
to the l ongest 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 e xon–intron structure o f the mouse and human NARG2
genes, and provide evidence that NARG2 encodes a nuclear
protein that i s 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]. T he results suggest that
NARG2 is a regulatory protein that i s present in the nucleus
of dividing cells and then down-regulated as progenitors exit
the cell c ycle and be gin to differentiate.
Experimental procedures
cDNA library screening
Isolation of mouse NARG2 cDNA by cDNA microarray
analysis originally identifie d NARG2, an EST ( AA472833)
that is expressed at higher than normal levels in the
developing brain of NMDAR1 knockout mice [11]. PCR
primers w ere d esigned based on the sequence o f t his 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], w e 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:
Abbreviations:NMDA,N-methyl-
D
-aspartate; EST, expressed
sequence tag; NLS, nuclear localization signal; h,human;m,mouse.
Present addresses: *Department of B ioc hemist ry, St. Marianna
University, Sugao, Miyamae-ku Kawasaki, Kanagawa, Japan;
Department of Neurological Surgery, Emory University School of
Medicine, Atlanta, GA, US A.
(Received 1 0 August 2004, revised 29 September 2004,
accepted 4 October 2004)
Eur. J. Biochem. 271, 4629–4637 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04414.x
these primers to screen a m ouse testis cDNA library
(Origene, Rockville, MO) for full-length NARG2 cDNA.
The screen yielded two clones, clone 3H (construct
pCMV6-NARG2–3H) and clone 2C (construct pCMV6-
NARG2–2C). The coding region of 3H was sequenced on
both strands and t he 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 lucife rase cDNA in Lucif-
erase T7 control plasmid DNA (Promega, Pittsburgh, P A)
with an Eco RI(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) NA RG2 fragment from
pCMV6-NARG2–3H into the EcoRI and SnaBI sites
of pCS2+M T [15,16]. The nuclear localization signal
(NLS)-deleted NARG2 mutant constr uct, pCS2+M T-
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 t herefore the r esulting PCR f ragment
lacks the NLS sequence. This fragment was cloned into
pBluescript I I S K and the sequence was verified. The 2 00 bp
fragment containing the m utated site was e xcised by Msc I/
BbsI digestion and used to replace the corresponding wild-
type s equence of pBS-NARG2-Pst930, which contains the
930 bp PstI fragment of p CMV6-NARG2–3H. Subse-
quently, the 519 bp mutant EcoNI/SanDI fragment was
excised f rom t he resulting plasmid and used t o r eplace
the corresponding fragment of pCS2+M T-NARG2 to
generate pCS2+MT-NARG2DNLS.
In vitro translation of NARG2 was performed in the pre-
sence o f Redivue [
35
S]methionine (Amersham, Pitscataway,
NJ) using a TNT T7 Quick coupled transcription/translation
system (Promega) and pT7-NARG2. Post-translational
modifications take place in this r eticulocyte-lysate based
system, including acetylation [17], isoprenylation [18],
myristolation [19,20], O-linked glycosylation [21] and phos-
phorylation [22]. The translation product w as 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 ( i.nlm.
nih.gov). EST and open reading frame a nalyses, as well as
Saccharomyces ce revisiae and Drosophila melanogaster
genomic analyses, were also carried out using the NCBI/
NIH website. T he Caenorhabditis e legans, Fugu rubripes
(pufferfish) and zebrafish genomic databases are available
at , genome.jgi-psf.org, and zfBl-
astA.tch.harvard.edu, respectively. Ot her p rograms u sed f or
data analysis inc lude nucleotide a lignment,
CLUSTALW
(); amino acid align-
ment,
CLUSTALW
and
MULTIPLE ALIGN SHOW
(http://
www.ualberta.ca/stothard/javascript/); protein sequence
analysis,
PREDICTPROTEIN
( />Predictprotein/); N LS analysis,
PREDICTNLS
(http://cubic.
bioc.columbia.edu/predictnls/). Exon–intron boundar ies
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
; .
