From the Department of Obstetrics and Gynecology,
Center for Research on Reproduction and Women's Health, School of
Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
19104-6142 and the § Department of Engineering, Yokohama
National University, Yokohama 240-8501, Japan
Received for publication, October 24, 2000, and in revised form, December 19, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Trax (Translin-associated factor X) has been
shown to interact with TB-RBP/Translin by its coimmunoprecipitation and
in yeast two-hybrid assays. Here we demonstrate that Trax is widely
expressed, does not bind to DNA or RNA, but forms heterodimers with
TB-RBP under reducing conditions. The heterodimer of TB-RBP and Trax inhibits TB-RBP binding to RNA, but enhances TB-RBP binding to specific
single stranded DNA sequences. The in vitro interactions between TB-RBP and Trax are confirmed by similar interactions in the
yeast two-hybrid system. Cell fractionation and confocal microscope
studies reveal that Trax is predominantly cytoplasmic. In contrast,
TB-RBP is present in both the nuclei and cytoplasm of transfected cells
and uses a highly conserved nuclear export signal to exit nuclei. In
addition to a leucine zipper, two basic domains in TB-RBP are essential
for RNA binding, but only one of these domains is needed for DNA
binding. Trax restores DNA binding to TB-RBP containing an altered form
of this domain. These data suggest that Trax-TB·RBP
interactions modulate the DNA- and RNA-binding activity of
TB-RBP.
The process of mammalian spermatogenesis is highly organized
spatially and temporally. Highly controlled transcription and protein
expression occur in each developmental stage. During the haploid
interval, spermiogenesis, the spermatids become transcriptionally inactive, although there is a need for the synthesis of many proteins essential for the formation of spermatozoa (1). The sex chromosomes encode numerous genes essential for gametogenesis. Because the spermatids are haploid cells, they contain either the X or Y
chromosome. Thus, intercellular transport of mRNA in the haploid
cells is a critical process to ensure genetic equivalency.
The testis brain RNA-binding protein
(TB-RBP)1 was identified and
cloned on the basis of its ability to bind H and Y sequence elements in
the 3'-untranslated repeats of mouse protamine 1 and 2 mRNAs (2).
TB-RBP is the mouse orthologue of human Translin, a single-stranded
DNA-binding protein that binds consensus sequence breakpoint junctions
of chromosomal translocations in lymphoid malignancies (3). The
TB-RBP/Translin consensus binding sequences are also found in
TLS-CHOP reciprocal translocations, in therapy-related translocations in acute myeloid leukemias, and in BCR-ABL
translocations in chronic myeloid leukemia (4-6). Sequence analysis in
a meiotic recombination hot spot region of human chromosome 16 shows
TB-RBP/Translin binding sequences near the breakpoint (7).
TB-RBP/Translin has also been proposed to act as a single-stranded
DNA-binding transcription factor, which activates early response gene
expression in the brain (8).
TB-RBP/Translin also functions as a RNA-binding protein mediating
intracellular and intercellular mRNA transport (9, 10). RNA binding
of TB-RBP has been observed in brain and testis, and the binding is
dependent upon Y and H sequence elements (2). Many testis- and
brain-specific mRNAs have Y and H consensus sequences, and specific
RNA·TB-RBP interactions have been demonstrated for testis mRNAs
encoding protamine 1 and 2 and AKAP 82 and in brain for myelin basic
protein mRNA, Using Translin as bait in a yeast two-hybrid assay, a protein of
unknown function, Translin-associated factor X (Trax), was identified
(7). TB-RBP/Translin and Trax are encoded by single-copy genes that are
evolutionarily conserved. In addition to having highly conserved
sequences in mammals, they are also found in frogs (Xenopus
laevis), plants (Arabidopsis thaliana and Oryza sativa), insects (Drosophila), and yeast
(Schizosaccharomyces pombe) (17). This extraordinary
conservation from yeast to mammals suggests that these molecules play
important biological functions. In the adult mouse, TB-RBP mRNA is
widely expressed, with its highest levels in testis and brain (15). The
subcellular localization of TB-RBP protein is both developmentally and
subcellularly regulated during spermatogenesis. During meiosis, the
TB-RBP protein primarily localizes in the nuclei of pachytene
spermatocytes, whereas in late meiotic prophase and in all subsequent
stages of germ cell differentiation, it is in the cytoplasm (10).
TB-RBP/Translin also is predominantly a nuclear protein in malignant
lymphoid cell lines but not in nonlymphoid cell lines (3). Treatment of
nonlymphoid cells with DNA-damaging agents was reported to cause a
shift of the TB-RBP/Translin from the cytoplasm into nuclei (18).
TB-RBP and Trax share a 28% identity at the protein level with a
conservation of 38% in the C-terminal regions. Both have putative
leucine zipper domains at the C terminus of TB-RBP and in the
mid-region of Trax (3, 7). Interaction between TB-RBP and Trax has been
demonstrated by coimmunoprecipitation (19) and in the yeast two-hybrid
assay (7, 8). Yeast two-hybrid and in vitro binding studies
indicate that TB-RBP dimers are the minimum unit needed for DNA or RNA
binding (20). Unlike TB-RBP (20), when Trax is used as bait in the
yeast two-hybrid system, it does not select itself, suggesting it does
not homodimerize. TB-RBP/Translin contains two putative basic domains
in the N-terminal region from amino acids 56 to 64 and from amino acids
86 to 97. Changes in the amino acid sequence in the 86-97 region
abolish the DNA-binding activity of Translin (21).
Analysis of the Trax sequence has suggested it contains a putative
nuclear localization signal (NLS) (7). The subcellular localization
changes in meiotic and post-meiotic cells (10, 15) and in nonlymphoid
cells after DNA damage (18) indicate a need to shuttle TB-RBP between
the nucleus and cytoplasm. Comparison of TB-RBP sequences with known
nuclear export signals (NES) suggests the presence of a putative
leucine-rich Rev-like NES sequence in its C terminus. Although the
Rev-like NES is one of the most commonly described NES (22, 23), none
of the Rev-like NES-containing proteins have been shown to interact
with cellular mRNAs.
To date, little is known of the biological function(s) of Trax. Because
heterodimeric partners often modulate the activity of proteins that can
homodimerize such as c-Fos/c-Jun (25), we set out to determine
whether TB-RBP·Trax heterodimers could alter the nucleic acid
recognition properties of TB-RBP. Here we show that Trax alone does not
bind to either DNA or RNA, but Trax does form heterodimers with TB-RBP.
