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The DNA兾RNA-binding protein MSY2 marks specific
transcripts for cytoplasmic storage in mouse male
germ cells
Juxiang Yang*, Sergey Medvedev
†
, P. Prabhakara Reddi
‡
, Richard M. Schultz
†
, and Norman B. Hecht*
§
*Center for Research on Reproduction and Women’s Health and †Department of Biology, University of Pennsylvania, Philadelphia, PA 19104;
and ‡Department of Pathology, University of Virginia Health System, School of Medicine, Charlottesville, VA 22908
Edited by Ryuzo Yanagimachi, University of Hawaii, Honolulu, HI, and approved December 20, 2004 (received for review June 30, 2004)
During spermatogenesis, male germ cells temporally synthesize
many proteins as they differentiate through meiosis and become
spermatozoa. The germ cell Y-box protein, MSY2, constituting
⬇0.7% of total protein in male germ cells, binds to a consensus
promoter element, and shows a general lack of RNA-binding
specificity. Combining immunoprecipitation and suppressive sub-
tractive hybridization, we identified populations of germ cell
mRNAs that are not bound or bound by MSY2. The former popu-
lation is enriched in cell growth and ubiquitously expressed
mRNAs, whereas the latter population is enriched for stored or
translationally delayed, male gamete-specific transcripts. Chroma-
tin precipitation assays reveal that most of the MSY2 target mRNAs
are transcribed from genes containing the Y-box DNA-binding
motif in their promoters. In transgenic mice, mRNAs encoding
exogenous GFP are directed or not directed into the MSY2-bound
fraction by promoters containing or lacking the Y-box motif,
respectively. We propose that MSY2 marks specific mRNAs in the
nucleus for cytoplasmic storage, thereby linking transcription and
mRNA storage兾translational delay in meiotic and postmeiotic male
germ cells of the mouse.
mRNA storage 兩spermatogenesis 兩transcription and translation
linkage 兩Y-box protein
During spermatogenesis, diploid spermatogonia differentiate
into meiotic spermatocytes that then transform into haploid
spermatids and species-specific shaped spermatozoa. This process
requires a precise temporal regulation of gene expression with high
levels of protein synthesis because meiotic cells increase many fold
in volume at pachytene where DNA recombination and repair
occurs. After two meiotic divisions, the haploid spermatid is also
highly active in protein synthesis as it reorganizes its nucleus and
synthesizes an axoneme and tail for the spermatozoon. Analyses of
testes cDNA libraries have detected ⬎18,000 sequence clusters with
⬎2,000 sequence clusters being specifically from germ cells (1).
Y-box proteins, consisting of variable N and C termini and a
highly conserved cold shock domain, are a family of evolution-
arily conserved DNA- and RNA-binding proteins that function
in both transcription and translation (2, 3). Y-box proteins
recognize the DNA motif, CTGATTGGC兾TC兾TAA, sequences
present in promoters of many germ cell-expressed genes. Al-
though Y-box proteins are reported to recognize specific se-
quences in vitro (4, 5), in cells, they appear to repress translation
by packaging mRNAs (4). The functions of Y-box proteins may
vary, depending on protein:RNA ratios (6). In in vitro assays,
high levels of Y-box proteins block translation (7, 8), whereas
lower amounts stimulate translation (9).
A germ cell Y-box protein, FRGY2, was first identified in
Xenopus laevis oocytes where it couples transcription and transla-
tion of maternal mRNAs derived from intron-less genes (2). This
discovery has led to the proposal that specific intronless oocyte
mRNAs are packaged as FRGY2-RNPs that allows a stable
association of FRGY2 and the mRNA in the nucleus and cytoplasm
(10). In contrast, mRNAs that require splicing of introns release
FRGY2.
In mice and humans, a number of Y-box proteins, MSY1,
MSY2a, MSY2b, and MSY4, have been identified in germ cells
(11–13). Based on sequence similarities, the mouse protein, MSY2,
and the human protein, Contrin (14), are orthologs of FRGY2.
MSY2 constitutes ⬇2% of total oocyte protein, is present in
diplotene-stage oocytes and fully grown oocytes, and is totally
degraded by the late two-cell stage, suggesting it stabilizes and兾or
regulates translation of maternal mRNAs (15). Consistent with this
proposal is that reducing MSY2 levels in mouse oocytes results in
reduced fertility (16). In the testis, MSY2 is highly abundant in both
meiotic and postmeiotic germ cells (12). In in vitro assays, FRGY2
binds to Y-box sequences in the promoter of the mouse protamine
(P)2 gene and stimulates transcription (17). The predominantly
cytoplasmic location of MSY2 in male germ cells suggests it serves
to stabilize and兾or regulate the translation of paternal mRNAs.
The abundance of MSY2 in male germ cells and its general lack
of RNA-binding specificity raise the question of how many meiotic
or postmeiotic male germ cell mRNAs avoid random translational
inactivation by MSY2, i.e., is there a mechanism to target MSY2 to
a specific population of germ cell mRNAs? We report here that
mRNAs transcribed from a Y-box promoter are preferentially
bound by MSY2, thereby linking transcription and mRNA storage兾
translational delay in male germ cells.
Materials and Methods
Polysome Gradient Fractionation and Analysis. Polysomal gradients
were prepared as described (18). RNA was purified from 20% of
each fraction and protein extracts from the remainder. Northern
blotting with
32
P-labeled P2 cDNA was used for gradient calibra-
tion. Immunoblotting was performed as described (18).
