Download Identification of Target Messenger RNA Substrates for the Murine

BIOLOGY OF REPRODUCTION 66, 475–485 (2002)
Identification of Target Messenger RNA Substrates for the Murine Deleted
in Azoospermia-Like RNA-Binding Protein1
Xinfu Jiao, Panayiota Trifillis,3 and Megerditch Kiledjian2
Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey 08854-8082
ABSTRACT
azoospermic males contain a deletion on the Y chromosome in a region encoding DAZ. The DAZ gene is Y chromosome linked and is present only in Old World monkeys
and hominoids. In addition, these species and all other
mammals contain an ancestral autosomal DAZ-like gene,
DAZL, which is approximately 80% identical to DAZ [4–
7]. More recently, a predecessor to the DAZL gene was also
identified [8], indicating that there are multiple members in
the DAZ family of proteins. All members of the DAZ protein family are expressed exclusively in germ cells [3, 8–
10]. The significance of DAZL in spermatogenesis was
demonstrated by a homozygous disruption of the murine
DAZL (mDAZL) gene in mice, which resulted in the absence of germ cells in both sexes [10]. Gametogenesis progressed only up to the mitotic spermatogonial stage of development, suggesting a requirement of mDAZL for the
development and survival of germ cells [10]. Similarly, the
Drosophila homologue of mDAZL, termed Boule, is essential for spermatogenesis; primary spermatocytes fail to
progress through the G2/M transition in mutant flies [11].
Expression of the human DAZ transgene in mDAZL knockout mice confers partial rescue of spermatogenesis, demonstrating an overlap in the function of both proteins [12].
Germ cells undergo spermatogenesis in association with
the somatic Sertoli cells. Sertoli cells serve as support units
for the developing germ cells and provide nutrition and
fate-determining signals [13]. Much of this influence is
thought to be mediated through direct Sertoli-germ cell
contact, which is required for spermatogenesis [14]. Several
molecules, including b1,4-galactosyltransferase [15], Ncadherin [16], and testis-specific protein 1 (Tpx-1) [17],
have been identified as candidate adhesion proteins. Antibodies to all 3 proteins inhibit Sertoli-germ cell binding to
various degrees in vitro. Chromosomal breakpoints within
the Tpx-1 gene have also been identified within infertile
men [18], further implicating a functional role for this protein in spermatogenesis. Tpx-1 is a 243-amino acid testisspecific protein expressed exclusively in germ cells and is
a member of the cysteine-rich secretory protein family [19].
The secreted protein subsequently associates with the spermatogenic cell surface [20]. Tpx-1 gene expression appears
to begin at the pachytene stage and persists throughout
spermatogenesis to the elongating spermatid stage [20–22].
Expression of the Tpx-1 protein is delayed relative to the
mRNA and is more intense in later stages of spermatogenesis, suggesting its expression is translationally regulated
[20].
A critical level of gene expression is governed by posttranscriptional regulation. Messenger RNA processing occurs in association with RNA-binding proteins within ribonucleoprotein complexes [23]. The processing events include capping at the 59 end, removal of intervening sequences by splicing, polyadenylation at the 3 9 end,
transport of the mRNA out of the nucleus, and ultimately
translation into protein [24–27]. Inherent throughout this
process is the turnover of the mRNA. All mRNAs have a
The murine autosomal deleted in azoospermia-like protein
(mDAZL) is a germ cell-restricted RNA-binding protein essential
for sperm production. Homozygous disruption of the mDAZL
gene results in the absence of germ cells beyond the spermatogonial stage. Progress into the function of DAZL in spermatogenesis has been hampered without identification of the cognate
mRNA substrates that it binds to and regulates. Using the isolation of specific nucleic acids associated with proteins (SNAAP)
technique recently developed in our lab, we identified mRNAs
from testis that were specifically bound by mDAZL. One mRNA
encoded the Tpx-1 protein, a testicular cell adhesion protein
essential for the progression of spermatogenesis. A 26-nucleotide region necessary and sufficient to bind mDAZL was found
within additional mRNAs isolated by the screen. These included
mRNA encoding Pam, a protein associated with myc; GRSF1, an
mRNA-binding protein involved in translation activation, and
TRF2, a TATA box-binding protein-like protein involved in transcriptional regulation. Each mRNA containing the mDAZL binding site was specifically bound by mDAZL. A similar sequence
is also present in the Cdc25A mRNA, a threonine/tyrosine phosphatase involved in cell cycle progression. The mDAZL and
Cdc25A homologues are functionally linked in Drosophila and
are necessary for spermatogenesis. Our demonstration that Tpx1 and Cdc25A mRNAs are bound by mDAZL suggests that
mDAZL regulates a subset of mRNAs necessary for germ cell
development and cell cycle progression. Understanding how
mDAZL regulates the target mRNAs will provide new insights
into spermatogenesis, strategies for therapeutic intervention in
azoospermic patients, and novel approaches for male contraception.
developmental biology, gene regulation, spermatogenesis
INTRODUCTION
Approximately 2% of males worldwide are affected by
infertility resulting from the absence of sperm production
(azoospermia). Cytological analysis of patients with azoospermia revealed a deletion on the Y chromosome in a significant proportion of these patients, implying a genetic
component to the phenotype in some individuals [1]. This
region was proposed to contain an azoospermia factor
(AZF). Subsequent analysis of this region revealed 3 distinct genes as AZF candidates [2], 1 of which was termed
deleted in azoospermia (DAZ) [3]. Approximately 10% of
Supported by the Johnson and Johnson Discovery Award and NIH grant
HD39744 to M.K.
2
Correspondence: Megerditch Kiledjian, Department of Cell Biology and
Neuroscience, Rutgers University, 604 Allison Rd., Piscataway, NJ 088548082. FAX: 732 445 0104; e-mail: [email protected]
3
Current address: PTC Therapeutics, South Plainfield, NJ 07080.