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 fibrobla st cells (ATCC, CRL-6509) were main-
tained in Dulbecco’s modified Eagle’s medium containing
5% (v/v) fetal bovine serum, and 3 · 10
5
cells 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 F uGENE6 transfection reagent (Roche,
Florence, SC). The cells were fixed with 3.7% (v/v) formal-
dehyde/NaCl/P
i
for 10 min and then blocked and permea-
bilized in 0.1% (v/v) Triton X-100 and 10% (v/v) goat serum
in NaCl/P
i
for 10 m in. For immunostaining, 9E10 mouse
anti-(c-Myc) monoclonal ascites fluid ( Sigma, St Louis, MO)
was u sed at a dilution of 1 : 1000, followed by Alexa 488-
conjugated goat anti-(mouse I gG) I gG 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 t he cytoplasm and the nucleus using
METAMORPH
software (Universal Im aging Co rporation, Marlow, B uck-
inghamshire, UK). For each cell (11 for w ild-type NARG2,
and 19 for NARG2 lacking the NLS), a ratio of average pixel
intensity i n t he cytoplas m d ivided 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 p rotocol did not
detect endogenous c-Myc p rotein 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 bacterio logical grade c ulture dishes
(Asahi Techno Glass Corp., Funabashi, Japan) at 1 · 10
5
cellsÆmL
)1
in the presence of 1 l
M
retinoic a cid. These
aggregate cultures were maintained for 4 days, trypsinized,
and then replated on t issue culture dishes in the absence of
retinoic acid. Two days after replating the medium was
replaced with fresh medium containing 5 lgÆmL
)1
cytosine

arabinoside ( Sigma); c ultures 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 p robe
corresponding to nucleotides 102–365 of AA472833 [11,25].
4630 N. Sugiura et al.(Eur. J. Biochem. 271) Ó FEBS 2004
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 Alt o, C A), each
using 2 lg poly(A)
+
RNA p er lane, w ere p rocessed in
parallel. The intactness of the RNA samples and equival-
ence in amounts from l ane 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 · 10
6
cpmÆmL
)1
of
32
P-labeled
cDNA probe in ULTRAhyb buffer (Ambion, Austin, TX)
at 4 2 °Cfor18 h.TheNARG2 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 42 °C for 15 min.
Results
NARG2 cDNA and protein
EST AA472833 was originally identified by cDNA micro-
array a nalysis 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 m NARG2 further, we screened a mouse
cDNA library and obtained two full-length clones, 2C and
3H. S equence analysis of c lone 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. 1 A). The open reading frame of clone 2C lacks a
130 b p s equence 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 p redicted, but may b e explained b y
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 a
s1
-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 w as performed to investigate both t he
origin of the d ifference i n sequence b etween mouse c lones
2C and 3H, a s well as to begin to e valuate the evolutionary
significance of N ARG2. Human NARG2 (hNARG2)
cDNA was identified in the GenBank database
(AL832046, AK055752), a nd hNA RG2 shares 74% amino
acid identity with mouse NARG2 (mNARG2) (Fig. 1A).
However, neither mNARG2 nor hNA RG2 have significant
similarity with other known genes. Only one gene encoding
NARG2 was identified in both human and mouse:
mNAR G2 is p resent on chromosom e 9D, a nd hNARG2 is
present on chromosome 15q21.3. The NARG2 gene is
present o n 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 c orrespond to 2 kb
of the 3¢ end of the cDNA, and a third is present o n
chromosome 3 that corresponds to th e full-length cDNA.
All three putative pseudogenes display high nucleotide
homology to NARG2 cDNA, lack open reading frames
long enough to encode NARG2 (< 2 50 amino acids), and
lack intervening sequences that would correspond to
introns. N o NARG2 ortholog was identified in lower
organisms such as S. cerevisiae, C. elegans, D. m elanogas-
ter, pufferfish and zebrafish.
The human and mouse NAR G2 genes have highly
conserved exon–intron structures including 16 exons, 11 of
which are identical in size i n t he 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 bu t a bsent i n clone 2C corresponds to exon
12, and suggests that this exon can be eliminated by
alternative splicing. In addition to the alte rnative splicing
that yields the d ifferent mouse mRNAs described a bove, the
human NARG2 gene generates an a lternatively spliced
mRNA that lacks bot h exon 4 and e xon 5 (e.g. BE814990).
