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J. Biol. Chem., Vol. 275, Issue 29, 22001-22008, July 21, 2000
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From the Department of Human Biological Chemistry and Genetics,
University of Texas Medical Branch, Galveston, Texas 77555-0645
Received for publication, February 8, 2000, and in revised form, May 5, 2000
Glucocorticoids regulate the expression of the
G1 progression factor, cyclin D3. Cyclin D3 messenger
RNA (CcnD3 mRNA) stability decreases rapidly when murine T lymphoma
cells are treated with the synthetic glucocorticoid dexamethasone.
Basal stability of CcnD3 mRNA is regulated by sequences within the
3'-untranslated region (3'-UTR). RNA-protein interactions occurring
within the CcnD3 3'-UTR have been analyzed by RNA electrophoretic
mobility shift assay. Three sites of RNA-protein interaction have been mapped using this approach. These elements include three
pyrimidine-rich domains of 25, 26, and 37 nucleotides. When the cyclin
D3 3'-UTR was stably overexpressed, the endogenous CcnD3 mRNA was
no longer regulated by dexamethasone. Likewise, overexpression of a
215-nucleotide transgene that contains the 26- and 37-nucleotide
elements blocks glucocorticoid inhibition of CcnD3 mRNA expression.
These observations suggest that the 215-nucleotide 3'-UTR element may
act as a molecular decoy, competing for proteins that bind to the
endogenous transcript and thereby attenuating glucocorticoid
responsiveness. UV-cross-linking experiments showed that two proteins
of approximate molecular weight 37,000 and 52,000 bind to this 3'-UTR element.
Malignant lymphoid cells of thymic origin cease to proliferate and
often die when exposed to glucocorticoids (1, 2), and glucocorticoids
are an important tool for treatment of leukemias and other malignant
and nonmalignant lymphoproliferative diseases. Glucocorticoids have
also been proposed to be responsible for triggering apoptosis of
CD4+/CD8+ cells in the thymus (reviewed in Ref. 3). We have analyzed
glucocorticoid effects on murine T lymphoma P1798 cells in an attempt
to elucidate the molecular mechanisms that account for glucocorticoid
inhibition of cell proliferation and induction of cell death. The data
indicate that glucocorticoids induce G0/G1
arrest by decreasing the expression of two critical cell G1
progression factors: c-Myc (4) and cyclin D3 (5). P1798 cells utilize
cyclin D3 as the principal D-type cyclin in G1/S phase
transition (6). Cyclin D2 is barely detectable and cyclin D1 is
undetectable in P1798 cells (5). Consequently, inhibition of cyclin D3
blocks activation of Cdk4/Cdk6 and precludes progression through
G1 phase (5). Simultaneous overexpression of c-Myc plus
cyclin D3 prevents cell cycle arrest and apoptosis of
glucocorticoid-treated cells, although neither c-Myc nor cyclin D3
alone will suffice to protect cells (7).
Glucocorticoids control cyclin D3 expression in T-lymphoid cells by
decreasing the stability of CcnD3 mRNA (5). CcnD3 mRNA is quite
stable in mid-log phase P1798 cells, as determined by measuring
mRNA abundance after treatment with actinomycin D. However, the
rate of degradation of CcnD3 mRNA increases within 2 h after addition of glucocorticoids, to the extent that a 50% decrease in
mRNA abundance occurs within 60-90 min after addition of
actinomycin D to glucocorticoid-treated cells (5). This
posttranscriptional mechanism of gene expression, coupled with the
short half-life of the cyclin D3 protein, ensures a rapid response to
glucocorticoids. There are several examples of glucocorticoid effects
on mRNA stability (8-10), but the molecular mechanisms that
underlie such events are largely unknown.
During the last decade, several RNA motifs have been identified that
control the degradation rate of specific mRNAs. Iron-responsive elements (reviewed in Ref. 11) influence the stability of the transcripts of several genes that are involved in iron metabolism. AU-rich sequences (10, 12, 13), and polypyrimidine tracts (14-16) are
known to control the stability of some mRNAs. Several families of
RNA-binding proteins have been implicated in controlling the stability
of mRNAs. The heterogeneous nuclear ribonucleoprotein (hnRNP)1 complexes involved
in maturation of mRNAs contain more than 20 protein species, many
of which are involved in mRNA stability control (17, 18).
Vertebrate homologues of the Drosophila embryonic lethal
abnormal vision protein family regulate the stability of mRNAs
encoding tumor necrosis factor- The experiments described in this paper were designed to test the
hypothesis that the 3'-UTR of CcnD3 mRNA contains specific protein-binding elements that are involved in glucocorticoid regulation of degradation of the mature transcript. We have undertaken to analyze
RNA-protein interactions within the 3'-UTR, to map the nucleotide
components that are required for such interactions, and to characterize
the RNA-binding proteins with which these elements interact. Our
results indicate that there is a protein-binding element within the
3'-UTR of CcnD3 mRNA. This element interacts with at least two
proteins, and this interaction is necessary for glucocorticoid-mediated
destabilization of CcnD3 mRNA.