Immunoprecipitation of MSY2-Bound mRNAs. Nonpolysomal frac-
tions (tubes 2–8), containing 6 – 8 mg of adult CD-1 mouse testis
cytoplasmic protein extract, were pooled, diluted 5-fold with TBS-T
buffer (20 mM Tris䡠HCl, pH 7.4兾137 mM NaCl兾0.1% Tween
20兾0.1% Empigen BB兾protease inhibitor兾RNasin), and incubated
with Protein A agarose beads (100
lof50%slurry)for1hat4°C.
After centrifugation, the supernatants were incubated with 50
gof
anti-MSY2 (15) for 1 h and 200
l of protein A agarose beads for
4 h at 4°C. After centrifugation (400 ⫻gfor 5 min), the supernatants
were incubated with 20
g of anti-MSY2 and 60
l of Protein A
agarose beads for 2 h at 4°C. After centrifugation, the pellets were
combined, washed four times with 5 ml of TBS-T buffer, and RNA
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: SSH, suppressive subtractive hybridization; ChIP, chromatin immunopre-
cipitation; Pn, protamine n;TPn, transition protein n; RNP, ribonucleoprotein.
§To whom correspondence should be addressed at: Center for Research on Reproduction
and Women’s Health, University of Pennsylvania School of Medicine, 1310 Biomedical
Research Building II兾III, 421 Curie Boulevard, Philadelphia, PA 19104. Email: nhecht@
mail.med.upenn.edu.
© 2005 by The National Academy of Sciences of the USA
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0404685102 PNAS
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DEVELOPMENTAL
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was purified (18). Polysomal RNA was purified directly from tubes
12–18. Poly(A) mRNAs were prepared by using the Qiagen (Va-
lencia, CA) Oligotex mRNA minikit.
Suppressive Subtractive Hybridization (SSH). One microgram of
MSY2 bound and polysomal (hereafter, this fraction will be called
nonbound because it lacks MSY2) mRNAs was used for SSH with
the Clontech PCR-select cDNA subtraction kit. After two rounds
of subtractive hybridization and selective amplification of differen-
tially expressed genes, MSY2-bound and -nonbound subtracted
cDNAs were generated by forward and reverse SSH. Two hundred
clones of each cDNA library were randomly chosen for PCR
amplification.
Differential screening was carried out by using the Clontech
PCR-Select differential screening kit. PCR products were hybrid-
ized with radiolabeled probes generated from the MSY2-bound
and -nonbound subtracted cDNAs. The relative differential expres-
sion of each clone was quantified by densitometry and expressed as
the ratio of the signal of the homologous to the heterologous
subtracted probes. Clones with a differential expression ratio of ⬎5
were sequenced and subjected to a BLAST search.
Quantitative RT-PCR Analysis of Selected Genes. The specificity of
MSY2-bound and -nonbound mRNAs identified above was con-
firmed by real-time RT-PCR. All primers (see Table 3, which is
published as supporting information on the PNAS web site) were
checked by PCR to ensure they generated single bands of the
predicted size. PCR was performed by using the SYBR green PCR
master mix and the ABI 7900 HT thermal cycler at typical
amplification parameters (at 50°C for 2 min and at 95°C for 10 min,
followed by 40 cycles of 95°C, for 15 s and at 60°C for 1 min) and
differences were displayed as the cycle of threshold value for each
gene.
Chromatin Immunoprecipitation (ChIP). The ChIP assay was carried
out by using a ChIP kit (Upstate Biotechnology, Lake Placid, NY)
following the manufacturer’s protocol. Testes were incubated with
collagenase to separate seminiferous tubules (19). After washing
twice, formaldehyde (1%) was added, and the preparation was
incubated at 37°C for 20 min. After adding 0.125 M glycine to stop
the cross-linking, male germ cells were dissociated, centrifuged, and
rinsed in cold PBS containing protease inhibitor mixture (Roche
Molecular Biochemicals). The cells were then incubated at 4°C for
10 min in swelling buffer (10 mM potassium acetate兾15 mM
magnesium acetate兾0.1MTris䡠HCl,pH7.6兾protease inhibitor),
and disrupted in a Dounce homogenizer. Germ cell nuclei were
resuspended in sonication buffer (1% SDS兾10 mM EDTA兾50 mM
Tris䡠HCl, pH 8.0) and incubated on ice for 10 min. The samples
were sonicated on ice to generate DNA fragments of 200 – 800 bp.
After centrifugation, the chromatin solution was incubated with
salmon sperm DNA兾Protein A agarose beads for1hat4°C,and
anti-MSY2 (2
g) and ssDNA兾agarose beads were added to
precipitate the immune complexes. After reversing the cross-
linking by incubating at 65°C for4hinafinalconcentration of 0.2
M NaCl, the eluted immune complexes were extracted with phenol-
chloroform, and the purified DNA was analyzed by quantitative
real-time RT-PCR. The time and conditions of cross-linking are
important as variable results were obtained when cells were disso-
ciated before cross-linking, perhaps because of changes in their
physiological states.
To analyze each promoter sequence, GENOME BROWSER GATE-
WAY software was used (which can be accessed at http:兾兾
genome.ucsc.edu). Primers were designed to investigate from ⫺800
bp to their transcription start site (see Table 4, which is published
as supporting information on the PNAS web site), and sequences
around the Y-box sequence were preferentially chosen. Equal
amounts of input DNA were added before immunoprecipitation
and amplified with each sample as controls for differences in
amplification efficiencies and DNA quantities. Fold differences
were determined by comparing the ⌬⌬ cycle of the threshold of
target genes from the DNA immunoprecipitated with anti-MSY2
to controls where no antibody was added after normalization for
amplification efficiency (determined from the input DNA ampli-
fication).