1
Received: 18 May 2001.
First decision: 20 June 2001.
Accepted: 27 September 2001.
Q 2002 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
475
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JIAO ET AL.
distinct half-life that is dictated by both cis elements and
trans factors [28]. The DAZ family of proteins contains a
ribonucleoprotein (RNP)-motif type of RNA-binding domain (RBD) at their amino terminus, and they bind RNA
[29–31]. Their localization to the cytoplasm and association
with polysomes suggests a functional role in mRNA stability or translational regulation [10, 29]. This suggestion
is further supported by the functional link between Boule
and Twine in Drosophila. Twine is a Cdc25 phosphatase
necessary for the G2/M transition in spermatocytes whose
translation is dependent on the presence of the Drosophila
DAZL homologue, Boule [32].
To begin addressing the components involved in spermatogenic defects arising in the absence of the DAZ family
of proteins, we set out to identify the substrate target
mRNAs bound by mDAZL. We recently described the isolation of specific nucleic acids associated with proteins
(SNAAP) approach, which employed copurification of unknown mRNAs bound by an RNA-binding protein and subsequent identification of the mRNA by reverse transcription
(RT) and polymerase chain reaction (PCR) using differential display technology [33]. We now report the identification of a testis-specific substrate mRNA bound by mDAZL
and the delineation of the mDAZL-binding sequence. The
presence of this sequence in other mouse mRNAs enabled
identification of a subset of mRNAs that are bound by and
potentially regulated by the DAZ family of proteins, including Cdc25A phosphatase. Identification of DAZLbound substrate mRNAs broadens and illustrates the potential spectrum of processes in gametogenesis regulated by
this family of proteins and provides valuable insight into
the function of this RNA-binding protein family in the pathogenesis of azoospermia.
MATERIALS AND METHODS
Preparation of Testis Total Extract
Testes were extracted from 6-wk-old Balb/C mice, washed twice in
PBS and placed into lysis buffer (20 mM Hepes, pH 7.6, 1.5 mM MgCl2,
10 mM KCl, 0.5 mM dithiothreitol [DTT], 2 mg/ml leupeptin, 0.5% aprotinin). The tissue was diced with a razor blade followed by sonication.
Insoluble matter was removed with a 5-min spin at 15 000 3 g at 48C,
and supernatant was collected, supplemented with 5% glycerol, and frozen
in aliquots at 2708C.
Generation of Glutathione S-Transferase Fusion Proteins
The pGEX-mDAZL plasmid encoding the glutathione S-transferase
(GST)-mDAZL fusion protein was constructed by placing the mDAZL
coding region into the pGEX-6P-1 vector (Pharmacia, Piscataway, NJ).
The coding region was reverse transcribed and amplified from mouse testis
RNA with primers that placed BamHI and EcoRI restriction sites on the
59 and 39 ends, respectively, and was inserted into the same sites within
the vector. The GST-aCP1 and GST-RBD expression plasmids have previously been described [33, 34]. Fusion proteins were expressed in Escherichia coli BL21 cells, and extract was prepared and treated with micrococcal nuclease to degrade bacterial RNA as previously described [33].
SNAAP Screen
The SNAAP screen was carried out as described by Trifillis et al. [33].
Micrococcal nuclease-treated GST-fusion protein (50 mg) was bound to 40
ml of glutathione beads at 48C for 15 min in a 1-ml total volume containing
RNA-binding buffer (RBB; 10 mM Tris-HCl, pH 7.5, 1.5 mM MgCl2, 250
mM KCl, 0.5 mM DTT, 2 mg/ml leupeptin, and 0.5% [v/v] aprotinin) with
0.5% Triton X-100 (RBB/0.5% TX). Unbound protein was removed with
four 1-ml washes with RBB/0.5% TX and two 1-ml washes with RBB.
The washed beads containing the fusion protein were resuspended in 350
ml of RBB and incubated with 300 mg of testis total extract precleared
with 20 ml of glutathione Sepharose beads. Binding was carried out at 48C
for 1 h followed by a rinse with RBB/0.25% TX and a 10-min wash in
RBB/0.25% TX containing 1 mg/ml heparin. The beads were subsequently
rinsed 4 times in RBB/0.25% TX and bound RNA was isolated by boiling
for 3 min in 200 ml Tris-EDTA/1% SDS as previously described [33].
Copurifying RNAs were identified by the differential display technique
(GenHunter, Nashville, TN) as previously described [33] using the 3 anchored 39 primers H-T11A, H-T11C, and H-T11G and 12 distinct 59 primers.
The 59 primers used were H-AP1 through H-AP8 (GenHunter), DAP28
(GTTTTCGCAG), DAP29 (GATCCAGTAC), DAP30 (GATCACGTAC),
and DAP31 (GATCTGACAC). The bands representing RNAs specifically
bound by mDAZL were cloned into the pGEM-T vector (Promega, Madison, WI) and sequenced.
Electrophoretic Mobility Shift Assays
Electrophoretic mobility shift assays were carried out with approximately 0.5 ng of in vitro transcribed [32P]UTP uniformly labeled RNA
(;10 000 cpm) per reaction. Binding reactions were carried out in RBB
with the indicated amount of GST-fusion protein in a 20-ml total volume.
Following a 20-min binding reaction at room temperature, 1.5 units of
RNase T1 (Roche, Indianapolis, IN) was added to the reaction and incubated for 10 min at room temperature. Addition of RNase T1 degrades
regions of the RNA not complexed with protein and enables a clearer
identification of RNA-protein complexes. Heparin is subsequently added
to a final concentration of 2 mg/ml for 10 min at room temperature to
minimized nonspecific RNA-protein interactions. Competitor RNA or oligonucleotide was added at the beginning of the reaction where indicated.
The complexes were resolved on a 5% polyacrylamide gel (60:1 acrylamide:bis) in 0.53 Tris-borate-EDTA buffer at 8 V/cm.