The heterodimer of TB-RBP and Trax is unable to bind to RNA, but binds
to DNA. Thus, the heterodimerization modulates the substrate
specificity in a very unique manner. Cell fractionation and
transfection studies reveal that most of Trax is in the cytoplasm.
TB-RBP appears to use a Rev-like nuclear export signal to exit the
nucleus and requires two basic regions in its N terminus in addition to
its leucine zipper to bind RNA.
Expression of Trax Protein in Escherichia coli--
A cDNA
encoding the complete open reading frame of human Trax was subcloned
in-frame with a thioredoxin-6X His-S-peptide N-terminal tag in a pET32a
vector (Novagen) and transformed into BL21(DE3) cells. The expressed
fusion protein was purified by nickel-nitrilotriacetic acid-agarose
column chromatography, and the thioredoxin-6X His tag was removed by
thrombin digestion to obtain Trax with an N-terminal S-peptide tag.
Northern and Western Blotting--
Total RNA preparations were
hybridized with 32P-labeled Trax cDNA under conditions
previously described (15). For Western blotting, tissue extracts were
prepared from sexually mature CD-1 male mice using the protocol of Wu
et al. (2). Aliquots (30 µg) of protein were
electrophoresed in 10% SDS-polyacrylamide gels, and the proteins were
transferred onto nylon membranes. The membranes were incubated
overnight with TBS containing 5% nonfat dry milk at 4 °C and then
incubated with a polyclonal antibody to Trax (1:2000) in TBS containing
0.25% nonfat dry milk for 1 h at RT. After washing, the membranes
were incubated with protein A conjugated with horseradish peroxidase,
and Trax was detected with the enhanced chemiluminescence protocol of
Amersham Pharmacia Biotech.
In Vitro Interactions between TB-RBP and Trax--
Recombinant
mouse TB-RBP (200 ng) was incubated with recombinant human
S-peptide-tagged Trax for 30 min at 4 °C in 200 µl of TBS-T (20 mM Tris, pH 7.6, 137 mM NaCl containing 1%
Tween 20) or in 200 µl of 20 mM HEPES, pH 7.5, containing
1.5 mM MgCl2 with or without 5 mM
DTT. S-protein-agarose beads (20 µl) were added to the mixture, and
the incubation was continued for 30 min at 4 °C with gentle shaking.
The mixture was centrifuged for 1 min at 1000 rpm at 4 °C, and the
pellet was washed three times with TBS-T (1 ml) interspersed with
centrifugation at 1000 rpm. The pellets were boiled in SDS loading
buffer for 3 min, and proteins were resolved on a 10%
SDS-polyacrylamide gel. The gel was stained with SYPRO Orange protein
dye (Bio-Rad), and the proteins were visualized by fluorescence
according to the manufacturer's protocol.
RNA and DNA Mobility Shift Assays--
Electrophoretic mobility
shift assays were performed with a DNA probe, Bcl-CL1, or an RNA probe,
transcript c, as described previously by Wu et al. (20). For
the RNA gel shifts, the RNA·protein complexes were routinely digested
with T1 RNase (1 unit/assay) and incubated with heparin (5 mg/ml).
Bcl-CL1 was labeled with [ Site-directed Mutagenesis of Putative Domains of
TB-RBP--
Site-directed mutagenesis of the two basic domains and a
putative nuclear export signal of TB-RBP was carried out using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA)
according to the manufacturer's protocol. The wild-type TB-RBP, TB-Nb
mutant, and TB-Cb mutant cDNAs were subcloned into a pET28a vector
for protein expression in E. coli.
Wild type TB-RBP and TB-RBPNES mutant cDNAs were
subcloned in-frame to the C terminus of GFP in the pEGFP C2 vector
(CLONTECH). We named the fusion proteins from these
constructs EGFP-TB-RBP and EGFP-TB-RBPNES, respectively.
The Trax cDNA was subcloned into the pEBFP C1 or pDsRed1-N1
vectors, which produce fusion proteins with the blue fluorescence
protein at the N terminus of Trax (EBFP-TRAX) or the red fluorescence
protein at the C terminus of Trax (TRAX-DsRed), respectively. The Trax
cDNA was also cloned into the pEGFP C2 vector.
Yeast Two-hybrid Assays--
The complete cDNAs of TB-RBP,
its mutant alleles, and Trax were subcloned into the
EcoRI/SalI sites of pBD-GAL4cam and
pAD-GAL4 (Stratagene, La Jolla, CA). Pairs of binding domain and
activation domain plasmid constructs were cotransfected into the yeast
strain YRG-2. Transformants were selected on SD medium lacking leucine and tryptophan. Protein·protein interactions were detected by growth
on SD medium lacking leucine, tryptophan, and histidine, and by the
5-bromo-4-chloro-3-indolyl Cell Culture, Transfections, and Confocal Fluorescence
Microscopy--
NIH 3T3 cells were grown in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum and
streptomycin. Cells were transiently transfected with the plasmid
constructs described above using FuGENE 6 reagent (Roche Molecular
Biochemicals) according to the manufacturer's protocol. Cells were
grown on two-well chamber slides (Lab-Tek) and fixed 18 h
post-transfection using 4% paraformaldehyde in PBS, washed three times
in PBS and mounted using Vectashield mounting medium (Vector Scientific).
Expression of Trax in Mouse Tissues--
To determine the extent
of Trax expression in various mouse tissues, a Northern blot
hybridization was performed with the complete open reading frame of
Trax as probe (Fig. 1A). In
the mouse, Trax is present as a single transcript of about 2.5 kb. As
previously reported for TB-RBP (15), brain and testis contain the
highest levels of Trax mRNA, whereas heart, kidney, liver, lung,
and spleen contain lower amounts of Trax mRNA. These differences are not due to RNA degradation or unequal loading, because
hybridization of the blot to a
To determine the protein levels of Trax in mouse tissues, a Western
blot was performed using a polyclonal rabbit anti-Trax antibody to
detect a protein band of 34 kDa that comigrates with recombinant Trax
(Fig. 1C, lane 8). As seen for the mRNAs, the highest levels of Trax expression are seen in testis and brain. In
general, Trax protein levels reflect the Trax mRNA levels, and the
level of Trax protein in the mouse tissues examined was similar to the
pattern reported for TB-RBP (2, 15). Quantitation of Western blots
using recombinant Trax and recombinant TB-RBP as standards revealed
TB-RBP to be present at about a 2- to 3-fold molar excess compared with
Trax in these tissue extracts (data not shown).