Cell Culture, Treatment, and RNA Isolation--
Murine
T-lymphoma P1798-S20 cells were maintained in suspension culture in
RPMI 1640 medium supplemented with 3 mM glutamine, 25 mM HEPES, 20 mM 2-mercaptoethanol, and 2%
fetal bovine serum, at 37 °C in 5% CO2. Mid-log phase
cultures containing 5-8 × 105 cell/ml were used for
all experiments. Dexamethasone was dissolved in 70% ethanol as a 0.1 mM stock, and this was diluted into medium to a final
concentration of 0.1 µM dexamethasone and 0.07% ethanol in all experiments. Ethanol has no effect on P1798 cultures at this
concentration. RNA isolation was performed using TRIzol reagent from
Life Technologies Inc. Northern blotting was carried out under standard
conditions, and all Northern blotting data were normalized to 18 S RNA.
Autoradiographic data were quantified using a Lynx 5000 digital workstation.
Preparation of Cytosolic Protein Extracts (S100)--
Cytosolic
protein extracts were prepared using the method described by Sun and
Antony (24). Cells were washed with 5 packed cell volumes of
phosphate-buffered saline by centrifugation at 2000 rpm in a Beckman
JA-14 rotor at 4 °C. Cells (5 × 107) were
incubated at 4 °C in Buffer A (10 mM HEPES, pH 7.9, containing 1.5 mM MgCl2, 10 mM KCl,
0.5 mM dithiothreitol) for 10 min at a ratio of 5 ml of
Buffer A per ml of packed cell volume. The cells were centrifuged (5 min at 2000 rpm in a Beckman JA-14 rotor), suspended in 2 ml of Buffer
A per ml of packed cells, and then lysed by 10 strokes of a glass
Dounce homogenizer (B pestle). Cell lysis was confirmed by microscopy.
The lysate was centrifuged at 2000 rpm for 10 min at 4 °C. The
resultant supernatant fraction was mixed with 0.11 supernatant volumes
of Buffer B (300 mM HEPES, pH 7.0, containing 10 mM MgCl2, 1.4M KCl), and this solution was centrifuged for 60 min at 100,000 × g. The supernatant
fraction was recovered and dialyzed for 8 h against 20 volumes of
Buffer C (20 mM HEPES, pH 7.9, containing 100 mM KCl, 0.2 mM EDTA 20% (v/v) glycerol, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM
dithiothreitol). Aliquots (100 µl) were frozen at -80 °C, and once thawed, they were never reused.
Transcription in Vitro--
The MAXIscript kit from AMBION Inc.
was used for the synthesis of cyclin D3 3'-UTR probes.
[ RNA Mobility Shift Assays--
RNA-protein binding reactions and
electrophoresis of the complexes formed were carried out using a
variation of the method described by Leibold and Munro (25). Binding
reactions were carried out with various amounts of cytosolic extract
and 15 pg of 32P-labeled RNA probe in 30 µl of Buffer D
(containing 10 mM HEPES, pH 7.6, 3 mM
MgCl2, 40 mM KCl, 2% glycerol, 1 mM dithiothreitol, and 5 mg/ml heparin). Cytosolic protein
extracts were diluted in Buffer C (described above) to equalize the
volumes among different reactions in each experiment. Reactions
performed with the CcnD3 3'-UTR probe were incubated for 20 min at
30 °C, and then 20 units of ribonuclease T1 were added and the
reaction was incubated for another 20 min at 30 °C. Under these
conditions, the ribonuclease T1 digestion is incomplete. In one
experiment, proteinase K was added to a final concentration of 2 mg/ml
for 20 min at 30 °C.
Reactions performed with the BC probe (1575-1789) were incubated for
30 min at 30 °C and contained 3.3 ng/µl of yeast tRNA as a
nonspecific competitor, and there was no ribonuclease T1 digestion of
the BC-protein complex prior to electrophoresis. Binding conditions
were otherwise identical to those described for the full-length 3'-UTR.
In competition experiments, labeled probe was mixed with the unlabeled
probes before addition of the cytosolic extracts. Electrophoresis of
RNA-protein complexes was carried out in 5% nondenaturing
polyacrylamide gels. Autoradiography was performed at -80 °C.
Ribonuclease T1 Mapping of RNA-Protein
Complexes--
RNA-protein complexes were resolved by electrophoretic
mobility shift. These complexes were excised from polyacrylamide gels, and the labeled RNA was isolated by phenol-chloroform extraction. The
isolated RNA fragments were digested in 10 mM Tris-HCl, pH 7.4, containing 1 mM EDTA and 20 units of ribonuclease T1
for 60 min at 37 °C. Ribonuclease T1-resistant fingerprint fragments were separated by electrophoresis in 8 M urea and 0.1%SDS
on 20% polyacrylamide gels.
UV Cross-linking of RNA/Protein Complexes--
The method used
to cross-link the BC probe to the RNA-binding proteins was performed as
described by Chang et al. (26) with some modifications.
Binding reactions were carried out with various amounts of cytosolic
extract and 32P-labeled RNA probe in Buffer D containing
0.1 µg/µl of yeast tRNA. After 20 min at 30 °C the samples were
transferred to ice and irradiated for 30 min with a UV light source
(254 nm, 15 W) at a distance of approximately 4 cm. After irradiation,
RNA was digested with 1 mg/ml of ribonuclease A for 1 h at
37 °C. The UV cross-linked products were separated on a 12%
SDS-polyacrylamide gel and detected by autoradiography.