RT-PCR and Riboprobe Generation
Mouse Tpx-1 full-length cDNA and the 39 untranslated region (UTR)
were amplified from mouse testis RNA RT product using the Advantage
cDNA Polymerase Mix (Clontech, Palo Alto, CA) and cloned into the
pGEM-T vector (Promega) to generate pGEM-Tpx cDNA and pGEM-Tpx
39 UTR, respectively. The Tpx-1 full-length cDNA was amplified with
primers 59-CCTCCGTGAGCAACGATAACC-39 and 59-GGTAGCTTGAGTTCTTTATTGAAAG-39. The Tpx-1 3‘ UTR was amplified with 59TAACATGCCCAGTGTGCAGC-39 and 59-ATTGAAAGAAGTGATTATCTGTG-39. RNA transcripts corresponding to the 59 half of the Tpx1 cDNA were generated by T7 RNA polymerase from the pGEM-Tpx
cDNA plasmid linearized with ApaLI (fragment A). Fragments within the
39 UTR were generated by SP6 RNA polymerase from the pGEM-Tpx 39
UTR plasmid linearized with NcoI (fragment B), AvaII (fragment C), and
AseI (fragment D). Fragment E was transcribed by T7 RNA polymerase
from a PCR-generated template (59-TAATACGACTCACTATAGGGTAATATCTTTCAGAAC-39 and 59-CAATTCCAAAGTTGTTATAC-39).
Uniformly 32P-labeled riboprobes were generated and gel purified on an
8% denaturing polyacrylamide gel as previously described [35].
Gene-specific PCR amplifications in Figure 1B were carried out for
Tpx-1 with 59-TAACATGCCCAGTGTGCAGC-39 and 59-ATTGAAAGAAGTGATTATCTGG-39 and for Dmc1 with 59-TAGGTGGGGAATTGGTACAG-39 and 59-ATGCTGGATGAGAAGTCTGG-39. PCR amplifications in Figure 5 were carried out with the following primer sets: for
Pam (protein associated with myc), 59-ACACGCAGATCCTTTGTCTA39 and 59-GTTTTATGACATGTACATTC C-39; for clone D2, 59-AATCGCCCAAATGGTGACAC-39 and 59-AGCACCTCCTCGTTAAATAC39; for GRSF1 (G-rich specificity factor 1), 59-TACAGCTTTATCCACGATC-39 and 59-AGAAACTTGGCCTCCCTAAG-39; and for Trf2
(TATA box-binding protein related factor 2; also referred to as TLP or
TRP), 59-ACTCAAACTTGGTTGGTTAG-39 and 59-AACTTTTATTTGTGCACATTCAA-39. Amplification of the Cdc25A RT product was carried out with 59-GAAGTTCCGCACCAAGAGC-39 and 59-GTTAAGGTCATCCACGAGG-39. The Cdc25C RT product was amplified with 59ATGAAGCATCTGGGCAGTCC-39 and 59-GAGAACGGCACATTCGAGGG-39.
Riboprobes used in Figure 5B were transcribed with T7 RNA polymerase
from PCR-generated templates using primers that introduce a T7 promoter at
the 59 end. The primer sets are as follows: Pam, 59-TAATACGACTCACTATAGGGGTAATAAGTATCACACTGTATA-39 and 59-CTTAACCTAGAAGTTTAAATGA-39; clone D2, 59-TAATACGACTCACTATAGGGGTGGCGTGACAAGGACAG-39 and 59-CTAAATATTTCCATAAGCCTC-39;
GRSF1, 59-TAATACGACTCACTATAGGGATCACACCAAAGCAACAG
A-39 and 59-CTATCCCTAAGTAATCAAATAC-39; and Trf2, 59-TAATACGACTCACTATAGGGTAGTCTTTTACATTTTTGTATGC-39 and 59-GATCTTTTCCAAACTTTTATTTG-39.
Oligonucleotides used in the binding studies were end labeled with g-
SUBSTRATE mRNA BOUND BY mDAZL
477
32P-dATP using T4 polynucleotide kinase (New England Biolabs, Beverly,
MA), and unincorporated nucleotides were removed with a G-25 spin
column (Pharmacia).
Binding of in vitro transcribed RNA or oligonucleotides to the GST
fusion protein were carried out as described above for the SNAAP screen
except the nucleic acid was used directly without testis extract.
RESULTS
Tpx-1 mRNA Is Specifically Bound by mDAZL
The mDAZL protein is necessary for spermatogenesis
and is presumed to regulate a subset of mRNAs essential
for this process. We utilized the SNAAP technique [33] to
identify mRNAs from mouse testis extract that were specifically bound by an mDAZL protein fused to the GST
domain (GST-mDAZL). The immobilized GST-mDAZL
fusion protein was incubated with testis total extract, and
mRNP complexes bound by GST-mDAZL were extracted.
Testis total extract, which contains both protein and RNA,
was used rather than just purified mRNA to mimic the natural repertoire of RNA-binding proteins and mRNA. Use
of total extract provides the natural complement of competitor proteins, increases the specificity of the protein-cognate mRNA binding, and enables the identification of true
substrate mRNA [33]. RNA bound by GST-mDAZL and
RNA bound to an unrelated control protein were each individually isolated and subjected to RT-PCR using differential display analysis [36] (see Materials and Methods).
Messenger RNAs specifically bound by GST-mDAZL were
determined by a direct comparison of the amplified band
profiles derived from GST-mDAZL-bound mRNA with
those obtained from mRNA bound to unrelated control
RNA-binding proteins.