To define the subcellular distribution of Trax, Western blot analyses
of cytoplasmic and nuclear extracts from mouse testis and from NIH 3T3
fibroblasts were performed (Fig. 1D). In extracts from
testis and transfected cells, Trax is predominantly found in the
cytoplasm (Fig. 1D, lanes 1 and 3),
although lower levels are seen in the nuclear fractions (Fig.
1D, lanes 2 and 4). Similar results
are seen with confocal microscopy (see below).
TB-RBP Forms a Heterodimer with Trax in Vitro under Reducing
Conditions--
Because Trax coprecipitates with TB-RBP with an
affinity-purified antibody to mouse recombinant TB-RBP (19) and the
proteins interact in yeast two-hybrid assays (7), we set out to
determine the conditions needed for Trax-TB·RBP interactions. An
S-peptide-tagged recombinant Trax protein was mixed with recombinant
TB-RBP and incubated in a series of buffers. Proteins precipitated with
S-protein-agarose beads were then separated on SDS-polyacrylamide gels
and stained using a SYPRO-Orange dye (Fig.
2). Trax and TB-RBP do not stably interact in buffers such as TBS or HEPES at neutral pH (Fig. 2, lanes 1 and 2). The presence of divalent cations
such as Ca2+ or Mg2+ in PBS, TBS, or HEPES
buffer also does not facilitate interaction. However, the addition of 5 mM DTT in HEPES buffer induces the proteins to interact at
roughly a ratio of 1:1 suggesting a heterodimer, although a larger
oligomer may also be formed (Fig. 2, lane 3). The
heterodimer is maintained when the DTT level is diluted to 0.5 mM, but prolonged dialysis of the protein mixture against HEPES buffer in the absence of DTT dissociates the heterodimer (data
not shown).
Trax Inhibits TB-RBP·RNA Interactions but Enhances DNA
Binding--
To determine whether the TB-RBP·Trax interaction alters
the nucleic acid binding of TB-RBP, gel shift assays were performed. RNA electrophoretic mobility shift assays were performed with transcript c and DNA electrophoretic mobility shifts assays with the
single-stranded DNA, Bcl-CL1, two probes previously used to characterize Translin/TB-RBP binding to nucleic acids (20). Although
Trax itself does not bind to RNA (Fig. 6, lane 2), the addition of increasing amounts of Trax under conditions where heterodimers are formed leads to about a 75% decrease in RNA binding at approximately a 1:1 ratio of Trax and TB-RBP (Fig.
3A, lane 5). The
free RNA in these samples is degraded by the T1 RNase incubation
routinely performed for the RNA gel shifts. (Fig. 3, lanes
2-5). In the absence of T1 RNase digestion, the RNA probe remains
undegraded (Fig. 6).
The addition of increasing amounts of Trax to TB-RBP enhances binding
to one of the DNA target sequences for TB-RBP, Bcl-CL1 (Fig.
3B). Severalfold increases in DNA binding are seen at a Trax:TB-RBP ratio of 2:1 (Fig. 3B, lane 4). A
higher level of Trax (4:1) does not elicit any further increase in DNA
binding (Fig. 3B, lane 5). As seen for the RNA
binding assay, Trax alone does not bind DNA (Fig. 7, lane
2).
The TB-RBP·Trax Heterodimer Is the Cause of the Changes in RNA
and DNA Binding--
To demonstrate that it is the heterodimer of Trax
and TB-RBP that is causing changes in TB-RBP binding to RNA and DNA,
heterodimers were prepared using thioredoxin-tagged Trax and
recombinant TB-RBP. This heterodimer migrates more slowly than either a
TB-RBP homodimer or a Trax·TB-RBP heterodimer, because of the
additional amino acids at its N terminus. Gel shift assays carried out
using this protein nucleic acid complex reveal that it is the
heterodimer of Trax and TB-RBP that binds poorly to RNA (Fig.
4, lanes 4 and 5)
and the heterodimer that binds well to specific single-stranded DNA
probes such as Bcl-CL1 (Fig. 4, lanes 9 and
10).
Mutations in Either of Two Basic Domains, Nb and Cb, of TB-RBP
Prevent RNA Binding--
The TB-RBP protein has been shown to contain
a leucine zipper important for DNA and RNA binding (20, 21) and two
putative basic domains, one of which has been reported to be essential for DNA binding (21) (Fig. 5). Mutations
in the basic domains of TB-RBP were made to analyze the TB-RBP-nucleic
acid binding in greater detail and to determine their effect on RNA
binding. Using radiolabeled transcript c, recombinant Trax, and
recombinant TB-RBP as controls, electrophoretic mobility shift assays
were performed to evaluate nucleic acid binding changes caused by
altering these two basic domains of TB-RBP. As previously seen, the
addition of Trax to TB-RBP reduces TB-RBP RNA binding (Fig.
6, compare lane 3 to
lane 4). A mutation of the N-basic domain of TB-RBP (TB-Nb)
drastically reduces the RNA binding in the absence (Fig. 6, lane
5) or presence of Trax (Fig. 6, lane 6). The
protein·RNA complex formed by the TB-Nb mutant also migrates more
rapidly than the wild type TB-RBP·RNA complex. A mutation of the
C-basic domain of TB-RBP (TB-Cb) leads to the complete abolition of RNA binding (Fig. 6, lane 7). The addition of Trax to the TB-Cb
protein does not show any effect on the reduction in RNA binding of
TB-Cb (Fig. 6, lane 8). We conclude that both the N-basic
and C-basic domains of TB-RBP are essential for RNA binding and the
addition of Trax does not substantially alter its RNA interactions with these mutated proteins.
Mutation in the C-basic Domain of TB-RBP Abolishes DNA Binding but
Trax Restores DNA Binding--
DNA gel shift assays were carried out
using an TB-RBP and Trax Interact in Vivo--
To verify that TB-RBP,
TB-Nb, TB-Cb, TB-NES, and Trax interact in vivo,
a yeast two-hybrid assay was employed (Fig.
8). As previously reported, TB-RBP
readily forms homodimers in yeast (20) (Fig. 8A) and
heterodimers with Trax (Fig. 8B) (7). Trax, however, does
not homodimerize (Fig. 8C). The TB-Nb, TB-Cb, and
TB-NES mutants can also homodimerize, dimerize with
wild-type TB-RBP, and heterodimerize with Trax in yeast (Fig. 8,
D-L), suggesting that the changes in nucleic acid binding
we detect by the gel mobility shift assays are specific and mediated by
changes produced in the heterodimer as a result of mutations in TB-RBP
not due to nonspecific interactions.