Analysis of mRNA Distributions in Polysomal Gradients from Trans-
genic Mice. Adult testes extracts were fractionated and immuno-
precipitated as above. Poly(A) mRNAs from the MSY2-bound
fraction and polysomes were isolated, and, after addition of rabbit
␣
-globin mRNA (1 pg) for normalization, aliquots (1
g) were
reverse-transcribed and quantified by real-time RT-PCR.
Results
MSY2 Is Highly Abundant in Male Germ Cells and Present in Nonpoly-
somal Ribonucleoproteins (RNPs). To quantify the amount of MSY2
in testis, an enriched population of dissociated spermatogenic cells
was extracted with RIPA buffer (0.15 mM NaCl兾0.05 mM Tris䡠
HCl, pH 7.2兾1% Triton X-100兾1% sodium deoxycholate兾0.1%
SDS). By using recombinant MSY2 to create a calibration curve,
⬇0.7% of total soluble protein was MSY2 (Fig. 1). Because MSY2
is solely expressed in the meiotic and postmeiotic male germ cells
(20), this result represents a minimal estimate of its cellular
concentration. Considering its abundance and sequence-
independent binding properties, the question is raised as to how
mRNAs selectively escape MSY2 inactivation.
Immunoblotting of a polysomal testis gradient revealed MSY2
predominantly sediments in the nonpolysomal fraction (tubes 3–9)
(Fig. 2A) as seen in Nycodenz gradients (21). The gradient was
calibrated by hybridization with a P2 cDNA because the longer
form of P2 mRNA is nonpolysomal (left arrow) whereas the shorter
form is polysomal (right arrow) (18) (Fig. 2B).
Identifying Populations of mRNAs Bound or Not Bound to MSY2.
Although MSY2 is an abundant RNA-binding protein in male germ
cells, little is known of the populations of mRNAs it binds. To
determine whether immunoprecipitation could fractionate
mRNAs into MSY2-bound and -nonbound populations, real-time
RT-PCR was used to compare twice immunoprecipitated nonpoly-
somal mRNAs to polysomal mRNAs. For a successful fraction-
Fig. 1. MSY2 is abundant in male germ cells. MSY2 was quantified in an
extract from dissociated spermatogenic cells by using an affinity-purified
antibody to MSY2. Enriched populations of dissociated germ cells were ho-
mogenized in RIPA buffer that solubilizes ⬎85% of total MSY2. Recombinant
MSY2 was used as a control calibration protein.
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www.pnas.org兾cgi兾doi兾10.1073兾pnas.0404685102 Yang et al.
ation, we would expect that known targets of MSY2 such as the
protamines (5) would be enriched in the bound fraction, whereas
mRNAs expressed in somatic cells of the testis should not be bound.
The P1 and P2 mRNAs were 10-fold-enriched in the MSY2-bound
pellet, whereas the Sertoli cell-expressed clusterin mRNA and the
somatic and early germ cell-expressed Gapdh mRNA were 10-fold-
enriched in the polysomal fractions. Because MSY2 is expressed
only in germ cells, the lack of binding to clusterin mRNA indicated
that MSY2 was not binding to mRNAs adventitiously in extracts.
These experiments demonstrated the feasibility to use immunopre-
cipitation to define mRNA populations in the MSY2-bound non-
polysomal and polysomal fractions.
To identify the in vivo targets of MSY2, we combined immuno-
precipitation and SSH. cDNA inserts from randomly chosen clones
of the MSY2-bound subtracted cDNA library (MSY2-bound) and
the polysomal mRNA cDNA library (MSY2-nonbound) were
amplified for differential screening confirmation (see Table 5,
which is published as supporting information on the PNAS web site)
and sequenced. Clones of genes expressed in cell types lacking
MSY2, such as clusterin from Sertoli cells, were excluded from
analysis.
Forty-eight clones encoding MSY2-bound mRNAs and 50 clones
of MSY2-nonbound mRNAs were identified (Table 1). Although
we do not know which cell types express and whether they are
posttranscriptionally regulated for all of the 98 clones, many germ
cell-specific mRNAs known to undergo storage and translational
delays, such as P1 and 2 (22), transition proteins (TPs) 1 and 2 (23),
A kinase anchor protein 4 (24), and outer dense fiber 2 (25), were
detected in the MSY2-bound group (Table 1). In contrast, many
mRNAs in the MSY2-nonbound group, e.g., osmotic stress protein,
cyclin A2 (26), calmodulin 2 (27), lactate dehydrogenase C4 (28),
and tubulins are concomitantly transcribed and translated. Anno-
tation analysis was performed by using the DAVID database (which
can be accessed at http:兾兾apps1.niaid.nih.gov兾david) followed by
GOCHARTS, allowing classification into subsets according to biolog-
ical processes and molecular function. Groups with three or more
mRNAs were sorted and summarized (Fig. 3). Notably, 15 male
gamete-specific mRNAs were detected in the MSY2-bound
mRNAs, supporting the hypothesis that stored germ cell-specific
mRNAs are MSY2-bound (Table 1 in bold). All of the male
gamete mRNAs examined (eight) by using real-time RT-PCR were
confirmed to be enriched in MSY2 RNPs (see Supporting Text,
which is published as supporting information on the PNAS web
site). The nonbound group was enriched for mRNAs that are not
stored and often encoded constitutively expressed proteins involved
in cell growth and maintenance (Table 1 in bold), purine nucleotide
binding, nucleic acid, protein, and carbohydrate metabolism, and
protein translation (Table 1).