The PCR profile derived using the H-T11G 39 primer and
a set of four 59 primers (H-AP5 to H-AP8) is shown in
Figure 1A. A comparison of the bands obtained from RNA
bound to the individual proteins permits a determination of
which bands correspond to RNA specifically bound by
mDAZL (lane 2) but not 2 other RNA-binding proteins,
aCP1 (lane 3) and the hetergeneous nuclear RNP U RBD
(lane 4). As expected, several of the bands were equally
associated with 2 or all 3 RNA-binding proteins, indicating
that they were nonspecifically bound and were not further
pursued. Four bands appeared to be bound specifically by
mDAZL (arrows). Isolation and sequencing of these bands
revealed that the top 2 bands (A and B) both corresponded
to distinct fragments within the Tpx-1 39 UTR. Band C
corresponded to a novel sequence, and band D corresponded to an mRNA encoding the actin capping protein b1 subunit, Cappb1 [37]. Because a combination of 59 primers
was used, the distinct Tpx-1 bands, A and B, resulted from
amplification by 2 different 59 primers (H-AP8 and H-AP6,
respectively). Tpx-1 was also identified a third time with
another set of primers (data not shown). Tpx-1 is an abundant mRNA; however, despite its abundance significantly
lower levels were reproducibly associated with the control
proteins (bands A and B in lanes 3 and 4). In total, 22
bands were identified with this assay system as substrates
that were reproducibly bound by mDAZL but not by the
control protein. Seven of the isolated mRNAs corresponded
to previously identified genes, and 15 did not correspond
to known genes in the GenBank database. In addition to
Tpx-1 and Cappb1, the remaining known mRNAs were
Pam [38]; GRSF1 [39]; Trf2 [40–43]; minor histocompatability antigen precursor H47 [44]; and proteosome a7/C8
subunit (Pa7/C8; GenBank accession no. AF055983).
Tpx-1 is a testis-specific protein [19] involved in testic-
FIG. 1. Isolation of Tpx-1 mRNA as a substrate for mDAZL. A) Mouse
testis mRNA bound by GST-mDAZL (lane 2), GST-aCP1 (lane 3), or GSTRBD (lane 4) were isolated, reverse transcribed with H-T11G, amplified
by PCR with the same 39 primer and a combination of four 59 primers
(H-AP5 to H-AP8), and resolved by denaturing 5% PAGE. Bands that were
reproducibly bound by mDAZL and not the control proteins in 3 independent experiments are denoted by an arrow. The indicated bands were
extracted, cloned, and sequenced. Band A corresponds to the 277-nucleotide 39 termini of the Tpx-1 mRNA, band B is a 231-nucleotide fragment
of the Tpx-1 mRNA 39 terminus, band C is a 169-nucleotide segment of
a novel gene, and band D is a 104-nucleotide fragment of Cappb1. DNA
size markers are shown on the left in nucleotides. B) Mouse testis mRNA
bound to GST-mDAZL, GST-aCP1, and GST-RBD were reverse transcribed
with an oligo(dT) primer and amplified by PCR using primers specific for
mouse Tpx-1 (lanes 2–5) or mouse Dmc1 as a negative control (lanes 7
and 8). RT-PCR amplification products using mouse testis total RNA are
shown in lanes 5 and 8. Migration of the Tpx-1 and Dmc1 products are
indicated by the arrows, and the DNA size markers are shown on the left
in base pairs.
ular cell adhesion, and at least 1 of its functions is to mediate the binding of spermatogenic cells to Sertoli cells during spermatogenic differentiation in vitro [20]. The testis
specificity, its repeated reproducible detection as substrate
of mDAZL by SNAAP, and its function made Tpx-1 an
interesting candidate gene to analyze. The specific binding
of mDAZL to the Tpx-1 mRNA was further confirmed
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JIAO ET AL.
FIG. 2. Murine DAZL binds the Tpx-1 mRNA 39 UTR. A) GST pull-down
assays were carried out with 4 mg of the fusion protein and 32P-labeled
Tpx-1 RNA segments. RNA bound to GST-mDAZL (mDAZL) or GST-RBD
(RBD) were eluted and resolved by denaturing PAGE. A fraction of each
RNA was included in the input lanes. The identity of each fragment is
indicated (C). B) GST pull-down assays were carried out as described
above using the full length Tpx-1 39 UTR (lanes 1–3), the 39 UTR lacking
the 61-nucleotide fragment E (lanes 4–6), or a control RNA derived from
the firefly luciferase coding region (lanes 7–9). C) The RNAs used in A
and B are schematically shown with the 59 UTR, coding, and 39 UTR
regions indicated. The RNA fragments are labeled with the corresponding
letter and size in nucleotides. The binding results are summarized on the
right: 1, binding; 2, lack of binding.
(Fig. 1B). Testis extract was incubated with GST-mDAZL,
and bound mRNA was eluted, reverse transcribed, and amplified by PCR using Tpx-1 cDNA primers. Tpx-1 was specifically bound by mDAZL (lane 2) but not by the 2 control
RNA-binding proteins (lanes 3 and 4). To ensure that
mDAZL is not a promiscuous RNA-binding protein that
associates with all mRNA, the PCR was also carried out
with primers to the abundant germ cell-restricted Dmc1
mRNA [45]. Despite the presence of this mRNA in testis
total extract (lane 8), it was not bound by mDAZL (lane
7). We concluded that mDAZL can specifically bind the
Tpx-1 mRNA.
Identification of the mDAZL Binding Site
The approach employed above enables identification of
mRNAs that are specifically bound by mDAZL but does
not provide information with regard to the binding site. The
Tpx-1 mRNA is 1481 bases long. The mDAZL binding site
was determined using a similar GST-mDAZL copurification
strategy as described above except RNAs consisting of distinct segments of the Tpx-1 mRNA were generated in vitro
and used. RNAs corresponding to the 59 UTR and most of
the coding region or the 39 UTR of the Tpx-1 cDNA were
transcribed in the presence of 32P-UTP. The labeled RNAs
were subsequently incubated with GST-mDAZL or the control GST-RBD, and copurifying RNAs were isolated and
resolved on a polyacrylamide gel. As shown in Figure 2A,
the mDAZL-binding site is not contained within the 59 portion of the mRNA; insignificant binding was detected to
this RNA (lane 2). In contrast, efficient binding of mDAZL
to the 39 UTR of Tpx-1 was detected (lane 5). The binding
specificity is further underscored by the failure of the GSTRBD control protein to bind this RNA (lane 6). These data
demonstrate that the binding site for mDAZL is contained
within the Tpx-1 39 UTR. This binding is direct and additional proteins are not required for mDAZL to bind the
Tpx-1 mRNA since recombinant protein in the absence of
testis extract was used.