Exon 10 is considerably larger than the other exons, and
most of the d ifferences between the mouse a nd human gene
products are localized to this exon (Fig. 1A). To quantify
the differences between mouse and human NARG2, non-
synonymous (amino acid altering), synonymous (silent),
and insertion/deletion nucleotide d ifferences per c odon [23]
were plotted along the a mino acid sequence (Fig. 2). Based
on the slope, it i s clear that nonsynonymous substitutions
have accumulated faster in exon 10 than i n 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. T he overall
dn/ds ratio for NARG2 is < 1, indicating that, on balance,
this protein i s under evolutionary pressure to r esist amino
acid substitutions, a nd that it is likely to h ave a functional
importance in multiple species. However, the dn/ds ratio is
higher for exon 10 (0.40) than f or the other exons (0.13). As
the ds value is similar for exon 10 (0.62) and the other e xons
(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, a nd raising the possibility that parts of e xon 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 presen t in many transcription factors
including Kruppel, Hunchback and Antennapedia [14], as
well as other DNA-binding proteins [28,29]. There are 11
repeatsof(S/T)PXXinmNARG2(seveninexon10),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 c anonical stretch
of basic amino acids near the C-terminus of the protein
(KKRKKIRR, amino acids 764–771 of the mouse sequence
Ó FEBS 2004 NARG2, a novel nuclear (S/T)PXX protein (Eur. J. Biochem. 271) 4631
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 l ocalizes to the nucleus, and the role o f 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 u sing a p rimary antibody against
c-Myc, followed by a n Alexa 488-conjugated secondary
antibody.
Wild-type NARG2 localized almost exclusively to the
nucleus in mostcells (Fig. 3B). A few cells expressed NARG2
at abnormally high levels, in which case it was localized to the

cytoplasm a nd excluded f rom the nucleus (< 20%; data not
shown). This latter result m ay be an artefact resulting from
excessive and pathological accumulation of the protein, a s
moderate expression of a short NLS–MT fusion protein
results in nuclear localization w hile 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 a nd mouse N ARG2. (A) A lignmen t of dedu ced amino ac id sequences of h NARG2 and mN ARG2 . Identities and conserved amino
acid substitutions are in black and grey sh aded back grounds, resp ectively. T he most divergent regio n of the protein, e ncoded by exon 1 0, is indicated
between arrowheads. The cano nical NLS is und erlined. (S/T)PX X repeats are ind icated by asterisks (p resent in both mN ARG2 and hNA RG2),
filled circles (mouse-specific), and o pen circles (human-specific). (B) Exon–intron structure of hNARG2 and mNARG2. Exon n umbers and sizes ( bp)
are indicated. Coding regions are indicated by fi lled boxes (above) or vertical bars (below).
4632 N. Sugiura et al.(Eur. J. Biochem. 271) Ó FEBS 2004
cytoplasmic to nucle ar signals w ere 0.37 ± 0.0 7 and
0.49 ± 0.10 for wild-type NARG2 and for N ARG2 lacking
the NLS, respectively, P < 0.005). The results indicate that
NARG2 is usually locali zed to t he nucleus, t hat the canonical
NLS p lays a s upporting role in nuclear localization, and that
there is p robably an N LS 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,
NARG2 is expressed at the highest levels in neonates, and is
subsequently down-regulated [11]. In the adult mouse,
NARG2 is expressed at v ery low levels in all t issues

examined, with the most appreciable levels of expression
observed in the kidney, testes, liver and brain [11]. N orthern
blot analyses demonstrate a similar e xpression pattern for
NARG2 in humans (Fig. 4). S ignificant NARG2 expression
was d etected in fetal k idney, 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 e xpression w as
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 mo use are underlined.