To confirm the differential expression we detected by SSH,
quantitative real-time RT-PCR was performed. Although we did
not assay all 98 of the mRNAs identified, of 16 mRNAs assayed, all
(11 MSY2-bound mRNAs and 5 -nonbound mRNAs) were en-
riched in the nonpolysomal and polysomal fractions, respectively
(see Fig. 5, which is published as supporting information on the
PNAS web site). Analyzing the MSY2-bound mRNAs, we detected
variations in enrichment with mRNAs encoding proteins such as
P1, P2, A kinase anchor protein 4, SP-10, and testis–brain RNA-
binding protein showing greater than a four-cycle enrichment,
whereas MSY2, MSY4, and TP2 showed a two-cycle enrichment
(see Fig. 5). Nonbound mRNAs, such as the osmotic stress protein,
cyclin A1, calmodulin 2, and lactate dehydrogenase C, showed a
four-cycle enrichment in the MSY2-nonbound fraction. Based on
these distributions, we believe the SSH cDNA libraries are distin-
guishing between populations of MSY2-bound and -nonbound
mRNAs.
Y-Box Sequences Are Present in the Promoter Regions of Genes Whose
mRNAs are MSY2-Bound. The distinct populations of MSY2-bound
and -nonbound mRNAs in male germ cells raises the question as to
why specific mRNAs are recognized by MSY2. The answer we
believe lies, in part, in the multifunctionality of MSY2. MSY2 is
both a DNA-binding protein recognizing the consensus sequence
(CTGATTGGC兾TC兾TAA) and a mostly sequence-independent
RNA-binding protein. By using GENOMATIX software, we analyzed
the Y-box sequences in 800 bp of the promoters of the genes we
identified. Analysis of 97 genes revealed a greater frequency of
Y-box sequences in the promoter regions of the MSY2-bound
mRNAs (82.9%) than in promoter regions of -nonbound mRNAs
(48.9%) (see Table 1 and Supporting Text). When we compare the
male gamete-specific mRNAs to the cell and growth and mainte-
nance mRNAs (Fig. 3), Y-box sequences are present in the pro-
moters of 12 of 15 (80%) MSY2-bound mRNAs versus 8 of 19
(42%) of the MSY2-nonbound mRNAs. Considering that we do
not know which Y-box sequences are functional in cells and many
of the Y-box containing genes may not be expressed in germ cells,
these numbers can only be interpreted as representing a trend to be
experimentally tested with additional assays (see below).
ChIP Reveals MSY2 Binds to Promoters Containing a Y-Box Sequence.
To establish which Y-boxes are bound by MSY2 in vivo,weused
ChIP to assay MSY2 binding to a representative subset of promot-
ers from Table 1 that contain or lack the consensus Y-box motif. As
positive controls, we know that there are two Y-box sequences in
the ⫺200-bp region of the P2 promoter that are functional in in vitro
assays (17) and P1 and P2 mRNAs are bound by MSY2 (5). The
promoter fragments for P1 and P2 and three other genes whose
mRNAs are bound by MSY2 (SP-10, acrosin, and GSTM5) were
significantly enriched after precipitation with anti-MSY2 (Fig. 4).
In contrast, clusterin, cyclin A2, PCNA, and DDX56, genes whose
promoters contain Y-boxes but whose mRNAs are not bound by
MSY2, were not enriched in the ChIP-MSY2 precipitate. Other
genes whose promoters lack Y boxes including lactate dehydroge-
nase C, calmodulin 2, Eif4E, Eif5A, DDX48, cystatin C, and
actin-related protein-10 were also not selectively precipitated. Of
particular note, although Sertoli cells do not express MSY2, the
promoter for clusterin, a Sertoli cell protein that contains a Y-box
sequence (at ⫺206 to ⫺192), was not preferentially precipitated by
MSY2. As a negative control, the precipitated promoters were not
immunoprecipitated with anti-HSF-3

antibodies (data not
shown). Thus, by analyzing a subset of the genes from Table 1, we
find a statistically significant in vivo association between MSY2 and
promoter regions of genes whose mRNAs are bound by MSY2,
Fig. 2. Sucrose gradient fractionation of testis RNP particles. The gradients
were fractionated into 18 tubes and divided for protein and RNA analysis (T,
top; B, bottom). (A) Immunoblot of MSY2. The open arrow indicates the
position of MSY2. (B) Northern blot of P2 mRNA. Gradients were centrifuged
for3hat100,000 ⫻grpm to maximally sediment the nonpolysomal RNPs. As
a result, most of the polysomes are near the bottom (tubes 12–18). A 60%
sucrose cushion prevented their pelleting.
Yang et al. PNAS
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February 1, 2005
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DEVELOPMENTAL
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which is consistent with MSY2 linkage of transcription and mRNA
storage in vivo.
GFP mRNA Partitions in the MSY2-Bound Fraction if It Is Transcribed
from a Y-Box Promoter. To test directly the hypothesis thataYbox
could direct an exogenous mRNA into the MSY2-bound popula-
tion of mRNAs in a functional assay, we assayed three different
lines of transgenic mice expressing GFP. Because the promoter of
acrosin contains a Y box sequence at ⫺69 to ⫺58 bp and acrosin
mRNA is bound by MSY2 (Table 1 and Supporting Text), we
analyzed transgenic mice expressing GFP from a 2.4-kb acrosin
promoter (29). Like P2 and acrosin mRNAs (data not shown), GFP
mRNA was enriched in the MSY2-bound fraction (Table 2).
Control mRNAs encoding clusterin and Gapdh were enriched in
the nonbound fractions.