Tetracycline-repressible Expression Vectors and Cell
Lines--
pUHD10-4 was constructed by replacing the
polylinker of pUHD10-3 (26) with a multiple cloning site that contains unique SacII (442), EcoRI (449), EcoRV
(457), SalI (462), AccI (463), PstI
(473), BglII (475), XbaI (481), and
BamHI (487) sites. Numbers in parentheses are positions
relative to the XhoI site of the parental pUHD10-3.
pUHD10-5 was made by replacing the polylinker of pUHD10-3
with a multiple cloning site that contains unique sites for
SacII (446), NotI (446), EcoRV (455),
SphI (469), AvrII (470), and PacI
(480). pUHDZeo4 was made by ligating the
XhoI/PvuII fragment of pZeo (InVitrogen) with the
XhoI/PvuII fragment of pUHD10-4.
pUHDZeo5 was made by ligating the XhoI/NaeI fragment of pUHD10-5 into the
XhoI/PvuII fragment of pZeo. These pUHDZeo
plasmid conveys stable resistance to the antibiotic zeocin. Modified
tetracycline-repressible expression vectors and their nucleotide
sequences are available upon request.
The 1878-base pair EcoRI fragment containing full-length
CcnD3 cDNA was cloned into pUHDZeo4 to create p4ZD3FL
(full-length), which we have designated tetD3FL. p4ZD3FL was digested
with XbaI to excise the 3'-UTR and then ligated to form
p4ZD3D3P (
p5z
P1798 cells were initially transfected to stably express the
tetracycline transactivator from pUHD15-1neo (27). G418-resistant clones were screened for transient expression of pUHD13-3
(tet/luciferase). Secondary transfections with appropriate expression
vectors were carried out. Some of the expression vector harbor linked
zeocin resistance genes; others were co-transfected with pZeo. Stable, zeocin-resistant clones were selected and analyzed for expression of
the appropriate transgenes.
Cyclin D3 mRNA is very stable in P1798 cells treated with
actinomycin D. As shown in Fig.
1A, there was little or no
decrease in CcnD3 mRNA within the first 4 h after addition of
actinomycin D to mid-log phase cells. In other experiments, we observed
no decrease in CcnD3 mRNA after 24 h in the presence of
actinomycin D (5), which leads us to suspect that actinomycin D affects the turnover of CcnD3 mRNA. Data that will be presented below are
consistent with this supposition. Addition of dexamethasone accelerated
CcnD3 mRNA degradation, to the extent that 50% decrease in CcnD3
mRNA abundance was observed within about 2 h after addition of
actinomycin D to cells that had been exposed to dexamethasone for
2 h (Fig. 1A). These data suggest that glucocorticoids
regulate the rate of degradation of CcnD3 mRNA.
The great majority of mRNA stability control mechanisms reported to
date have been found in the 3'-UTRs of the respective transcripts.
Consequently, our first objective was to analyze the turnover of cyclin
D3 mRNA derivatives with and without the 3'-UTR.
Tetracycline-repressible expression systems were developed to permit
analysis of mRNA abundance without resort to nonspecific inhibitors
of transcription, such as actinomycin D. The development of the
appropriate cell lines is described under "Materials and Methods,"
and Fig. 1B shows the results obtained upon analysis of two
cell lines that stably express full-length cyclin D3 mRNA (tetD3FL)
or cyclin D3 mRNA from which the 3'-UTR had been deleted (tetD3 The rapid turnover of full-length transgenic tetD3 mRNA,
(t1/2 of about 2 h) is inconsistent with the
data shown in Fig. 1A, in which the abundance of endogenous CcnD3 mRNA remained relatively constant for several hours after addition of actinomycin D. These observations suggested that
actinomycin D might interfere with degradation of cyclin D3 mRNA.
The rate of degradation of transgenic tetD3FL mRNA was measured in
cells that had been treated with actinomycin D for 2 h prior to
addition of tetracycline. As shown in Fig. 1C
(diamonds), no significant decrease in the abundance of the
full-length tetD3 transcript was observed within 4 h after
addition of tetracycline to actinomycin D-treated cells.
This apparent stability should be contrasted with the rapid rate of
degradation that occurs upon addition of tetracycline to
tetD3FL-expressing cultures in the absence of actinomycin D (Fig.
1C, circles). We conclude that actinomycin D has an effect
on the basal stability of cyclin D3 mRNA, but this phenomenon has
not been pursued.
Cells that express tetD3 derivatives were treated for 24 h with
dexamethasone, as shown in Fig. 1B. Neither the full-length tetD3 transcript (lane 3) nor the 3'-truncated tetD3 The data shown in Fig. 1B suggest that overexpression of the
cyclin D3 3'-UTR from a transgene (tetD3FL) interfered with
glucocorticoid regulation of the endogenous CcnD3 mRNA expression.