Trax Is Predominantly a Cytoplasmic Protein--
To determine the
subcellular locations of TB-RBP and Trax, transfections were performed
in NIH 3T3 mouse fibroblasts using a fusion protein of TB-RBP and green
fluorescence protein and Trax with a blue or red fluorescence protein.
Confocal fluorescence microscopy reveals that Trax is predominantly
cytoplasmic with a high concentration localized around the nucleus
(Fig. 9C). TB-RBP is also
mostly in the cytoplasm, although a reproducible low level is seen in
nuclei, but not in nucleoli (Fig. 9, B and D).
Cotransfections of TB-RBP with Trax also show a predominantly
cytoplasmic localization for the two proteins. Identical results are
obtained whether the fluorescence protein tag is on the N or C terminus
of Trax (Fig. 9, D-F). The staining of the
GFP-Trax-transfected cells with the fluorescent Golgi/ER marker BODIPY
558/568 suggests that much of the Trax is Golgi/ER-associated (Fig.
9G). This is supported by studies where the disruption of
the Golgi with brefeldin A leads to a more diffused cytoplasmic
localization of Trax (Fig. 9H).
TB-RBP Has a Functional Leucine-rich Nuclear Export Signal--
By
sequence comparison of TB-RBP with other nuclear-cytoplasmic shuttling
proteins such as HIV Rev and c-Abl (22, 23), we noted a putative
nuclear export signal N-terminal to the leucine zipper of TB-RBP (Fig.
5). This sequence shows high sequence homology to other leucine-rich
NES sequences present in a number of shuttling proteins (Fig.
10A) and is highly conserved
from Drosophila to humans (Fig. 10B). To test the
functionality of this sequence, site-directed mutagenesis was used to
disrupt the putative nuclear export signal (from LASELSRLSVN to
LASEQSRLSVN) (Fig. 10A). The mutated TB-RBP,
TB-RBPNES, was then fused to GFP and transfected into NIH
3T3 cells. In contrast to wild type TB-RBP, which is mostly seen in the
cytoplasm (Fig. 9, B and D), the
GFP-TB-RBPNES mutant protein localizes mostly in the
nucleus with little staining in the nucleoli (Fig. 9I).
Trax is Widely Expressed--
As previously shown for TB-RBP (2,
15), Trax is widely expressed in mouse tissues with high levels of
mRNA and protein in brain and testis (Fig. 1). By Northern
blotting, we detect one transcript of about 2.5 kb, in agreement with
the 2.7-kb mRNA reported by Aoki et al. in humans (7).
In general, this pattern of expression of Trax in mouse tissues is
similar to the expression pattern observed for TB-RBP (2, 15),
suggesting a functional relationship between these two similar proteins.
Trax and TB-RBP Form Heterodimers--
To begin to define the
functional relationship between Trax and TB-RBP, we have examined the
interactions of recombinant TB-RBP with recombinant Trax. Trax and
TB-RBP interact poorly under nonreducing conditions, whereas in the
presence of DTT they form a heterodimer or oligomer at roughly a 1:1
ratio (Fig. 2). We believe this interaction is physiologically
significant, because Trax was initially isolated as a Translin/TB-RBP
interacting protein using TB-RBP as a bait in yeast two-hybrid assays
(7) and Trax protein coimmunoprecipitates with TB-RBP with an
affinity-purified monospecific anti-TB-RBP antibody (19). A homodimer
is the minimal structural unit of TB-RBP that is sufficient for TB-RBP
binding to either single-stranded DNA or RNA (20). Heterodimerization
of Trax and TB-RBP appears to require the reduction of the cysteine
disulfide linkage that stabilizes the TB-RBP homodimer. Incubation of
the heterodimer at reduced levels of DTT results in the formation of
TB-RBP homodimers, indicating that heterodimer formation is reversible
(data not shown). Under the reducing environment of cells, the
interconversion of heterodimers and homodimers should occur spontaneously.
Trax Inhibits TB-RBP RNA-binding Interactions and Enhances DNA
Binding--
Using RNA and DNA gel shift assays, we have found that
Trax does not bind RNA or DNA by itself and interactions between Trax and TB-RBP reduce the RNA binding of TB-RBP in a
concentration-dependent manner (Fig. 3A). In
contrast, heterodimerization increases DNA binding of the complex.
Heterodimerization of various transcription factors and coactivators
often results in changes in DNA sequence specificities and their
transcriptional activation. The Drosophila proteins,
spineless and tango, interact in a manner
similar to their mammalian orthologues, aryl hydrocarbon receptor and
aryl hydrocarbon receptor nuclear translocator, causing changes in DNA
binding specificity (24). Similar effects are known for c-Jun/c-Fos
heterodimers (25). Many of the heterogeneous nuclear ribonucleoproteins
(hnRNPs) play a variety of roles in DNA and RNA metabolism. In in
vitro assays, the hnRNPs A1, A2/B1, D, and E bind G-rich
single-stranded DNA overhangs similar to the G-biased strands or
G-strands found in telomeres. The hnRNP A1 appears essential for
maintenance of telomere length in mouse cell lines (26) while having
important roles in pre-mRNA splicing and mRNA export (27). To
date, little is known about how the specificity for RNA and
single-stranded DNA binding is regulated in hnRNPs. The G-strand
binding protein of Chlamydomonas reinhardtii (Gbp1p) was
recently reported to contain atypical RNP motifs and to bind RNA
sequences and single-stranded DNA as monomers (28). The homodimeric
form of the protein was reported to lose RNA binding completely,
whereas the dimeric Gbp1p shows a strong preference for single-stranded
DNA. The decrease in RNA binding when the TB-RBP homodimer is replaced
by a heterodimer of TB-RBP and Trax offers many regulatory
possibilities in the release of transported or stored mRNAs in
post-meiotic male germ cells (10). In light of the cessation of
transcription in these cells, post-transcriptional regulation of
mRNAs plays a prominent role in cellular differentiation (1).
DNA and RNA Binding Domains of TB-RBP--
Many proteins,
including the hnRNPs A1, A2/B1, D, and K and Gbp1p have shared nucleic
acid binding domains for single-stranded DNA and RNA (26-28). TB-RBP
contains two putative basic domains from amino acids 56 to 64 and from
86 to 97. We confirm the observation of Aoki and colleagues (21) that
mutation of the Cb domain (amino acids 86-97), but not the Nb domain
(amino acids 56-64), leads to loss of DNA binding (Fig. 7). In
addition, mutation of either of these two domains abolishes RNA binding
(Fig. 6). This suggests that more sequence elements in TB-RBP may be
needed for RNA binding than for DNA binding.