To evaluate more critically the relationship between Y boxes and
Table 1. MSY2-bound兾nonbound mRNAs and promoter analysis of genes identified from immunoprecipitation and suppressive
subtractive hybridization
GenBank
accession no. MSY2-bound mRNAs Y box
GenBank
accession no. MSY2-nonbound mRNAs Y box
X14003 Protamine 1 ⫹NM㛭009828 Cyclin A2 ⫹
NM㛭008933 Protamine 2 ⫹BC005778 Proliferating cell nuclear antigen protein ⫹
NM㛭013694 TP2 ⫹NM㛭009446
␣
-Tubulin 3 ⫹
AF087517 A kinase anchor protein 4 (AKAP4) ⫹AK011136 ATP-dependent RNA helicase DDX56 ⫹
U31992 Acrosomal protein SP-10 ⫹Y00094 Ras-related YPT1 protein ⫹
D00754 Acrosin ⫹AK033235 ATP dependent RNA helicase DDX 19 ⫹
AF073954 Y-box protein MSY2 ⫹NM㛭008228 Histone deacetylase 1 ⫹
NM㛭013615 Outer dense fiber 2 (Odf2) ⫹AB023062 Actin-like 7b ⫹
BC008206 GST, M5 ⫹BC051444 Calmodulin 2 –
NM㛭009350 Testis nuclear RNA binding protein (Tnbr) ⫹BC002227 ARP10 actin-related protein 10 –
AB120716 Spergen-1 ⫹AF356520 Axonemal dynein heavy chain 8 long form –
NM㛭009407 TP1 ⫺NM㛭007628 Cyclin A1 –
AF246224 Y-box protein MSY4 – BC050769
␣
-Tubulin 7 –
BC048680 AKAP-like sperm protein ⫺BC012401 Translocating membrane protein 1 –
AB116526 Spetex-1 ⫺NM㛭009211 Actin regulator of chromatin Smarcc1 –
NM㛭007808 Cytochrome C, somatic ⫹NM㛭011568 RNA and export factor-binding protein 1 –
M62867 Y-box protein MSY1 ⫹NM㛭011401 Solute carrier family 2 member 3 –
NM㛭198623 Ubiquilin 3 ⫹NM㛭138669 Dead(Asp-Glu-Ala-Asp) box peptide 48 –
NM㛭026449 N-acetyl galactosaminyl transferase ⫹AF294327 Ran-binding protein兾karyopherin beta3 –
AK029613 Peroxiredoxin 5 ⫹BC027629 NAD(P)H dehydrogenase, quinone 2 ⫹
NM㛭009611 Actin-like 7a ⫹BC049803 Rho-interacting protein 3 ⫹
BC054413 Ubiquitin A-52 ribosomal protein fusion form1 ⫹NM㛭009840 Chaperonin subunit 8 (
)⫹
BC043124 Dipeptidylpeptidase 8 ⫹BC050927 Heat-shock 70-kDa protein 5 ⫹
BC052770 Carnitine deficiency associated gene ⫹BC016619 Pyruvate kinase, muscle ⫹
J03750 P9 DNA-binding protein ⫹BC013509 Succinate DH complex subunit B ⫹
NM㛭011462 Spindlin ⫹BC054386 Aldehyde dehydrogenase family 1 ⫹
AF294328 Ankyrin-like protein ⫹BC025481 Ribosomal protein F ⫹
AK005361 Membrane-interacting protein RSG16 ⫹BC009655 Ribosomal protein L3 ⫹
AK015685 DNA J-like protein ⫹NM㛭027444 HMG-box transcription factor BBX ⫹
BC043023 Bernardinelli–Seip congenital lipodystrophy 2 ⫹AK077878 HepA-related protein ⫹
NM㛭019464 SH3 domain GRB2-like B1 ⫹AK015901 Tubulin

-2 chain homolog ⫹
NM㛭029782 Similar to calreticulin 3 ⫹NM㛭008187 Gene trap locus 3 ⫹
AK075758 Similar to CLIP-170-related protein ⫹NM㛭054004 TBP-interacting protein 120A ⫹
AK077071 Serine protease ⫹BC016080 Ribophorin 1 ⫹
AK038289 GRAM domain-containing protein ⫹BC050797 Similar to T-complex protein 1 ⫹
AK007091 Elongation factor-like protein ⫹NM㛭011020 Osmotic stress protein –
BC034193 Oncoprotein-induced transcript 1 ⫹X04752 Lactate dehydrogenase-C –
NM㛭025356 Ubiquitin-conjugating enzyme E2D3 ⫹BC007152 Translation elongation factor 2 –
AK005938 Similar to PNG protein ⫹AK076067 Translation initiation factor 4E-like protein –
BC049963 UBX domain-containing protein 2 ⫹BC024899 Translation initiation factor 5A –
NM㛭199019 Similar to SPPL2b; presenilin-like protein 1 ⫹BC002072 Cystatin C –
NM㛭010191 Farnesyl diphosphate farnesyl transferase 1 ⫹AK013955 Mitochondrial processing peptidase

–
AK019488
␣
兾

hydrolase structure protein ⫹BC052093 Disintegrin and metalloproteinase domain 1a –
AF234179 Testis–brain RNA-binding protein (TB-RBP) – BC046233 poly(A)-binding protein, cytoplasmic 1 –
BC060971 Synaptophysin-like protein, variant 1 – NM㛭021713 Melanocyte proliferating gene –
AK014926 Hypothetical actin and actin-like protein – BC004651 GM2 ganglioside activator protein –
BC033444 1-Acylglycerol-3-phosphate O-acyltransferase 3 – BC019578 Peroxiredoxin 4 –
XM㛭356583 Similar to solute carrier family 26 * AK077968 Supernatant malic enzyme –
XM㛭135742 Similar to integral membrane transporter –
D84391 L1-repetitive element –
The genes in bold in the MSY2-bound and -nonbound groups represent male gamete-specific genes and cell growth- and maintenance-related genes,
respectively. Sequences and sites of the Y boxes are presented in Supporting Text.