The specific sequence bound by mDAZL was further
identified using smaller segments of the Tpx-1 39 UTR. A
200-nucleotide region (fragment C) was specifically bound
by mDAZL (Fig. 2A, lanes 8 and 9), but fragment D, which
is shorter by 61 nucleotides on the 39 end, was not (Fig.
2A, lane 11), indicating that this 61-nucleotide region is
necessary for binding. Binding analysis carried out with the
61-nucleotide fragment E showed that this region alone
could be bound by mDAZL (Fig. 2A, lane 14). Therefore,
fragment E is necessary and sufficient for mDAZL binding.
Fragment E also contains the primary mDAZL binding site
within the Tpx-1 mRNA; a 39 UTR containing a deletion
of this 61-nucleotide region was not bound by mDAZL
(Fig. 2B, fragment F, lane 5) nor was an unrelated RNA
(Fig. 2B, lanes 8 and 9).
Specific binding of mDAZL to the 61-nucleotide fragment E was also detected using electrophoretic mobility
shift analysis and is not restricted to the protein copurification assay. In vitro transcribed fragment E was incubated
with an increasing amount of mDAZL protein followed by
treatment with RNase T1. The RNase step removes regions
of the RNA that are not bound by protein and retains a
minimal RNA-protein ribonucleoprotein complex. As
shown in Figure 3A, 2 complexes were detected with purified recombinant GST-mDAZL protein (lanes 2–4) that
are lacking with the 2 control recombinant RNA-binding
proteins (lanes 5–10). The lower band was nonspecific and
unrelated to the GST-mDAZL protein; it was also present
with the control proteins. Although the exact nature of the
2 upper bands has not been addressed, they most likely
came from a monomer and homodimer of GST-mDAZL
binding to the RNA, because mDAZL has been shown to
homodimerize [30]. The binding of GST-mDAZL is specific to the 61-nucleotide fragment E; it is competed by self
RNA (Fig. 3B, lanes 2–5) but not by an unrelated RNA
(Fig. 3B, lanes 6–8). These data demonstrate that with at
least 2 distinct binding assay systems, mDAZL can specifically bind the 61-nucleotide fragment E contained within
the Tpx-1 39 UTR.
The binding site within fragment E was further narrowed
using oligonucleotides. In many cases, RNA-binding proteins can bind single-stranded DNA in addition to RNA
[23, 33, 46]; therefore, delineation of the mDAZL binding
site within fragment E was carried out using deoxyoligonucleotides spanning both halves of this region. Each oligonucleotide was 32P-labeled at the 59 end and tested for
its ability to be bound by GST-mDAZL. Testing the 59 and
39 fragments of this region individually demonstrated that
the 39 half (fragment E2) was specifically bound by mDAZL whereas the 59 half (fragment E1) was not (Fig. 4A;
SUBSTRATE mRNA BOUND BY mDAZL
479
compare lanes 2 and 5). Therefore, the mDAZL-binding
region is contained within the 31-nucleotide 39 segment.
Further fine tuning of the essential sequences for mDAZL binding within the 31-nucleotide region was carried
out by a competition assay. The 61-nucleotide fragment E
RNA was radiolabeled and used in an electrophoretic mobility shift assay in the presence of a series of competitor
oligonucleotides that each contained 7 nucleotide substitutions. As expected, the 31-nucleotide fragment E2 oligonucleotide competed for mDAZL binding (Fig. 4B, lanes 3
and 4). The mutant oligonucleotide containing substitutions
at the 59 end (E2m1; Fig. 4B, lanes 5 and 6) was still able
to compete for GST-mDAZL binding, implying that this
region is not critical for binding, because the mutant oligonucleotide is still competent to bind the protein and sequester it from the labeled Tpx-1 RNA. However, mutations
in the remaining regions of the sequence seem to have an
adverse effect on binding. Mutant E2m2 (Fig. 4B, lanes 7
and 8), E2m4 (Fig. 4B, lanes 11 and 12), and E2m5 (Fig.
4B, lanes 13 and 14) were less effective in their ability to
compete for GST-mDAZL binding, and mutant E2m3 had
an intermediate competition phenotype. These mutant oligonucleotides have lost their ability to be bound by GSTmDAZL, suggesting that the 26-nucleotide region spanning
nucleotides 1120–1145 of the Tpx-1 RNA are critical for
GST-mDAZL binding. This region is referred to hereinafter
as the mDAZL-binding site (DBS).
Presence of the DBS in Other mRNAs
Sequence searches of the clones identified by the
SNAAP screen were carried out using the ClusterW pairwise alignment [47] to determine whether additional
mRNAs also contained the 26-nucleotide DBS sequence.
All of the remaining 6 previously identified mRNAs also
contained a DBS-like sequence in the 39 UTR, as did 1 of
the unknown genes (clone D2, GenBank accession no.
AK014447)) present as multiple expressed sequence tags
(ESTs) in the database. Pam is a 510-kDa protein of unknown function [38], GRSF1 is an RNA-binding protein
that functions in translation initiation [39], Trf2 is a TATA
box-binding protein (TBP)-like protein highly expressed in
testis [40–43], and Cappb1 is a component of a heterodimeric actin capping protein [37]. The H47 mRNA encodes
a minor histocompatability antigen precursor possibly involved in graft rejection [44], and Pa7/C8 is an mRNA
encoding a putative murine proteosome subunit listed in
GenBank. An alignment of the DBS sequences contained
within the isolated substrate mRNAs is shown in Figure 5.