A modest change in the electrophoretic mobility of the DNA·protein
complex is seen when Trax interacts with the TB-Nb protein and Trax
dramatically alters the binding of TB-Cb to DNA (Fig. 7). Enhanced DNA
binding observed upon heterodimer formation of wild type TB-RBP or the
TB-Cb mutant with Trax may be due to improved interaction of the
N-basic domain with DNA. Thus, modulation of TB-RBP nucleic acid
binding by Trax may be through conformational changes in TB-RBP that
are induced by Trax. These changes in protein·protein interactions
are likely due to heterodimer formation, because similar interactions
are seen in vivo using the yeast two-hybrid assay. All of
the mutant alleles interact as homodimers or with Trax as a partner for
a heterodimer. These interactions indicate that, in vivo,
the introduced mutations do not significantly alter the interacting
capabilities of the fusion proteins (29). Although interaction strength
was not quantified, we detect differences among the TB-RBP alleles.
Wild type TB-RBP and TB-RBPNES interact strongly as
homodimers and heterodimers, whereas TB-Nb interacts noticeably weaker.
These results suggest that the effects on nucleic acid binding in the
gel mobility shift assays are predominantly due to the mutations in
TB-RBP, not gross alteration of protein·protein interactions.
Subcellular Locations of TB-RBP and Trax--
Using confocal
microscopy of N-terminal GFP-tagged TB-RBP in transfected NIH 3T3
cells, we detect the majority of TB-RBP in the cytoplasm with low
levels in nuclei. No TB-RBP is seen in nucleoli, suggesting that TB-RBP
is not directly involved with ribosomal RNA transcription or transport.
It has been reported that treatment of HeLa cells with mitomycin C or
etoposide induces endogenous TB-RBP to move into nuclei (18). However,
treatment of the transfected NIH 3T3 cells with doxorubicin, a compound that causes DNA double-strand breaks (at concentrations to 30 µM) does not alter the location of the TB-RBP fusion
protein in NIH 3T3 cells (data not shown).
Trax has been proposed to have a bipartite nuclear localization signal
in its N terminus that could facilitate the movement of TB-RBP/Translin
into the nucleus (7). Western blot analyses of cytoplasmic and nuclear
protein fractions from mouse testis and NIH 3T3 cells indicate that
Trax is predominantly a cytoplasmic protein (Fig. 1D),
although a low level of Trax is found in the nuclear fractions (Fig.
1D, lanes 2 and 4). Although we cannot exclude
the possibility that Trax transiently enters and rapidly exits nuclei
as part of a transport function, most of the "steady-state" levels
of Trax protein in our Western blot assays appear to be cytoplasmic.
Comparison of the Trax protein sequences of S. pombe, Drosophila melanogaster, A. thaliana, mouse, rat,
and human indicate that, although Trax is a very conserved protein, the
putative NLS sequence is situated in a region of the protein with lower sequence conservation.
We find that Trax fusion constructs either tagged with blue
fluorescence protein at their C terminus or with red fluorescence protein at their N terminus localize predominantly to the cytoplasm of
3T3 cells (Fig. 9, C, E, and G).
Although we find colocalization of GFP-TB-RBP and BFP Trax in the
cytoplasm of doubly transfected cells (Fig. 9, D-F), we
only detect substantial amounts of TB-RBP in the nucleoplasm of the
cells. The Trax fusion protein localizes predominantly around the
nuclei in the transfected cells, reminiscent of a Golgi/ER
distribution. Staining the fixed cells with BODIPY 558/568 reveals a
colocalization of Trax with the Golgi/ER (Fig. 9G).
Moreover, Trax delocalizes from the Golgi into the cytoplasm following
disruption of the Golgi with brefeldin A (Fig. 9H). Although
the retention of the Trax fusion protein in Golgi may be preventing
Trax from utilizing its putative NLS for nuclear entry, following the
disruption of the Golgi, we do not see any substantial increase of Trax
in the nucleus. This may be due to rapid transport into and out of the
nuclei, levels of nuclear Trax too low to be detected by these
methodologies, or the presence of cytoplasmic retention sequences in
Trax that override the NLS sequences (30). Subcellular fractionation of
Translin and Trax in cerebellar extracts also suggests the two proteins
are enriched in the cytoplasm (31).
Other cytoplasmic proteins such as the Fanconi anemia complementation
group A gene product, FAA, also contain a putative bipartite NLS
sequence (30). A hybrid protein containing the NLS of SV40 large T
antigen and FAA also localizes in the cytoplasm of transfected human
293 cells, showing a specific cytoplasmic retention. The subcellular
distribution of the Drosophila Cubitus interruptus protein, which mediates Hedgehog signaling, appears
to be regulated by opposing bipartite NLS and cytoplasmic
targeting/retention signals (32). Trax may also contain similar
cytoplasmic retention sequences that override its putative NLS. From
our confocal studies, Trax appears to predominantly localize to the
Golgi/ER, although we cannot exclude low levels of Trax in the nuclei
(Fig. 9G). Studies using Trax as bait in yeast two-hybrid
assays have primarily detected Trax interactions with cytoplasmic and
Golgi resident proteins.2
The selective localization and movement of proteins between the nucleus
and cytoplasm is often influenced by protein phosphorylation. A GFP
fusion of MAPKAP kinase 2, which contains a bipartite NLS, exits the
nuclei of transfected cells under stress following phosphorylation (33). Phosphorylation of the adenomatous polyposis coli protein is
required for its nuclear import (34). We have found that recombinant
Trax can be phosphorylated in testis germ cell nuclear extracts
(unpublished data). Whether a phosphorylated (or nonphosphorylated) form of Trax localizes transiently in the nucleus, thereby helping TB-RBP to enter the nucleus as proposed by Aoki et al. (7), remains to be determined.