The abundance of the tetD3FL transcript, when maximally derepressed by
withdrawal of tetracycline, never exceeded five times that of the
endogenous CcnD3 mRNA (Fig. 1B), suggesting that the
effect prevailed at relatively low concentrations of the transgenic
3'-UTR. We concluded that the transgenic D3 3'-UTR was acting in trans
to block glucocorticoid-mediated destabilization of CcnD3 mRNA. To
test this prediction, a number of P1798 cell lines were generated that
stably expressed the
Glucocorticoid-mediated Destabilization of Cyclin D3 mRNA
Involves RNA-Protein Interactions in the 3'-Untranslated Region of
the mRNA*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(19), N-Myc (20, 21), and the
cyclin-dependent kinase inhibitor p21Cip1 (22). Regulation
of replication-dependent histone mRNAs involves
specific RNA-protein interactions within the 3'-UTR. Processing and
degradation of those mRNAs are mediated by a 26-base stem-loop
structure and a Mr 31,000 protein (23). The
general conclusions that one may advance are that 1) mRNA stability
is frequently determined by RNA-protein interactions, and 2) those
interactions frequently occur within the 3'-UTR.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]CTP (800Ci/mmol, NEN Life Science Products,
Inc.) was used to label the probes. The full-length 3'-UTR of murine
CcnD3 mRNA from nucleotide 933 to 1871 was amplified by polymerase
chain reaction and cloned into InVitrogen pCRII to yield pCRII/D3UTR. This plasmid was digested with BamHI, and run-off
transcription was performed from the T7 promoter to generate the D3
3'-UTR probe (specific activity 217.8Ci/µM). Nucleotides
933-1574 of the cyclin D3 cDNA were eliminated by digesting
pCRII/D3UTR with StuI and EcoRV and ligating the
blunt ends to generate a plasmid named pCRII/D3UTR
1-1574.
pCRII/D3UTR1-1574 was digested with AvrII (which cuts at
1789), and run-off transcription was performed with T7 RNA polymerase
to yield a probe corresponding to nucleotides 1575-1789. This probe
was named BC (specific activity, 54.4Ci/µM). The probes
were purified by electrophoresis in 8 M urea on 5% polyacrylamide gels.
3'-UTR), which is designated tetD3-UTR. The
215-nucleotide BC element was excised as a StuI/AvrII
fragment from pCRIID3FL and cloned into the
EcoRV/XbaI sites of pUHD4Zeo to form p4zBC. The
p4zBC transcript has a predicted size of about 250 nucleotides and is terminated by SV40 polyadenylylation signals. Transcribed from the
tetO/human cytomegalovirus major immediate early TATA box (HCMV)
chimeric promoter, the p4zBC transcript is presumed to be capped. We
have not been able to quantify this transcript, and we have not
ascertained that the transcript is either capped or polyadenylylated.
Gal was made by ligating the
NotI/AhaIII fragment of cytomegalovirus/Gal
(Promega) into the NotI/EcoRV sites of pUHDZeo5.
This expression vector is identified herein as -gal. p5BGD3UTR was made by inserting the
SpeI/XbaI fragment from pCRII/D33-UTR (containing
the 3'-UTR) into the AvrII site of
p5zGal. This transgene is called
-galD3UTR herein.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The 3'-UTR of CcnD3 mRNA affects turnover
of transgenic tetD3 transcripts. In the experiment shown in
A, wild type P1798 cells were treated with 1 µg/ml
actinomycin D (AcD). A second group (Dex then
AcD) was treated with dexamethasone for 2 h prior to addition
of actinomycin D at 0 h. Total RNA was extracted at the indicated
times, and CcnD3 mRNA was detected by Northern blotting. Two cell
lines were prepared that express full-length cyclin D3 mRNA
(tetD3FL) and cyclin D3 mRNA from which the 3'-UTR was
deleted (tetD3 UTR), and two representative
clones were analyzed for transgenic tetD3 and endogenous CcnD3 mRNA
expression using an RNase protection assay, as illustrated in
B. RNA was extracted from mid-log phase cultures
(lanes 1 and 4) or from cultures that had been
exposed to 0.1 µg/ml tetracycline for 24 h (lanes 2 and 5). The samples analyzed in lanes 3 and
6 were extracted from cells that had been treated for 6 h with 0.1 µM dexamethasone. In the experiment shown in
C, the abundance of transgenic full-length tetD3FL mRNA
(circles) and tetD3UTR mRNA (squares) was
measured as a function of time after addition of tetracycline to
control cells (filled symbols) or to cells that had been
treated with dexamethasone for 24 h (open symbols). A
third culture of cells that express tetD3FL was treated with 1 µg/ml
of actinomycin D for 2 h before addition of tetracycline, and the
abundance of tetD3 mRNA was measured as a function of time
thereafter (filled diamonds).
-UTR). The properties of the two clonal cell lines used in
these experiments are representative of at least four independently isolated clones that express either full-length or truncated D3 transgenes from the tet promoter. A ribonuclease protection assay was
developed to discriminate between the transgenic tetD3 transcripts and
the endogenous CcnD3 mRNA. Expression of both full-length and
3'-truncated tetD3 transcripts was repressed upon addition of
tetracycline (Fig. 1B, lanes 2 and 5), whereas
expression of the endogenous CcnD3 mRNA was not affected. The cell
lines illustrated in Fig. 1B were used to measure the
stability of full-length and truncated tetD3 transcripts, as shown in
Fig. 1C. The abundance of full-length tetD3 transcripts
decreased 50% within about 2 h after addition of tetracycline
(filled circles). In contrast, the tetD3 derivative that was
deleted of the 3'-UTR exhibited a slower rate of degradation
(filled squares). We estimate a T1/2 of degradation of about
12 h for the 3'-deleted transcript. These data suggest that there
are elements within the 3'-UTR of cyclin D3 mRNA that control basal
stability of the transcript.