Conserved sequences were used to derive a consensus that
reveals the binding site to be particularly rich in uracil and
adenine. The uracil-rich nature of the binding site is consistent with results of a previous report demonstrating preferential binding of mDAZL to poly(U) ribohomopolymers
[29] or a uracil-rich sequence [48].
We next tested whether other DBS-containing
mRNAs, in addition to Tpx-1, can also be bound by
mDAZL. Four of the remaining 7 DBS-containing
mRNAs where chosen for further study. The specific association of the DBS-containing mRNA with mDAZL
protein is shown in Figure 6A. Messenger RNAs from
all 4 genes tested (Pam, D2, GRSF1, and Trf2) could be
specifically copurified with mDAZL from testis extract
(lanes 2, 6, 10, and 14) but not by control proteins as
detected by RT-PCR analysis with gene-specific primers.
Binding was not detected to an unrelated mRNA, Dmc1
FIG. 3. Specificity of mDAZL binding to the 61-nucleotide fragment E of
the Tpx-1 39 UTR. A) An electrophoretic mobility shift assay was carried out
using 32P-UTP uniformly labeled fragment E RNA with an increasing amount
of protein ranging from 1 to 4 mg of GST-mDAZL (lanes 2–4), GST-RBD (lanes
5–7), or GST-aCP1 (lanes 8–10). Following RNase treatment, the RNA-protein
complex was resolved on a 5% polyacrylamide gel and visualized by autoradiography. The mDAZL complexes are indicated on the left. The input RNA
is shown in lane 1. B) An electrophoretic mobility shift assay was carried out
as described above with 2 mg of GST-mDAZL protein bound to 32P-UTP
uniformly labeled fragment E in the absence (lane 2) or presence (lanes 3–8)
of competitor RNA. Lane 1 contains the input RNA only, lanes 3–5 were
competed with unlabeled self RNA, and lanes 6–8 contained unlabeled RNA
generated from the polylinker region of pcDNA3 (control). Lanes 3 and 6
contain 1.25 pmol, lanes 4 and 7 contain 2.5 pmol, and lanes 5 and 8
contain 3.6 pmol of competitor RNA.
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JIAO ET AL.
FIG. 4. Identification of the DBS. A) GST
pull-down assays were carried out as described for Figure 2 except 32P-labeled
deoxyoligonucleotides were used as binding substrates. The sequence of fragment E
(Fig. 2) is shown as are the 2 oligonucleotides. A summary of the binding results is
shown on the right. The 31-nucleotide 39
fragment region (fragment E2) contains the
binding region. B) An electrophoretic mobility shift assay was carried out with uniformly labeled fragment E as described for
Figure 3 with 2 mg of GST-mDAZL protein
and competed with various unlabeled
deoxyoligonucleotides containing substitution mutations within fragment E2. Lanes
3, 5, 7, 9, 11, and 13 contain 4.5 pmol,
and lanes 4, 6, 8, 10, 12, and 14 contain
9 pmol of competitor RNA. The E2 fragment and the various substitution mutations within this fragment are schematically shown at the bottom. The boxed area
includes the substitution mutations, and
the dashes represent nucleotides that correspond to the wild-type sequence.
(lane 18). To determine whether binding was direct or
mediated through additional proteins, RNAs containing
the respective DBS regions were generated and tested.
32P-labeled in vitro-transcribed RNA was incubated with
GST-mDAZL in the absence of testis extract, and bound
RNA was isolated and resolved by denaturing PAGE. As
seen in Figure 6B, mDAZL can bind each RNA directly
and does not require additional proteins (lanes, 2, 5, 8,
11, and 14) but does not bind a control RNA (lane 17).
The RNAs are not promiscuously bound by mDAZL because they are not bound by an unrelated RNA-binding
protein (lanes 3, 6, 9, 12, and 15). The binding is mediated through the DBS because oligonucleotides consisting of the respective DBS sequences were sufficient
to bind mDAZL (data not shown). Furthermore, the 39
UTR of the remaining 3 DBS-containing genes were also
able to compete for mDAZL binding to the Tpx-1 RNA
in mobility shift assays (data not shown). These data support the conclusion that mDAZL can specifically bind
mRNAs containing a DBS and affirms the predictive potential of the DBS in identifying mDAZL substrates.
Murine DAZL Can Specifically Bind Cdc25A Phosphatase
The Drosophila homologue of mDAZL, Boule, is
functionally linked to the meiotic cell division cycle protein Cdc25 phosphatase (Twine) and appears to regulate
the expression of Twine mRNA translation. In mammals,
there are 3 Cdc25 genes, Cdc25A, Cdc25B, and Cdc25C
[49–51]. In the testis, Cdc25A is expressed in mitotic and
meiotic germ cells, Cdc25B is predominantly in somatic
cells, and Cdc25C is expressed in the late stages of germ
cell development [51–54]. To determine whether any of
the murine Cdc25 phosphatase mRNAs are potential ligands for mDAZL, we searched for a DBS sequence in
these mRNAs. A DBS-like sequence is present in the
Cdc25A 39 UTR but not in the remaining 2 genes. We
next determined whether mDAZL could bind the Cdc25A
SUBSTRATE mRNA BOUND BY mDAZL
mRNA. Total testis extract was incubated with GSTmDAZL, and copurifying RNAs were isolated, reverse
transcribed, and amplified by PCR with gene-specific
primers. Results from the 2 germ cell Cdc25 forms are
presented in Figure 7. Despite the presence of both
Cdc25A and Cdc25C mRNAs in the testis extract (lanes
5 and 9, respectively), the Cdc25A mRNA is efficiently
bound by mDAZL but the Cdc25C mRNA is not (compare lanes 2 and 6). The specificity of mDAZL binding
to the Cdc25A mRNA is further demonstrated by the lack
of detectable binding to this mRNA by the nonspecific
RNA-binding protein in lane 4. These data suggest a
functional role for mDAZL in the progression of the mitotic spermatogonia into the meiotic spermatocyte in
mammals, as is the case in Drosophila. A low, but reproducible, level of binding to Cdc25A was also detected
by the aCP1 protein (lane 3), which is an avid poly(C)binding protein [55–58]. Sequence search of the Cdc25A
mRNA revealed 3 poly(C)-rich regions in the mRNA that
could be potential aCP1 binding sites positioned at nucleotides 467–492, 1875–1891, and 2777–2800. The
functional significance of aCP1 binding to Cdc25A is
currently unclear.