*No promoter was found for the similar to solute carrier family 26 gene.
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mRNA storage, two lines of transgenic mice expressing GFP from
the same germ cell-specific SP-10 promoter (⫺408兾⫹28 and ⫺266兾
⫹28) were examined (30, 31). The SP-10 promoter has a Y-box
consensus element at ⫺338 to ⫺327, allowing us to examine the
distribution of GFP mRNA from the same promoter with or
without a Y box. GFP mRNA was enriched in the nonbound
fraction when driven by the ⫺266兾⫹28 promoter, whereas the
⫺408兾⫹28 shifted the GFP mRNA into the nonpolysomal fraction
(Table 2). This finding suggests that the subcellular translational
fate of the exogenous GFP mRNA is determined by the presence
or absence of a Y box in its promoter.
Discussion
Messenger RNA storage plays an especially important role in
regulating maternal mRNAs in oocytes. This process is also essen-
tial in meiotic and postmeiotic spermatogenic cells, because tran-
scription terminates in the early stages of haploid germ cell differ-
entiation. MSY2 is one of the most abundant DNA兾RNA-binding
proteins, constituting ⬇2% and 0.7% of total protein in oocytes
(15) and spermatogenic cells, respectively (Fig. 1). The Xenopus
ortholog of MSY2, FRGY2a兾b, functions in large RNP complexes
to stabilize, store, and suppress mRNA translation (2, 3). In mouse
testis, it is estimated that up to 75% of polyadenylated RNA is
complexed with MSY4 and MSY2 (11).
Here, we have combined immunoprecipitation with SSH to
obtain populations of mRNAs bound or not bound to MSY2. The
association of MSY2 with specific mRNAs is complex, and for
MSY2-bound mRNAs highly transient, because all are presumably
translated in later-stage cells. To minimize the complexity of
changing associations of MSY2 binding兾nonbinding as cells differ-
entiate, we compared the MSY2-bound mRNAs immunoprecipi-
tated from a nonpolysomal fraction to the mRNAs in polysomes, a
subcellular fraction with little, if any, MSY2. This comparison
provided an initial approach to the identification of MSY2 bound
mRNA and comparison with a steady-state population of unbound
mRNAs. Future studies will be needed examine the populations of
bound and nonbound mRNAs from individual populations of germ
cells as well as compare the bound兾unbound mRNAs in RNPs.
Despite the limitations of using total testes extracts and desig-
nating the polysomes as the unbound fraction, we identified a
sizable number of MSY2-bound marker mRNAs by using two
complementary but different techniques. We obtained similar
distributions of MSY2-bound and -nonbound mRNAs by using
SSH with testis extracts or by immunoprecipitation of extracts from
transgenic mice expressing GFP (Fig. 4). In both cases, many of the
mRNAs bound to MSY2 were gamete-specific meiotic and post-
Table 2. Real-time RT-PCR analysis of GFP mRNA distribution in
transgenic mice
mRNA MSY2-bound MSY2-nonbound
Acrosin-GFP-transgenic mice
Protamine 2 1 0.13 ⫾0.01
Clusterin 1 525 ⫾77.78
GAPDH 1 8.2 ⫾4.2
GFP 10.5 ⫾0.28
SP-10-GFP-transgenic mice (⫺266兾⫹28 promoter)
Protamine 2 1 0.14 ⫾0.01
Clusterin 1 189 ⫾74.95
GAPDH 1 32.45 ⫾10.67
GFP 1 1.85 ⫾0.54
SP-10-GFP-transgenic mice (⫺408兾⫹28 promoter)
Protamine 2 1 0.16 ⫾0.01
Clusterin 1 222 ⫾38.89
GAPDH 1 17.7 ⫾11.75
GFP 10.59 ⫾0.03
Quantification was performed with the ⌬⌬ cycle of threshold method
normalizing the amount of each mRNA to the MSY2-bound fraction. The bold
values indicate the GFP-enriched fractions.
Fig. 4. ChIP products were analyzed by using quantitative real-time RT-PCR.
Twenty-one-day-old CD1 mice were used to maximize ease of chromatin
sonication after DNA-protein crosslinking. Identical results were obtained
with sexually mature CD-1 mice. The promoter sequences from 16 genes
expressed in testis were analyzed. The data from three independent assays
represent the average of six determinations ⫾SEM and are presented as the
fold difference of target genes from the DNA immunoprecipitated with
anti-MSY2 compared with controls where no antibody was added (the control
level for each promoter was set as a value of 1) after normalization with input
DNA amplification. Assigning each sample a rank based on fold enrichment,
differences between ranks were analyzed by using the Kruskal–Wallis test.
The five genes marked with stars were significantly different from the others
(P⬍0.001).
Fig. 3. Clustering of mRNAs bound and nonbound to MSY2 identified with
SSH. The numbers in parentheses represent the number of mRNAs identified
in the category. Proteins with multiple functions or involved in several bio-
logical processes were counted in multiple annotation categories. Black bars,
MSY2-bound mRNAs; white bars, MSY2-nonbound mRNAs.
Yang et al. PNAS
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February 1, 2005
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vol. 102
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no. 5
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DEVELOPMENTAL
BIOLOGY
meiotic transcripts that are known to be stored and critical for germ
cell development. In contrast, many genes involved in cell growth
and general metabolism whose mRNAs are immediately translated
were not bound by MSY2. Interestingly, MSY2 protein binds to its
own mRNA, suggesting its autoregulation as seen for other RNA-
binding proteins such as
␣
-CP2 GFP mRNA (32) and PABP (33).