-UTR
derivative (lane 6) was regulated by glucocorticoids. The
stability of both full-length and tetD3-UTR transcripts was
determined by measuring the abundance of the RNAs in
dexamethasone-treated cells after addition of tetracycline. As shown in
Fig. 1C, dexamethasone did not affect the stability of
transgenic tetD3 transcripts (open circles and open
squares). This result was unanticipated, because we had previously
shown that glucocorticoids do not inhibit transcription of the cyclin
D3 gene (5) and that endogenous CcnD3 mRNA is degraded more rapidly
in dexamethasone-treated cells (Fig. 1A). We were also
surprised to note that dexamethasone failed to inhibit the expression
of the endogenous CcnD3 mRNA in cells that were transfected with
the full-length tetD3 cDNA expression vectors (Fig. 1B, lane
3), whereas CcnD3 mRNA was inhibited when dexamethasone was
added to cells that express the 3'-truncated derivative (lane 6).
-galactosidase gene, with or without the cyclin
D3 3'-UTR, under the control of a tetracycline-repressible promoter.
All of the clones that were subsequently analyzed for cyclin D3
expression (six clones) exhibited essentially the same properties as
the two clones for which -galactosidase activity data are shown in
Fig. 2A. Four such clones were
analyzed, and all showed essentially the same properties. The
open bars in Fig. 2A illustrate the properties of
those clones that express -gal, whereas the filled bars
illustrate the properties of those cells that express -galactosidase
fuse to the D3 3'-UTR chimera (-galD3UTR). Initially, we noted that the level of expression of the -galD3UTR chimeric gene was
consistently lower than that of authentic -gal. (Note that the
scales are different in Fig. 2A.) We have shown that the D3
3'-UTR increases the turnover of tetD3 transgenic RNAs, and we suspect
that this reproducible difference in -galactosidase activity may be
due to an increased turnover of -galD3UTR transcripts; however, the rates of degradation of the -galactosidase mRNAs have not been measured. We also observed that dexamethasone had no effect on -galactosidase expression, irrespective of the presence or absence of the D3 3'-UTR. This observation is consistent with the data shown in
Fig. 1B, which indicate that the presence or absence of the
D3 3'-UTR did not affect glucocorticoid regulation of the tetD3
transgenes. Finally, tetracycline inhibition of -galactosidase activity was >95% for the cell line illustrated in Fig. 2A
and for all cell lines used in the experiments described below. Because tetracycline regulates the transcription of the transgene, it is
reasonable to assume that the decrease in -galactosidase activity that can be observed upon addition of tetracycline is associated with
and attributable to a corresponding decrease in -galactosidase mRNA.
View larger version (16K):
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Fig. 2.
The CcnD3 3'-UTR attenuates glucocorticoid
regulation in trans. Cell lines were engineered
to express -galactosidase transgenes with and without the CcnD3
3'-UTR. Several clones were isolated and screened for tetracycline
regulation, and the data from two representative clones are shown in
A. The open bars illustrate the activity of
-galactosidase in cells that express wild type -gal mRNA, and
the filled bars represent the activity detected in cells
that express -galD3UTR chimeric transgenes. Note that the scale for
wild type -gal (left axis) is different from that for the
-galD3UTR chimera (right axis). Activity was measured in
control (mid-log phase) cells, in cells that had been treated for
24 h with dexamethasone (Dex), and in cells that had
been treated for 24 h with 0.1 µg/ml tetracycline
(tet). Clones that exhibited >90% repression of
-galactosidase activity after addition of tetracycline were selected
for further analysis. B and C illustrate the
results obtained when CcnD3 mRNA abundance was measured after
addition of dexamethasone to two clones that express -gal
transgenes. Mid-log phase cultures were used for all experiments. CcnD3
mRNA was measured in control cultures (filled circles)
or in cultures that had been treated for 24 h with 0.1 µg/ml
tetracycline. D-G show the results of similar experiments
in which CcnD3 mRNA was measured after addition of dexamethasone to
cultures that express -galD3UTR chimeric transgenes. Each panel
represents an independently isolated clone.
Glucocorticoid regulation of CcnD3 mRNA was measured in several
clones that express -gal and -galD3UTR transgenes, as shown in
Fig. 2. Fig. 2, B and C, illustrates the
properties of two clones that express authentic -gal mRNA. The
abundance of CcnD3 mRNA decreased rapidly upon addition of
dexamethasone, indicating that expression of -galactosidase had no
effect on glucocorticoid-mediated inhibition of CcnD3 mRNA
abundance (filled circles). Conversely, little or no
decrease in CcnD3 mRNA was observed when dexamethasone was added to
galD3UTR clones, as shown in Fig. 2, D-G (filled circles). These results indicate that cells that express the
-galactosidase/cyclin D3 3'-UTR chimera were resistant to
glucocorticoid inhibition of cyclin D3 expression. The abundance of
CcnD3 mRNA decreased rapidly when dexamethasone was added to
galD3UTR cells that had been treated with tetracycline (Fig. 2,
D-G, open circles), indicating that glucocorticoid
inhibition of CcnD3 mRNA expression was restored when transcription
of the -galD3UTR chimeric transgene was repressed. Tetracycline had
no effect upon glucocorticoid inhibition of cyclin D3 expression in
cells that express -galactosidase without the CcnD3 3'-UTR (Fig. 2,
B and C, open circles).