DISCUSSION
The presence of mDAZL throughout spermatogenesis
suggests that it regulates multiple target mRNAs, either simultaneously or at different stages of sperm development,
that are essential for proper spermatogenesis. In the present
study, we obtained evidence that the mDAZL protein can
481
FIG. 5. Alignment of the Tpx-1, Pam, Trf2, GRSF1, Cappb1, Pa7/C8,
H47, and clone D2 DBS sequences; a consensus sequence is shown at
the bottom. A preference for each position was designated when a particular nucleotide was represented 4 or more times or when 2 nucleotides
each were represented at least 3 times. N, any nucleotide.
specifically bind to at least 9 distinct mRNAs: Tpx-1, Pam,
GRSF1, Trf2, Cappb1, Pa7/C8, H47, clone D2, and
Cdc25A. We also isolated a consensus binding site for
mDAZL that will enable identification of additional potential mDAZL target mRNAs as sequences become available
in the databases.
The following observations suggest that one of the functions of mDAZL is to regulate the expression of the Tpx1 mRNA. First, mDAZL can directly bind to the 39 UTR
of the Tpx-1 mRNA. Second, the temporal expression pattern of both proteins is consistent with Tpx-1 being a downstream target of mDAZL. The mDAZL protein is present
FIG. 6. mDAZL-binding to RNA containing a DBS. A) Primer pairs specific for the
indicated genes were used to amplify reverse-transcribed mRNA that was copurified with GST-mDAZL (mDAZL) or the 2
controls, GST-aCP1 (aCP1) and GST-RBD
(RBD). Amplification of mRNA isolated
from total extract is shown (Total; lanes 5,
9, 13, 17, and 21). The mDAZL protein
can bind to the DBS-containing mRNA but
not the Dmc1 control mRNA. DNA size
markers are shown in lane 1, and the corresponding sizes are denoted on the left.
B) The DBS-containing regions of Txp-1
(61 nucleotides), Pam (97 nucleotides), D2
(100 nucleotides), GRSF1 (100 nucleotides), and Trf2 (100 nucleotides) or the
pcDNA3 polylinker (150 nucleotides; control) were transcribed in vitro in the presence of 32P-UTP. The RNA was incubated
with the indicated fusion proteins, and
specifically bound RNAs were eluted and
resolved by denaturing PAGE as described
for Figure 2.
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JIAO ET AL.
FIG. 7. Cdc25A is specifically bound by mDAZL. Messenger RNA copurifying with GST-mDAZL was tested for the presence of Cdc25A and
Cdc25C mRNAs with gene-specific primers as described for Figure 5A.
Cdc25A but not Cdc25C was bound by mDAZL. The DNA markers and
corresponding sizes are shown on the left.
in the early stages of the mitotic spermatogonia and is most
abundant during the pachytene stage of meiosis and thereafter [10, 59, 60]. Tpx-1 mRNA expression appears to begin at the pachytene stage and persists throughout spermatogenesis to the elongating spermatid stage [20–22].
Sperm cell development in mDAZL knockout mice ceases
within the mitotic spermatogonia stage and is unable to
progress into meiosis [10]. Third, the potential functional
significance for Tpx-1 in spermatogenesis was recently suggested by the identification of 3 infertile men that carry a
chromosomal breakpoint within the Tpx-1 gene [18]. Therefore, Tpx-1 could be critical for spermatogenesis, and the
demonstration that mDAZL specifically binds this mRNA
implies that expression of Tpx-1 could be regulated by
mDAZL perhaps by conferring stability onto the mRNA or
translational regulation.
Mice containing a homozygous disruption of the
mDAZL gene have sparse tubule populations just prior to
birth and an absence of germ cells at 9 wk of age [10].
These data indicate that the initial action of mDAZL is
in the mitotic spermatogonial stage because defects are
observed prior to birth. In mice, the meiotic stage of spermatogenesis does not initiate until about 8 days of age
[61]. Thus, it is unlikely that Tpx-1 is the initial target
mRNA for progression into meiosis. Therefore, what is
the significance of regulating Tpx-1 mRNA expression
by mDAZL in the pathogenesis of azoospermia? Tpx-1
appears to be important in the attachment of spermatogenic cells with Sertoli cells [17], and this attachment is
obligatory for spermatogenesis [13, 14, 62]. Aberrant expression of Tpx-1 could result in the loss of, or improper,
interactions between these 2 cell types. The demonstration that mDAZLnull/mDAZL1 heterozygous mice contain
4-fold higher amounts of abnormal sperm cells, which
accounts for 61% of the total sperm produced in these
animals [10], is consistent with a defect in Sertoli-germ
cell interactions. Sertoli cells are essential for the fatedetermining signals that regulate the incidence of apoptosis and the eventual phagocytosis of degenerating
spermatogenic cells [13, 63]. The influence of the Sertoli
cells may be diminished in the heterozygous animals,
which could account for the high incidence of abnormal
sperm that are not purged. Of the 2 proteins in addition
to Tpx-1 that are postulated to be essential for spermatogenic and Sertoli cell attachment, the N-cadherin mRNA
also contains a DBS-like sequence, suggesting that it too
could be regulated by mDAZL.