TB-RBP Contains a Nuclear Export Signal--
Previous studies have
indicated that TB-RBP exits nuclei of male germ cells toward the end of
meiosis (10), is associated with various mRNAs in germ cell
cytoplasmic extracts (11), and moves through the intercellular bridges
in haploid spermatids (10). Movement of proteins from the nucleus to
the cytoplasm utilizes specific nuclear export signal sequences. The
HIV I Rev-like NES sequence binds directly to a nuclear export receptor
CRM1/Exportin, which is involved in the export of various proteins such
as MAPKK, PKI-alpha, FMRP, and p53 in a RanGTP-dependent
manner (22). Various cellular RNA-binding proteins utilize other
shuttling signals such as the M9 signal in hnRNP A1, KNS signal in
hnRNP K, and HNS signal in HuR (23). Comparing TB-RBP with known NES sequences, we have found a highly conserved putative leucine-rich NES
sequence at its C terminus (Fig. 10). We believe this is a functional
sequence, because when we alter the NES of TB-RBP by site-directed
mutagenesis, the GFP-TB-RBP fusion protein accumulates in the nuclei
(Fig. 9I). This suggests that TB-RBP utilizes a Rev-like NES
to move from the nucleus to the cytoplasm. We propose that TB-RBP has a
role in mRNA binding and export in male germ cells and neuronal
cells where it associates with a specific subset of mRNAs. In the
cytoplasm, Trax interacts with TB-RBP producing a heterodimer with
reduced affinity for RNA leading to the release of mRNAs in the cytoplasm.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-calmodulin kinase II mRNA, Tau mRNA, and
BC1 RNA (11-13). Translationally suppressed mRNAs are bound to
microtubules by TB-RBP in cellular extracts (14) and recombinant TB-RBP
binds specific mRNAs in vitro to reconstituted microtubules (11). The presence of TB-RBP in the cytoplasmic bridges
that connect germ cells in a syncytium, together with the association
of TB-RBP with transported mRNAs and the cytoskeleton (10), suggest
that TB-RBP acts as a transport molecule in the testis for mRNAs in
intracellular (from nucleus to cytoplasm) and intercellular (between
spermatids) mRNA transport. The shift of subcellular localization
of TB-RBP in meiotic and post-meiotic mouse germ cells (10, 15), the
dendritic translocation of BC1 RNA and TB-RBP in rat hippocampal
neurons (13), and the role of TB-RBP for mRNA sorting in dendrites
(16) support this hypothesis.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP using T4
polynucleotide kinase, whereas transcript c was transcribed from a pGEM
3Z plasmid using SP6 RNA polymerase and [
-32P]CTP.
Recombinant proteins were incubated with 40,000 cpm of DNA or RNA probe
for 10 min at RT in 20 µl of binding buffer (20 mM HEPES,
pH 7.6, 3 mM MgCl2, 40 mM KCl, 2 mM DTT, 5% glycerol), and DNA·protein and RNA·protein
complexes were detected following electrophoresis in 4% polyacrylamide
gels in TBE buffer. All electrophoretic mobility shift assays were
performed after incubation of TB-RBP and Trax at 10 times the final
concentration of the proteins in 20 mM HEPES (pH 7.5)
containing 5 mM DTT and 1.5 mM
MgCl2 for 30 min followed by 10× dilution of the mixture
to reduce the DTT concentration to 0.5 mM.
-D-galactopyranoside
(X-gal) filter lift assay on SD medium lacking leucine and
tryptophan. Strength of interaction was determined by the addition of
3-amino-1,2,4-Triazole (Sigma Chemical Co., St. Louis, MO) to the
medium at concentrations of 5-100 mM. The transformation
and filter lift assay procedures were performed following the
manufacturer's instructions (Stratagene).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin cDNA reveals similar
amounts of undegraded mRNAs on the filter (Fig. 1B).
View larger version (30K):
[in a new window]
Fig. 1.
Analysis of expression of Trax.
A, Northern blot analysis of expression of Trax in various
mouse tissues. Total RNAs (10 µg) from brain, heart, kidney, liver,
lung, spleen, and testis (lanes 1-7, respectively) were
hybridized to the complete the open reading frame of a human Trax
cDNA. B, rehybridization of the blot from A
with a mouse -actin cDNA. C, Western blot analysis
for Trax protein expression in various mouse tissues.
Post-mitochondrial extracts (30 µg) from brain, heart, kidney, lung,
liver, spleen, and testis (lanes 1-7, respectively) were
separated by 10% SDS-polyacrylamide gel electrophoresis, transferred
to nylon membranes, and probed with an antibody to Trax. Recombinant
histidine-tagged human Trax (10 ng) is included as a control
(lane 8). D, Western blot analysis (30 µg) of
Trax expression in cytoplasmic and nuclear extracts from: testis
cytoplasm, lane 1; testis nuclei, lane 2; NIH 3T3
cytoplasm, lane 3; and NIH 3T3 nuclei, lane 4.
For C and D, equal protein loading in each lane
was confirmed by Coomassie staining.
View larger version (12K):
[in a new window]
Fig. 2.
Trax and TB-RBP form heterodimers.
Recombinant S-tagged human Trax (200 ng) was added to recombinant mouse
TB-RBP (200 ng) in TBS buffer at 4 °C. The proteins were
immunoprecipitated with S-protein-agarose beads for 30 min at 4 °C.
The pellets were washed three times with TBS containing 0.1% Tween 20, and the precipitate was suspended in 2× SDS-polyacrylamide gel
electrophoresis loading buffer. The proteins were separated in 10%
SDS-polyacrylamide gels and stained with SYPRO Orange. Trax and TB-RBP
in TBS + 0.1% Tween 20, lane 1; in 20 mM Hepes
buffer, pH 7.5, lane 2; and in 20 mM Hepes
buffer, pH 7.5, plus 5 mM DTT, lane 3. Lane 4 is a TB-RBP control (200 ng).
View larger version (59K):
[in a new window]
Fig. 3.
Trax alters nucleic acid binding of
TB-RBP. A, RNA gel shift. 32P-Labeled
transcript c alone, lane 1; transcript c and recombinant
TB-RBP (40 ng), lane 2; transcript c and recombinant TB-RBP
(40 ng) and recombinant Trax (10 ng), lane 3; transcript c
and recombinant TB-RBP (40 ng) and recombinant Trax (20 ng), lane
4; transcript c and recombinant Trax (40 ng) and recombinant Trax
(40 ng), lane 5. 40,000 cpm of transcript c were used in
each assay. Lanes 2-5 were treated with RNase T 1 (10 units) at RT for 10 min. B, DNA gel shift. 40,000 cpm of
32P-labeled BclCL1 DNA alone, lane 1; BclCL1 DNA
plus recombinant TB-RBP (40 ng), lane 2; BclCL1 DNA plus
recombinant TB-RBP (40 ng) plus recombinant Trax (40 ng), lane
3; BclCL1 DNA plus recombinant TB-RBP (40 ng) plus recombinant
Trax (80 ng), lane 4; and BclCL1 DNA plus recombinant TB-RBP
(40 ng) plus recombinant Trax (160 ng), lane 5.