Immunoprecipitation and cDNA microarrays have been used to
define specific populations of mRNAs complexed with the RNA-
binding proteins, HuB (34) and
␣
-CP2 (32). Relating clusters of
structurally or functionally related mRNAs that are bound by
MSY2 to clusters recognized by other RNA-binding proteins may
provide insight into a new level, coordinating gene expression
regulation in mammalian cells.
In Xenopus egg extracts, FRGY2 facilitates in vitro transcription
from oocyte-specific promoters containing Y-box sequences (17).
Transfection of FRGY2 into somatic cells activates transcription
from the Xenopus heat shock protein 70-kDa promoter and herpes
simplex virus tk promoter, creating mRNAs masked from transla-
tion (35). Specific protein–RNA interactions and the splicing
process within the nucleus influence the fate of mRNAs in the
cytoplasm (10, 36). Overexpression of FRGY2 protein in Xenopus
oocytes selectively represses translation of mRNAs transcribed in
vivo, but does not affect translation of mRNAs microinjected into
the oocyte cytoplasm (37). Many endogenous mRNAs that show
translational repression in Xenopus oocytes are synthesized from
intronless genes (36). The relief of this translational repression by
injection of anti-FRGY2 into nuclei suggests mRNAs undergoing
splicing evade this translational repression.
We find that in vivo the mammalian germ cell Y-box protein,
MSY2, also links transcription and mRNA storage兾stabilization in
male germ cells. Many of the genes whose mRNAs are bound to
MSY2 contain Y-box DNA sequences in their promoters. The
binding of MSY2 to DNA appears selective because ChIP assays
demonstrate that transcripts from genes that contain Y boxes in
their promoters (P1 and P2, SP-10, acrosin, and GSTM5) are in
MSY2 complexes, whereas promoters from genes such as lactate
dehydrogenase, calmodulin 2, and cystatin C that lack these se-
quences are not precipitated and their mRNAs are not bound.
Moreover, although the promoters of genes such as clusterin,
PCNA, and cyclin A2 contain Y-box sequences, they are not
precipitated and their mRNAs are not bound by MSY2. Real-time
RT-PCR assays of total testis RNA from wild-type and germ
cell-less (c-kit) mice indicates that some of the genes we identified
as containing a Y-box such as aldehyde dehydrogenase (family 1)
are enriched in somatic cells of the testis (data not shown), which
is consistent with their fractionation into the MSY2-nonbound
fraction (Table 1).
Our transgenic mice studies that specifically test whether the fate
of a marker mRNA is determined by the presence兾absence of a
Y-box sequence were consistent with a direct MSY2-mediated
linkage of transcription and mRNA inactivation in mouse male
germ cells. Clearly, this is only one level of many regulatory
mechanisms controlling gene expression in the testis because many
nonbound mRNAs contain Y boxes in their promoters and a few
translationally delayed mRNAs such as TP1 lack the sequences (at
least in the first 800 bp of their promoters). Although MSY2 is
predominately cytoplasmic in germ cells, low levels are detected in
male germ cell nuclei and FRGY2 stimulates transcription from the
mouse protamine promoter in vitro (17). Moreover, the association
of the somatic Y-box protein, YB-1, with Balbiani ring pre-mRNAs
in the nucleus is maintained in the cytoplasm (38). The selective
marking by MSY2 of mRNAs transcribed from promoters con-
taining Y-box sequences helps explain how a population of mRNAs
is preferentially bound or escapes complexing with MSY2 and
provides a mechanism whereby an RNA-binding protein with
apparently low binding specificity gains specificity.
Although we demonstrate that a Y-box sequence in the promoter
of genes expressed in male germ cells can direct their transcripts
into MSY2 complexes, this is likely to be only one of many ways that
MSY2 regulates mRNA translation. In the cytoplasm, Y-box
proteins form large RNP particles that allow long-term mRNA
storage and could restrict the recruitment of mRNA to the trans-
lational machinery. Although MSY2 generally binds RNA in a
sequence-independent manner in both male and female germ cells
(21, 39), certain RNA sequences such as UCCAUCA can be
preferentially recognized by MSY2 (5) and Selex assays indicate
preferential binding to the sequence AACAUC (4). We, however,
have not detected a differential enrichment of these sequences or
any specific secondary structure in our populations of MSY2-bound
or -nonbound mRNAs. Because MSY2 appears capable of binding
most, if not all, mRNAs, perturbations in the cellular levels of
MSY2 will likely prove useful in investigating how it fine tunes its
selective binding to mRNA. Reduction of MSY2 levels in mouse
oocytes reduces fertility (16), and overexpression in mouse testis of
another Y-box protein, MSY4, severely disrupts spermatogenesis
(40). Preliminary studies suggest that a targeted deletion of the
MSY2 gene in mice causes infertility in both males and females,
providing an additional model to define the mechanism linking this
highly abundant DNA兾RNA-binding protein and selective mRNA
expression in germ cells.
We thank G. Gerton (University of Pennsylvania, Philadelphia) for
generously providing the acrosin-GFP-transgenic mice and C. Williams
for assistance with statistical analysis. This work was supported by
National Institutes of Health Grant HD44449 (to R.M.S. and N.B.H.).