There is strong evidence that Cdc25A is also a substrate
for mDAZL regulation. The Drosophila Cdc25 homologue
Twine has been functionally linked to the Drosophila
mDAZL homologue Boule. Boule was recently shown to
be required for proper translational regulation of Twine
mRNA [32]; however, it is not known whether it regulates
Twine mRNA directly or indirectly. Our demonstration that
mDAZL can bind the Cdc25A mRNA suggests that mDAZL also regulates this mRNA in mice and that the regulation is direct. The Cdc25 proteins are threonine/tyrosine
phosphatases, which function on cyclin-dependent kinases
and influence the cell cycle [64]. Cdc25A is expressed in
spermatogonia and is at its highest level during the early
meiotic stages of spermatogenesis in the primary and secondary spermatocytes but is not present in the later stages,
suggesting that Cdc25A is important for the transition into
meiosis [51]. This function is similar to that postulated for
Twine in Drosophila germ cell development. We noticed a
DBS-like sequence within the 39 UTR of Twine (nucleotides 1610–1641), suggesting that Boule directly binds the
Twine mRNA and regulates its expression.
The Cdc25A and Cdc25C proteins are both approximately 55% similar to Twine at the protein level. However,
the similarity of the expression profile between Cdc25A
and Twine and the presence of a DBS in the 39 UTR of
both these mRNAs suggest that Cdc25A is the functional
homologue of Twine in mammals. This hypothesis is further supported by the recent demonstration that mice containing a knockout of the Cdc25C gene are fertile [65],
precluding Cdc25A as a critical mRNA regulated by mDAZL in fertility. Our demonstration that mDAZL binds
the 39 UTR of the Cdc25A mRNA indicates that this gene
is an essential mDAZL target in vertebrates and regulates
the progression of spermatogonia into spermatocytes.
aCP1, which has an affinity for Cdc25A, is also involved
in both mRNA stability [35] and translation [66–70].
Whether it has a functional role in the regulation of
Cdc25A needs to be determined. The binding of aCP1 to
the Cdc25A mRNA could also explain why this mRNA
was not identified as an mDAZL substrate in the initial
SNAAP screen. Bands that were common to both the target
mDAZL protein and the control aCP protein were not evaluated further and would have been scored as false negative.
An exhaustive screen for mDAZL substrates has not yet
been carried out.
In humans, the Y-chromosome-encoded DAZ protein is
expressed in the early spermatogonial stage, whereas the
autosomal DAZL protein is expressed in later stages, suggesting that each protein contributes to distinct aspects of
this process [71]. The presence in mice of only an autosomal copy suggests that mDAZL is involved in multiple
functions throughout gametogenesis. Perhaps 1 function is
to regulate the progression into meiosis by the control of
Cdc25A and the subsequent differentiation by the regulation of Tpx-1 expression.
Although Tpx-1 is the only testis-specific mDAZL substrate mRNA that we have thus far identified, the remaining
isolated mRNAs could also be essential in spermatogenesis.
SUBSTRATE mRNA BOUND BY mDAZL
Pam was originally identified as a protein associated with
c-myc [38]. Expression of a c-myc transgene in the mouse
testes results in the arrest of spermatogenic cell differentiation, leading to sterility [72]. Therefore, a protein that interacts with c-myc could have significant effects on the
function of c-myc in spermatogenesis. GRSF1 is a cytoplasmic RNA-binding protein originally identified by its
binding preference to guanosine-rich sequences [73]. It was
subsequently shown to upregulate the translation of influenza virus and is most likely involved in general translational control [39]. Misregulation of GRSF1 could adversely affect the translation of various genes essential for spermatogenesis. Trf2 is a TBP-like protein that can associate
with the basal transcription factor TFIIA and inhibit transcription of promoters containing a TATA-box and is
thought to activate transcription from promoters containing
nonconsensus TATA boxes [40–43]. Despite its apparent
ubiquitous low level of expression in all cells tested, Trf2
mRNA is overexpressed in the testes [41–43], suggesting
that it is an important component of gene expression in this
organ. While this manuscript was under review, Zhang et
al. [74] reported that mice containing a disrupted Trf2 gene
are sterile because of a spermiogenesis defect. This observation is consistent with our hypothesis that mDAZL binds
to and potentially regulates the expression of the Trf2
mRNA.
Of the 22 clones that were identified in the SNAAP
screen, all 8 with a known complete mRNA sequence contain a discernable DBS. The remaining 14 isolated clones
are novel and have no corresponding ESTs or incomplete
ESTs that do not appear to span the entire 39 UTR. Therefore, it is unclear whether they contain a DBS. It is also
conceivable that some of the mRNAs do not have a DBS
and copurified with mDAZL only because of their association with an mDAZL interacting protein. In such a scenario, mDAZL does not directly bind these mRNAs but is
associated with them through a larger multiprotein RNP
complex. A protein bound directly or indirectly to an
mRNA can still exert an influence on the regulation of that
mRNA. However, these clones have not been further characterized to independently confirm that they are true mDAZL substrates, and they could represent false-positive
results.
Identification of mDAZL substrate mRNAs will now
enable a detailed mechanistic assessment of the consequence of mDAZL binding to each target mRNA. The
DBS is contained in the 39 UTR of each DBS-containing
mRNA isolated thus far. The 39 UTR location of the DBS
and the cytoplasmic polysome association of mDAZL
[29] indicate that it regulates expression of its substrate
mRNAs by regulating translation or stability of the
mRNA. Both possible mechanisms are currently under
investigation for the individual substrate mRNAs. The
results of these investigations will provide valuable insights into the role of mDAZL and the related Y-chromosome homologue DAZ in azoospermia and may lead
to novel avenues for therapeutic intervention.
ACKNOWLEDGMENTS
We thank C. Tovar and B. Babiarz for providing mouse testes and D.
Denhardt for providing the DAP28–DAP31 primers. We are grateful to
N.D. Rodgers and Z. Wang for critical reading of the manuscript.
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