View larger version (56K):
[in a new window]
Fig. 4.
Changes in the nucleic acid binding of TB-RBP
are due to Trax-TB·RBP heterodimer formation. RNA and DNA gel
shifts were performed as described under "Experimental Procedures."
S-Trax, S-tagged Trax; Trx-Trax,
thioredoxin-tagged Trax. 40,000 cpm of 32P-labeled
transcript c alone, lane 1; recombinant TB-RBP (40 ng),
lane 2; recombinant TB-RBP (40 ng) and S-tagged Trax (40 ng), lane 3; recombinant TB-RBP (40 ng) and
thioredoxin-tagged Trax (40 ng), lane 4; and recombinant
TB-RBP (40 ng) and thioredoxin-tagged Trax (80 ng), lane 5. Lanes 6-10 are identical to lanes 1-5
except that 40,000 cpm of 32P-labeled BclCL1 DNA was
substituted for the 40,000 cpm of transcript c used in lanes
1-5. In lane 6, the radiolabeled BclCL1 DNA probe was
run out of the gel.
View larger version (9K):
[in a new window]
Fig. 5.
Schematic representation of putative domains
of TB-RBP. Nb represents a N-terminal basic domain,
Cb represents a second N-basic domain, NES
represents a leucine-rich nuclear export signal, and the leucine zipper
of TB-RBP is indicated. The mouse TB-RBP sequences for Nb, Cb, and NES
are indicated. Site-directed mutagenesis was used to alter the Nb
sequence to NAQEN, the Cb sequence to TFNEN, and the NES sequence to
LASEQSRLSVN.
View larger version (67K):
[in a new window]
Fig. 6.
Effect of Nb and Cb mutations on TB-RBP
binding to RNA. RNA gel shifts were performed as described in Fig.
3A with wild type TB-RBP (40 ng), lanes 3 and
4; Nb mutant TB-RBP (40 ng), lanes 5 and
6; and Cb mutant TB-RBP (40 ng), lanes 7 and
8. Recombinant Trax (40 ng) was added to lanes 2,
4, 6, and 8. To detect binding in
lanes 5 and 6, this gel was overexposed.
-32P-labeled Bcl-CL1 probe with control
recombinant TB-RBP and Trax and with the two TB-RBP proteins with
altered basic domains, TB-Nb and TB-Cb (Fig.
7). As previously demonstrated, DNA
binding of TB-RBP is enhanced by heterodimerization of TB-RBP with Trax
(Fig. 7, compare lane 3 to lane 4). The mutation
in the N-basic domain of TB-RBP does not appear to affect binding to
Bcl-CL1, although a more rapidly migrating DNA·protein complex is
seen (Fig. 7, lane 5). Interestingly, when Trax is added to
TB-Nb under conditions in which heterodimers are formed, no enhanced
DNA binding is seen (Fig. 7, lane 6). The mutation in the
second basic domain of TB-RBP, TB-Cb, abolishes DNA binding completely
(Fig. 7, lane 7). Surprisingly, the addition of Trax,
forming a Trax·TB-Cb heterodimer, restores DNA binding of the TB-Cb
mutant (Fig. 7, lane 8). These data suggest that Trax can
induce changes in mutant TB-RBP conformation, which influence its
binding to DNA.
View larger version (70K):
[in a new window]
Fig. 7.
Effect of Nb and Cb mutations on TB-RBP
binding to DNA. DNA gel shifts were performed as described in Fig.
3B with aliquots of the same protein samples analyzed in
Fig. 6. This gel was overexposed to detect any weakly binding
complexes.
View larger version (126K):
[in a new window]
Fig. 8.
TB-RBP and Trax interactions in a yeast
two-hybrid assay. The open reading frames of TB-RBP, its
mutant alleles, and Trax were cloned in-frame into both the pBDGAL4cam
(binding domain (BD)) and pADGAL4 (activation domain (AD)) plasmids.
Filter lift assays for -galactosidase activity were done on
cotransformants streaked on synthetic minimal (SD) medium plates
lacking leucine and tryptophan. All pairs interact except for BD
Trax × AD Trax. A, BD TB-RBP × AD TB-RBP;
B, BD TB-RBPxAD Trax; C, BD Trax × AD Trax;
D, BD TB-RBP × AD TB-Nb; E, BD TB-Nb × AD TB-Nb; F, BD TB-RBP × AD TB-Cb; G, BD
TB-Cb × AD TB-Cb; H, BD TB-RBP × AD
TB-NES; I, BD TB-NES × AD
TB-NES; J, BD Trax × AD TB-Nb;
K, BD Trax × AD TB-Cb; and L, BD Trax × AD TB-NES.
View larger version (61K):
[in a new window]
Fig. 9.
Confocal micrographs showing localization of
TB-RBP and Trax in transiently transfected mouse NIH 3T3
fibroblasts. The cells were observed 18-h post-infection.
A, control pEGFP transfection; B, transfection of
N-terminal fusion protein of EGFP and TB-RBP (pEGFP-TB-RBP);
C, transfection of N-terminal fusion protein of EBFP to Trax
(pEBFP-Trax); D-F, cytoplasmic colocalization of TB-RBP and
Trax. D, pEGFP-TB-RBP; E, pEBFP-Trax;
F, merge of D and E; G,
BODIPY and EGFP-Trax; H, BODIPY and EGFP-Trax and brefeldin
A (30 µM incubation for 1 h at 37 °C);
I, transfection with pEGFP-TB-RBPNES.
Magnification, × 40.
View larger version (29K):
[in a new window]
Fig. 10.
TB-RBP contains a highly conserved nuclear
export signal. A, nuclear export signals from known
shuttling proteins; B, TB-RBP nuclear export signals in
various species.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant HD28832 (to N. B. H.) and by Training Grant T32HD07305 (to J. D. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Both authors contributed equally to this work.
To whom correspondence should be addressed: Center for
Research on Reproduction and Women's Health, University of
Pennsylvania Medical School, 1310 Biomedical Research Building II/III,
421 Curie Blvd., Philadelphia, PA 19104-6142. Tel.: 215-898-0144; Fax:
215-573-5408; E-mail: nhecht@mail.med.upenn.edu.