1. Eddy, E. M. (2002) Recent Prog. Horm. Res. 57, 103–128.
2. Matsumoto, K. & Wolffe, A. P. (1998) Trends Cell Biol. 8, 318 –323.
3. Sommerville, J. (1999) BioEssays 21, 319 –325.
4. Bouvet, P., Matsumoto, K. & Wolffe, A. P. (1995) J. Biol. Chem. 270, 28297–28303.
5. Giorgini, F., Davies, H. G. & Braun, R. E. (2001) Mol. Cell. Biol . 21, 7010–7019.
6. Kohno, K., Izumi, H., Uchiumi, T., Ashizuka, M. & Kuwano, M. (2003) BioEssays 25, 691– 698.
7. Davydova, E. K., Evdokimova, V. M., Ovchinnikov, L. P. & Hershey, J. W. (1997) Nucleic
Acids Res. 25, 2911–2916.
8. Minich, W. B., Maidebura, I. P. & Ovchinnikov, L. P. (1993) Eur. J. Biochem. 212, 633– 638.
9. Minich, W. B. & Ovchinnikov, L. P. (1992) Biochimie 74, 477– 483.
10. Braddock, M., Muckenthaler, M., White, M. R., Thorburn, A. M., Sommerville, J.,
Kingsman, A. J. & Kingsman, S. M. (1994) Nucleic Acids Res. 22, 5255–5264.
11. Davies, H. G., Giorgini, F., Fajardo, M. A. & Braun, R. E. (2000) Dev. Biol. 221, 87–100.
12. Gu, W., Tekur, S., Reinbold, R., Eppig, J. J., Choi, Y. C., Zheng, J. Z., Murray, M. T. &
Hecht, N. B. (1998) Biol. Reprod. 59, 1266 –1274.
13. Tafuri, S. R., Familari, M. & Wolffe, A. P. (1993) J. Biol. Chem. 268, 12213–12220.
14. Tekur, S., Pawlak, A., Guellaen, G. & Hecht, N. B. (1999) J. Androl. 20, 135–144.
15. Yu, J., Hecht, N. B. & Schultz, R. M. (2001) Biol. Reprod. 65, 1260 –1270.
16. Yu, J., Deng, M., Medvedev, S., Yang, J., Hecht, N. B. & Schultz, R. M. (2004) Dev. Biol.
268, 195–206.
17. Yiu, G. K. & Hecht, N. B. (1997) J. Biol. Chem. 272, 26926–26933.
18. Yang, J., Chennathukuzhi, V., Miki, K., O’Brien, D. A. & Hecht, N. B. (2003) Biol. Reprod.
68, 853–859.
19. Wu, X. Q., Lefrancois, S., Morales, C. R. & Hecht, N. B. (1999) Biochemistr y 38, 11261–11270.
20. Oko, R., Korley, R., Murray, M. T., Hecht, N. B. & Hermo, L. (1996) Mol . Reprod. Dev. 44, 1–13.
21. Herbert, T. P. & Hecht, N. B. (1999) Nucleic Acids Res. 27, 1747–1753.
22. Kleene, K. C., Distel, R. J. & Hecht, N. B. (1984) Dev. Biol. 105, 71–79.
23. Heidaran, M. A. & Kistler, W. S. (1987) J. Biol. Chem. 262, 13309 –13315.
24. Morales, C. R., Lefrancois, S., Chennathukuzhi, V., El-Alfy, M., Wu, X., Yang, J., Gerton,
G. L. & Hecht, N. B. (2002) Dev. Biol. 246, 480– 494.
25. Morales, C. R., Oko, R. & Clermont, Y. (1994) Mol. Reprod. Dev. 37, 229 –240.
26. Ravnik, S. E. & Wolgemuth, D. J. (1996) Dev. Biol. 173, 69 –78.
27. Cruzalegui, F. H., Kapiloff, M. S., Morfin, J. P., Kemp, B. E., Rosenfeld, M. G. & Means,
A. R. (1992) Proc. Natl. Acad. Sci. USA 89, 12127–12131.
28. Kroft, T. L., Li, S., Doglio, L. & Goldberg, E. (2003) J. Androl. 24, 843– 852.
29. Nakanishi, T., Ikawa, M., Yamada, S., Par vinen, M., Baba, T., Nishimune, Y. & Okabe, M.
(1999) FEBS Lett. 449, 277–283.
30. Reddi, P. P., Flickinger, C. J. & Herr, J. C. (1999) Biol. Reprod. 61, 1256 –1266.
31. Reddi, P. P., Shore, A. N., Acharya, K. K. & Herr, J. C. (2002) J. Reprod. Immunol. 53, 25–36.
32. Waggoner, S. A. & Liebhaber, S. A. (2003) Mol. Cell. Biol. 23, 7055–7067.
33. Hornstein, E., Harel, H., Levy, G. & Meyuhas, O. (1999) FEBS Lett. 457, 209 –213.
34. Tenenbaum, S. A., Carson, C. C., Lager, P. J. & Keene, J. D. (2000) Proc. Natl. Acad. Sci .
USA 97, 14085–14090.
35. Ranjan, M., Tafuri, S. R. & Wolffe, A. P. (1993) Genes Dev. 7, 1725–1736.
36. Matsumoto, K., Wassarman, K. M. & Wolffe, A. P. (1998) EMBO J. 17, 2107–2121.
37. Bouvet, P. & Wolffe, A. P. (1994) Cell 77, 931–941.
38. Soop, T., Nashchekin, D., Zhao, J., Sun, X., Alzhanova-Ericsson, A. T., Bjorkroth, B.,
Ovchinnikov, L. & Daneholt, B. (2003) J. Cell Sci. 116, 1493–1503.
39. Yu, J., Hecht, N. B. & Schultz, R. M. (2002) Biol. Reprod. 67, 1093–1098.
40. Giorgini, F., Dav ies, H. G. & Braun, R. E. (2002) Development (Cambridge, U.K.) 129,
3669–3679.
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www.pnas.org兾cgi兾doi兾10.1073兾pnas.0404685102 Yang et al.