No. Exon size (bp)
Boundary sequences
Intron size (kb)
5¢ boundary 3¢ boundary
1>79
CCTGAG gt gggc … cttc ag TTAACT 0.89
2 142
GAACTG gt gagt … ccac ag GGATGT 1.4
3
105
a
TAACAG gt aata … ttcc ag ACGTAT 6.5
4 250 TATCTG gt atgt … taac ag GATATG 0.89
5
120 ACGGAG gt aaaa … tccc ag CAACTC 1.7
6
138 GTTTTG gt aaga … attt ag GGCAGT 0.52
7

117 CACTGT gt aagt … tttc ag GATATT 0.42
8
160 TGGAAG gt ttgt … ttta ag GTTCCA 0.74
9
182 GTGGAC gt atgt … tcca ag ATGCCC 3.0
10 1024
AAGGAC gt aagt … ttca ag GATTGC 0.57
11
176 AGAAGA gt aagt … ttgc ag CAATTT 4.5
12
130 ATGTTG gt gagt … tttt ag GGCATA 3.9
13
85 ACTCAA gt aagg … taat ag GATTTC 1.1
14
51 ATGTAG gt aagt … atgc ag CTTGCA 1.4
15
259 GCAAAG gt aaga … tcta ag GTTGGT 3.5
16 >600
Table 2. Exon–intron boundary sequences of th e hum an NARG2 . Exon sequen ces a re shown in u pperca se, introns in l owercase. Exons of the same
size in human and mo use are underlined.
No. Exon size (bp)
Boundary sequences
Intron size (kb)5¢ boundary 3¢ boundary
1 >120
CATGAG gt gggc … actc ag CTGAGT 0.93
2 133
GAATTG gt gagt … tcac ag GGATAT 1.8
3
105 CAACAG gt aata … ttcc ag ACGTAT 7.7
4

262
a
TACTTG gt atgt … aatc ag GATATG 1.3
5
120
a
ACAGAG gt aaaa … tctc ag AAAATT 9.9
6 138 GCATTG gt aagg … attt ag GGCAGT 1.2
7
117 CATTAT gt aagt … tttc ag GATATT 0.17
8
160 TTGCAG gt ttgt … ttta ag GTTCAA 1.2
9
182 ATGGAC gt atgt … cata ag ATGTCC 3.8
10 994
AAGGAC gt aagt … ttaa ag GATTGC 0.70
11
176 AGAAGA gt aagt … ttgc ag CAATTT 5.4
12
130 ATGTTG gt aagt … tttt ag GGCATA 6.3
13
85 ACTCAA gt aaga … tatc ag GATTTC 4.2
14
51 AAGTAG gt aagt … attt ag CTTGCA 3.2
15
259 CAAAAG gt aaga … tctt ag ATTGGT 4.7
16 >640
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).
Ó FEBS 2004 NARG2, a novel nuclear (S/T)PXX protein (Eur. J. Biochem. 271) 4633

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 c ycle and attain a
neuron-like phenotype in vitro [24]. Following treatment
with retinoic acid u nder s pecific culture c onditions, 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 a nd used RNase protection t o 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). T his p attern of expression is
opposite t o that found for neuronal markers. For example,
RNase protection analyses performed on aliquots of the
P19 R NA samples used here demonstrated strong
up-regulation of NMDAR1 following treatment with
retinoic acid [13]. Increased NMDAR1 expression with
NARG2 down-regulation in differentiating P19 cells concurs
with the pattern of regulation of thes e 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 subs titutions and non syn onymous su bstitutions p er
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. Th e rate of synonymous sub stit utions remains
relatively c onstant in all exons , i ncluding in exon 10. Figure gener-
ated by
SNAP
.
Fig. 3. Localization o f 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 nu clear localizatio n
sequence. Cells were tra nsie ntly trans fected with N ARG2 con structs
and the proteins were visualized by immunoflu orescence using a
primary antibody (anti-Myc) followed by an Alexa 488-conjugated
secondary antibody.
Fig. 4. Autoradiograms of Northern blot analyses o f NARG2 expres-
sion in human fetal and adult tissues. (A) Fetal tissues, by lane: 1,
19–23 week kidney; 2 , 18–24 week l iver; 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,smallintestine;10,
spleen; 11, stomach; 12, testes.