The data shown in Figs. 1 and 2 suggest that there are sequences within
the D3 3'-UTR that influence glucocorticoid regulation of the stability
of CcnD3 mRNA. These elements appear to act in trans, to the extent
that overexpression of the 3'-UTR from a transgene (tetD3FL or
-galD3UTR) interferes with regulation of the endogenous CcnD3
mRNA. The data are consistent with the hypothesis that sequences
within the transgenic 3'-UTR are acting as molecular decoys, titrating
cellular proteins that would otherwise interact with the endogenous
transcript to affect glucocorticoid regulation of CcnD3 mRNA stability.
RNA mobility shift assays were used to identify potential RNA-protein
interactions within the CcnD3 3'-UTR. The full-length CcnD3 3'-UTR was
transcribed in vitro in the presence of
-32P-labeled nucleoside triphosphates. The full-length,
labeled 3'-UTR transcript, ~1000 bases long, was incubated with S-100
cytoplasmic extracts, which were prepared from exponentially growing
P1798 cells. Ribonuclease T1 was added to degrade those regions of the transcript that were not protected by stable RNA-protein complexes. The
T1 resistance complexes were then resolved by electrophoresis on
nondenaturing polyacrylamide gels, as shown in Fig.
3. Lane 1 of Fig.
3A contains a ribonuclease T1-resistant RNA-protein complex
(arrow). Formation of this complex was precluded by addition of 120 ng of unlabeled CcnD3 3'-UTR (lane 2) However,
addition of 120 ng of unlabeled E. coli -galactosidase
mRNA failed to compete for binding of S100 proteins to the CcnD3
3'-UTR (lane 3).
|
Fig. 3B shows the results of an experiment in which labeled CcnD3 3'-UTR was incubated with increasing amounts of S100 protein. The results indicate that formation of the gel shift entity requires the presence of S100 protein (lane 1) and is not simply a ribonuclease T1-resistant fragment. The intensity of the shifted band increased as a function of the protein concentration in the binding reaction (lanes 2 and 3). Furthermore, the shifted band was abolished when the ribonuclease T1-resistant complex was treated with proteinase K before electrophoresis (lane 4), indicating that the labeled band that we observed results from formation of an RNA-protein complex.
An RNA mobility shift assay was performed, and the RNA-protein complex
was excised from the gel. The labeled RNA was isolated and digested to
completion with ribonuclease T1. The products of ribonuclease T1
digestion were separated by electrophoresis on 20% polyacrylamide gels
containing 8 M urea, as shown in Fig. 4A. Lane 1 contains
a 10-nucleotide DNA ladder, the largest fragment shown being 50 bases.
The RNA gel shift entity contains three predominant ribonuclease T1
resistant fragments, as shown in lane 2. The sizes of these
fragments, estimated by comparison to the DNA ladder, correspond to a
fragment of 35-40 bases and two fragments comprising a doublet of
25-30 bases. These fragments may be precisely identified by comparison
to the limit ribonuclease T1 digest of the labeled 3'-UTR (lane
3), and consist of ribonuclease T1 fingerprint fragments of 25, 26, and 37 nucleotides. The ribonuclease T1 pattern of digestion of the
3'-UTR can be predicted by searching for sequences that contain no GMP
residues. Therefore, we can assign the ribonuclease T1-resistant
fragments from the gel shift entity to specific positions in the CcnD3
3'-UTR. The three major fragments of 25, 26, and 37 nucleotides, which
were consistently obtained by this fingerprinting assay, were
designated A, B, and C, respectively. These fragments map to
T1-resistant sequences at 1168-1192 (A), 1611-1636 (B), and
1686-1722 (C). From the relative intensities of the ribonuclease T1-protected fragments, we estimate that about 90% of the binding activity is associated with the B element that centers around the
26-nucleotide T1-resistant fragment and the C element that contains the
37-nucleotide T1-resistant fragment. These two elements are located
adjacent to each other, within about 100 nucleotides (1611-1722). The
proximity of the B and C elements and the intensity of binding to these
sequences focused our attention on this part of the 3'-UTR. The
nucleotide sequence of the murine CcnD3 3'-UTR from 1574 to 1754 is
shown in Fig. 4B. The 26- and 37-nucleotide T1 fingerprint
fragments (B and C) are underlined. This region of the
3'-UTR is highly conserved among mouse, human, and rat cyclin D3
mRNAs. Comparison of the mouse and human sequences revealed 84%
identity from 1561 to 1714 nucleotides, compared with 87% identity
within the coding regions. The complete 3'-UTRs of mouse and human
CcnD3 mRNAs (including the BC domain) are 63% identical.