Published, JBC Papers in Press, January 26, 2001, DOI 10.1074/jbc.M009707200
2 V. M. Chennathukuzhi, Y. Kurihara, J. D. Bray, and N. B. Hecht, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TB-RBP, testis brain RNA-binding protein; Trax, Translin-associated factor X; NLS, nuclear localization signal; NES, nuclear export signal; TBS, tris-buffered saline; DTT, dithiothreitol; RT, room temperature; GFP, green fluorescence protein; BFP, blue fluorescence protein; PBS, phosphate-buffered saline; kb, kilobase(s); ER, endoplasmic reticulum; hnRNP, heterogeneous nuclear ribonucleoprotein.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Hecht, N. B. (1998) Bioessays 20, 555-561[CrossRef][Medline] [Order article via Infotrieve] |
2. | Wu, X. Q., Gu, W., Meng, X., and Hecht, N. B. (1997) Proc. Natl. Acad. Sci. U. S. A. 27, 5640-5645[CrossRef] |
3. | Aoki, K., Suzuki, K., Sugano, T., Tasaka, T., Nakahara, K., Kuge, O., Omori, A., and Kasai, M. (1995) Nat. Genet. 10, 167-174[Medline] [Order article via Infotrieve] |
4. | Kanoe, H., Nakayama, T., Hosaka, T., Murakami, H., Yamamoto, H., Nakashima, Y., Tsuboyama, T., Nakamura, T., Ron, D., Sasaki, M. S., and Toguchida, J. (1999) Oncogene 18, 721-729[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Jeffs, A. R.,
Benjes, S. M.,
Smith, T. L.,
Sowerby, S. J.,
and Morris, C. M.
(1998)
Hum. Mol. Genet.
7,
767-776 |
6. | Atlas, M., Head, D., Behm, F., Schmidt, E., Zeleznik-Le, N. H., Roe, B. A., Burian, D., and Domer, P. H. (1998) Leukemia 12, 1895-1902[CrossRef][Medline] [Order article via Infotrieve] |
7. | Aoki, K., Ishida, R., and Kasai, M. (1997) FEBS Lett. 20, 109-112 |
8. | Taira, E., Finkenstadt, P. M., and Baraban, J. M. (1998) J. Neurochem. 71, 471-477[Medline] [Order article via Infotrieve] |
9. | Hecht, N. B. (2000) Mol. Reprod. Dev. 56, 252-253[CrossRef] |
10. | Morales, C. R., Wu, X. Q., and Hecht, N. B. (1998) Dev. Biol. 201, 113-123[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Wu, X. Q.,
and Hecht, N. B.
(2000)
Biol. Reprod.
62,
720-725 |
12. | Han, J. R., Gu, W., and Hecht, N. B. (1995) Biol. Reprod. 53, 707-717[Abstract] |
13. | Kobayashi, S., Takashima, A., and Anzai, K. (1998) Biochem. Biophys. Res. Commun. 253, 448-453[CrossRef][Medline] [Order article via Infotrieve] |
14. | Han, J. R., Yiu, G. K., and Hecht, N. B. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9550-9554[Abstract] |
15. | Gu, W., Wu, X. Q., Meng, X. H., Morales, C., el-Alfy, M., and Hecht, N. B. (1998) Mol. Reprod. Dev. 49, 219-228[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Severt, W. L.,
Biber, T.,
Wu, X.-Q.,
Hecht, N. B.,
DeLorenzo, R. J.,
and Jakoi, E. R.
(1999)
J. Cell Sci.
112,
3691-3702 |
17. | Devon, R. S., Taylor, M. S., Millar, J. K., and Porteous, D. J. (2000) Mamm. Genome 11, 395-398[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Kasai, M.,
Matsuzaki, T.,
Katayanagi, K.,
Omori, A.,
Maziarz, R. T.,
Strominger, J. L.,
Aoki, K.,
and Suzuki, K.
(1997)
J. Biol. Chem.
272,
11402-11407 |
19. | Wu, X. Q., Lefrancois, S., Morales, C. R., and Hecht, N. B. (1999) Biochemistry 38, 11261-11270[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Wu, X. Q.,
Xu, L.,
and Hecht, N. B.
(1998)
Nucleic Acids Res.
26,
1675-1680 |
21. | Aoki, K., Suzuki, K., Ishida, R., and Kasai, M. (1999) FEBS Lett. 443, 363-366[CrossRef][Medline] [Order article via Infotrieve] |
22. | Henderson, B. R., and Eleftheriou, A. (2000) Exp. Cell Res. 256, 213-224[CrossRef][Medline] [Order article via Infotrieve] |
23. | Moroianu, J. (1999) J. Cell. Biochem. Suppl. 32-33, 76-83 |
24. | Emmons, R. B., Duncan, D., Estes, P. A., Kiefel, P., Mosher, J. T., Sonnenfeld, M., Ward, M. P., Duncan, I., and Crews, S. T. (1999) Development 26, 3937-3945 |
25. |
Zhou, X. F.,
Shen, X. Q.,
and Shemshedini, L.
(1999)
Mol. Endocrinol.
13,
276-285 |
26. | LaBranche, H., Dupuis, S., Ben-David, Y., Bani, M. R., Wellinger, R. J., and Chabot, B. (1998) Nat. Genet. 19, 199-202[CrossRef][Medline] [Order article via Infotrieve] |
27. | Weighardt, F., Biamonti, G., and Riva, S. (1996) Bioessays 18, 747-756[Medline] [Order article via Infotrieve] |
28. |
Johnston, S. D.,
Lew, J. E.,
and Berman, J.
(1999)
Mol. Cell. Biol.
19,
923-933 |
29. | Brent, R., and Finnley, R. L., Jr. (1997) Annu. Rev. Genet. 31, 663-704[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Kruyt, F. A.,
Waisfisz, Q.,
Dijkmans, L. M.,
Hermsen, M. A.,
Youssoufian, H.,
Arwert, F.,
and Joenje, H.
(1997)
Blood
90,
3288-3295 |
31. | Finkenstadt, P. M., Kang, W.-S., Jeon, M., Taira, E., Tang, W., and Baraban, J. M. (2000) J. Neurochem. 75, 1754-1762[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Wang, Q. T.,
and Holmgren, R. A.
(1999)
Development
126,
5097-5106 |
33. |
Engel, K.,
Kotlyarov, A.,
and Gaestel, M.
(1998)
EMBO J.
17,
3363-3371 |
34. |
Zhang, F.,
White, R. L.,
and Neufeld, K. L.
(2000)
Proc. Nat. Acad. Sci. U. S. A.
97,
12577-12582 |