4634 N. Sugiura et al.(Eur. J. Biochem. 271) Ó FEBS 2004
development in viv o, a nd is consistent with NMDA receptor
function playing a role in the down-regulation of NARG2
[11]. T he finding that some NARG 2 expression re mains 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, a s is t he case in vivo [11], NARG2 expression
is inversely related to the degree of differentiation in
cell culture.
Discussion
In a p revious study we identified NARG2 as one of a group
of three genes, NARG1 (now referred to as mNAT1),
NARG2 and 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 w ere previously uncharacterized in vertebrates.
Moreover, they share regulatory properties including high
levels of expression in the neonatal b rain, d ramatic down-
regulation during early postnatal development, and high
levels of expression in proliferating c ells. We now report that
NARG2 is a novel gene that encodes a nuclear protein that is
conserved in mammals, but appears t o be absent in lower
organisms. NARG2 contains repeats of ( S/T)PXX, a motif
present in many t ranscription factors as well as other
regulatory proteins that bind to DNA such as histones and
RNA polymerase II [14,2 8,29].
The classic monopartite N LS present n ear 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 t he monopartite N LS, i t 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 i n different ways and to different
degrees include E1a [36], hnRNP K [37], an d 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 sequen ce 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 p lay diverse roles i n determining a
specific phenotype. It will be interesting to d etermine
whether, in the absence of NMDAR1, t he increased le vels
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 m ouse, with 74% overall identity, suggesting t hat 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 a re excluded the
identity between mouse a nd human NARG2 rises to 86%,

indicating that mos t 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 e xons, which
have a dn/ds value (0.13) that is similar to the average of
that for m ouse–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 nones sential domains experience random drift in their
amino acid c omposition. The dn/ds ratio o f 0 .40 f or exon 10
indicates an a ccumulation of nonsynonymous changes
faster than in the rest of the coding sequence, and faster
than would b e expected by random drift. This suggests that
nonsynonymous su bstitutions are subject to positive selec-
tion in exon 10. Although dn/ds for exon 10 is not high
enough to be formally defined a s b eing subject to positive
selection ( dn/ds > 1; [39]), w e s uggest that a subset of
amino acids encoded by exon 10 may be under positive
selection for substitution s 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. b y amino acids that are
randomly drifting or being actively conserved [40]. A classic
Fig. 5. Down-regulation of NARG2 during n eu rona l differentiation of
mouse P19 embryonic carcinoma cells. Neuronal differe ntiation o f P19
cells was induced with 1 l
M
retinoic acid. T he cells were h arvested at
the indicated times and total RNA was extracted. Single-stranded
antisense

32
P-labeled riboprobe complementary to mNARG2 was
synthesized, isolated , a nd hybridiz ed with 5 lg of the in dicated t otal
RNA sample, digested with RNases, and fractionated by gel electro-
phoresis. An auto radiogram of th e g el is sho wn, with the sizes (bp) of
the undigested probe (arrow) an d the protected species (arrowhead)
indicated. As a control, 2 lg a liquots of the samples used for RNase
protection were run in parallel o n an e th idium bromide-stained
denaturing agarose gel. That e ach lane c ontains similar a mounts of
good quality total RNA is indicated by the r elative signals and i ntact
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 , und igested p robe; t RNA, negative c on trol; d, d ay.
Ó FEBS 2004 NARG2, a novel nuclear (S/T)PXX protein (Eur. J. Biochem. 271) 4635
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 a lso und er 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 n ervous 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 i n lower organ isms that r egulate c ell g rowth
and differentiation include dkk and krm. These genes
encode proteins that functionally cooperate to block Wnt/
b-c atenin signaling [43]. From an e volutionary 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-cateninpathwaybyregulatingproteins
that are critical for signaling. We hypothesize that NA RG2,
similar to the proteins encoded by dkk and krm,interacts
with an d modulates the function o f e volutionarily 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 R ajan Patel
for expert technical assistance, and Dr Oliver Wessely for his careful
reading o f the manuscript and helpfu l s uggestions. We are also grateful
to Dr David Turner and S tacy DeRuiter for vectors, cells, protocols
and a dvice, and to Dr Jeffrey Loeb, Dr Qunfang L i and Dr Kunio
Takishima for assistance with preliminary experiments.
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