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Overexpression of the D3 3'-UTR attenuates dexamethasone-mediated destabilization of the endogenous CcnD3 mRNA, and the transgenic full-length tetD3 transcripts are not destabilized by glucocorticoids (Fig. 1). Consequently, we were unable to use deletion mutants to identify the RNA sequences that cause loss of stability in the presence of glucocorticoids. However, the observation that the 3'-UTR attenuated glucocorticoid regulation in trans suggested an alternative way to functionally map the response element. We reasoned that overexpression of the 3'-UTR might titrate the proteins responsible for the dexamethasone-mediated destabilization, thereby preventing their association with the endogenous CcnD3 mRNA. This hypothesis predicts that overexpression of the RNA response element should attenuate glucocorticoid regulation of CcnD3 mRNA. Our binding data suggest that the B and/or C sequences might be the binding sites for those proteins. If that is so, then overexpression of the BC domain should inhibit the dexamethasone effects on the stability of the endogenous CcnD3 mRNA. We constructed a cell line that was designed to overexpresses the 215-nucleotide BC domain (nucleotides 1574 to 1789), as described under "Materials and Methods." A mixed population of these cells, designated P1798p4zBC, was treated with dexamethasone for various periods of time. Total RNA was extracted and Northern blotting was carried out to measure CcnD3 and c-Myc mRNA. These "BC" cells exhibited glucocorticoid inhibition of c-myc expression, as shown in Fig. 4C. However, glucocorticoids caused little or no change in the abundance of CcnD3 mRNA. Addition of dexamethasone to control cells that contain the "empty" expression vector (designated P1798p4z) caused a rapid decrease in the abundance of both CcnD3 and c-Myc mRNAs. Control and BC cells contained similar amounts of CcnD3 mRNA, and the basal turnover rates of CcnD3 mRNA (insofar as such rates can be estimated using actinomycin D) were similar in both populations of cells (data not shown).
Subsequent RNA gel shift experiments utilized a transcript that extends
from nucleotide 1575 to 1789 in CcnD3 mRNA. This transcript contains both the B and C elements and is designated the BC probe. The
25-nucleotide A element has not been studied further. Given the small
size of the BC probe, gel mobility shift assays could be performed
without the need for ribonuclease T1 digestion after the binding
reaction. In order to validate the use of the BC probe, a competition
experiment was performed, as shown in Fig.
5A. Lane 1 contains
free BC probe, without protein. Addition of S100 protein quantitatively
shifted the BC probe, as shown in lane 2. This complex was
not detected when a 100-fold molar excess of unlabeled BC transcript
was added to the binding reaction (lane 3). However, addition of a 100-fold excess of -galactosidase mRNA (lane
4) did not block formation of the RNA-protein complex. Note that the probe was completely shifted under the conditions that were employed in this experiment. If we add more probe to the binding reactions, we begin to observe another, more abundant binding activity
that is probably due to polypyrimidine tract binding protein (data not
shown). If we add less S100 protein, the signal becomes difficult to
detect. Consequently, we have been unable to establish conditions under
which we can perform mobility shift assays in probe excess.
|
A label transfer experiment was used to estimate the molecular weight of the proteins that bind to the BC elements of the CcnD3 3'-UTR. A binding reaction was performed using 32P-labeled BC probe and S100 cytoplasmic extract from control cells. The reaction was irradiated with UV light to cross-link the probe and any protein closely interacting with it. The probe was digested with ribonuclease A, which cuts 3' to every accessible pyrimidine so as to leave the RNA-binding protein labeled with a short oligonucleotide. The labeled protein was then resolved by SDS-polyacrylamide gel electrophoresis. The molecular weight of the RNA-binding protein(s) may be estimated by this approach, if one assumes that the mass of the bound oligonucleotide is insignificant. The results from such an experiment are shown in Fig. 5B. Lane 1 contains probe with no S100 protein. Two proteins were labeled when S100 protein was included in the binding reaction, as shown in the lane 2. Addition of a 100-fold molar excess of unlabeled BC transcript precluded formation of any labeled protein (lane 3). The molecular weights of the two proteins that are labeled by the BC element are estimated to be about 52,000 and about 37,000.
In the course of optimizing the UV cross-linking experiments, we
observed that the protein composition of the RNA-protein complex varied
as a function of protein concentration in the binding reaction, as
shown in Fig. 6. Both the
Mr 52,000 and 37,000 proteins were labeled when
80 µg of S100 protein was added to the 150-µl binding reaction
(lanes 1 and 2). When the binding reaction was carried out using 40 µg of protein, only the
Mr 52,000 protein was labeled by UV
cross-linking (lanes 3 and 4). These data suggest that the Mr 52,000 protein either is more
abundant or has higher affinity than the Mr
37,000 protein. We also observed no differences in protein labeling
when UV cross-linking was carried out using extracts from control,
mid-log phase cells (Fig. 6, lanes 1 and 3) or
from cells that had been treated with dexamethasone for 24 h
(lanes 2 and 4). Likewise, we observed no
significant difference in electrophoretic mobility of the BC-protein
complex when gel shift assays were performed using increasing
concentrations of S100 protein from control or dexamethasone-treated
cells (data not shown). We have examined more than five sets of
extracts from control and dexamethasone-treated cells and never
detected a significant difference in binding to the BC probe.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
We report here the mapping of an element that is involved in glucocorticoid regulation of CcnD3 mRNA turnover. Located within the 3'-UTR of CcnD3 mRNA, this element centers around two polypyrimidine tracts located within about 100 nucleotides of each other. This so-called BC element is highly conserved in mouse and human CcnD3 3'-UTR. The C element is predicted (GCG M Plot) to assume a stable (-55 kcal) stem-loop structure. Preliminary studies indicate that this transcript contains large nuclease S1-resistant domains, but the structure of this region remains to be elucidated. BLAST analysis of the BC element revealed no significant nucleotide sequence homology to other RNA elements; however, there are obvious similarities between the BC element and other polypyrimidine-rich regulatory elements that would not be revealed by a sequence-based comparison. The BC element binds two proteins, of approximate molecular weights 37,000 and 52,000. The binding data suggest that the Mr 52,000 protein either is more abundant or has a higher affinity. It is also possible that the formation of the BC-protein complex occurs by an ordered mechanism. Unfortunately, it is difficult to analyze the kinetics or thermodynamics of binding in the crude S100 preparations. These extracts contain abundant polypyrimidine tract-binding protein, which binds to the BC element and interferes with detection of the 37,000/52,000/BC complex when binding experiments are performed in probe excess.
CcnD3 mRNA that is deleted of the 3'-UTR is not destabilized by
glucocorticoids. Overexpression of the BC element attenuates glucocorticoid regulation of CcnD3 mRNA in trans. These
observations are consistent with the hypothesis that formation of a
ternary RNA-protein complex is necessary for destabilization of CcnD3 mRNA. Fig. 7 illustrates the working
hypothesis that we have developed to account for our observations. We
propose that the BC element interacts with the
Mr 37,000 and 52,000 proteins to form a ternary complex. When the BC element is overexpressed, it functions as a
molecular decoy, titrating the Mr 37,000 and
52,000 proteins from the endogenous transcript, thereby precluding
formation of the ternary complex and attenuating
glucocorticoid-mediated destabilization of CcnD3 mRNA in
trans.
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Interaction between the Mr 37,000 and 52,000 proteins and the BC element appears to be constitutive, because no change in binding was detected in extracts from glucocorticoid-treated cells. We propose that glucocorticoids induce another active principle, designated Dx in Fig. 7, that interacts with the ternary complex to form an unstable quaternary complex. Shoenberg and co-workers (28) have identified estrogen-induced ribonucleases that regulate mRNA stability in Xenopus liver, and it is conceivable that the principle that we have designated "Dx" could be a glucocorticoid-induced ribonuclease that is recruited to the ternary complex. Other hypotheses may be imagined, but the essential elements of any plausible hypothesis would seem to be that 1) two or more proteins must interact with each other and with the BC cassette to elicit dexamethasone-mediated destabilization, 2) the proteins must bind independently to the regulatory element and therefore distribute among the overexpressed BC cassettes, and 3) one or more of these proteins must be limiting in concentration. None of our data are inconsistent with any of these assumptions, although there is clearly a lot more to learn about how the Mr 37,000 and 52,000 proteins interact with the BC element.
Deletion of the 3'-UTR affects basal stability of tetD3 constructs, suggesting that there are elements within the 3'-UTR that control CcnD3 mRNA abundance in proliferating cells. However, overexpression of the BC element does not affect basal stability of endogenous CcnD3 mRNA. Actinomycin D blocks CcnD3 turnover in mid-log phase cells but does not prevent CcnD3 degradation in glucocorticoid-treated cells. These observations suggest that basal turnover and glucocorticoid-mediated degradation proceed by different mechanisms.
We have examined the properties of known RNA-binding proteins in an effort to identify p37 or p52. Our analysis has focused upon RNA-binding proteins of the appropriate size or those that are know to bind polypyrimidine tracts. Among these, hnRNP K (Mr 66,000), hnRNP I (62,000), hnRNP C1 (42,000), and hnRNP C3 (44,000) bind polypyrimidine tracks but are not of the right size (17, 18, 29-32). hnRNP A1 (Mr 36,000) binds AUUUA elements but not other polypyrimidine tracts in the 3'-UTRs of several mRNAs (33), and hnRNP H, with a molecular weight of 53,000, binds poly-G (34). Among the known poly-C-binding proteins (29, 30, 35), murine CUBP (Mr 48,000) may be a possible candidate for the Mr 52,000 CcnD3-binding protein that we have observed. This remains to be determined.
In summary, there is within the 3'-UTR of CcnD3 mRNA an element
that appears to be necessary but not sufficient for glucocorticoid regulation of mRNA degradation. This element is conserved among mammalian CcnD3 mRNAs and contains, but is probably not restricted to, two polypyrimidine tracts. These two tracts form part of the binding sites for two proteins. We assume that formation of the BC/37,000/52,000 complex does not cause destabilization in the presence
of glucocorticoids. Rather, we assume that this element is necessary
for interaction with some other, as yet unidentified, glucocorticoid-induced protein(s). The mechanism is likely to be
significantly more complex than that proposed in Fig. 7, and it may be
that destabilization will require not only other proteins but also
other nucleotide elements within the 3'-UTR or the coding sequence of
CcnD3 mRNA.
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FOOTNOTES |
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* 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.
To whom correspondence should be addressed. Tel.: 409-772-3361;
Fax: 409-747-4050; E-mail: athompso@utmb.edu.
Published, JBC Papers in Press, May 9, 2000, DOI 10.1074/jbc.M001048200
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ABBREVIATIONS |
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The abbreviations used are:
hnRNP, heterogeneous
nuclear ribonucleoprotein;
UTR, untranslated region;
-gal, -galactosidase.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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