From the Minerva Center for Calcium and Bone Metabolism, Nephrology Services, Hadassah University Hospital, Jerusalem il-91120, Israel
Received for publication, June 22, 2000, and in revised form, November 16, 2000
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ABSTRACT |
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Calcium and phosphate regulate parathyroid
hormone (PTH) gene expression post-transcriptionally by changes in
protein-PTH mRNA 3'-untranslated region (UTR) interactions, which
determine PTH mRNA stability. We have identified the protein
binding sequence in the PTH mRNA 3'-UTR and determined its
functionality. The protein-binding element was identified by binding,
competition, and antisense oligonucleotide interference. The sequence
was preserved among species suggesting its importance. To study its
functionality in the context of another RNA, a 63-base pair cDNA
PTH sequence was fused to the growth hormone
(GH) gene. There is no parathyroid (PT) cell line and
therefore an in vitro degradation assay was used to
determine the stability of transcripts for PTH, GH, and a chimeric
GH-PTH 63 nucleotides with PT cytosolic proteins. The full-length PTH
transcript was stabilized by PT proteins from rats fed a low calcium
diet and destabilized by proteins from rats fed a low phosphate diet,
correlating with PTH mRNA levels in vivo. These PT
proteins did not affect the native GH transcript. However, the chimeric
GH transcript was stabilized by low calcium PT proteins and
destabilized by low phosphate PT proteins, similar to the PTH
full-length transcript. Therefore, we have identified a PTH RNA-protein
binding region and shown that it is sufficient to confer responsiveness
to calcium and phosphate in a reporter gene. This defined element in
the PTH mRNA 3'-UTR is necessary and sufficient for the regulation
of PTH mRNA stability by calcium and phosphate.
Parathyroid hormone
(PTH)1 acts to maintain serum
calcium within a narrow physiological range (1). A 7-transmembranous
calcium-sensing receptor on the parathyroid (PT) cell recognizes
small changes in serum-ionized calcium and regulates PTH secretion (2).
Low serum calcium increases PTH secretion, PTH mRNA levels (3), and
if prolonged, PT cell proliferation (4). PTH then acts to correct serum
calcium by mobilizing calcium from bone and increasing renal
reabsorption of calcium. Phosphate also regulates the PT, with low
serum phosphate decreasing serum PTH, PTH mRNA levels, and PT cell
proliferation (4-8).
The effects of dietary calcium and phosphate on PTH gene
expression are both post-transcriptional (5, 9). The PTH cDNA consists of three exons coding for the 5'-UTR (exon I), the prepro region of PTH (exon II), and the structural hormone together with the
3'-UTR (exon III) (10, 11). The rat 3'-UTR is 239 nt long out of the
712 nt of the full-length PTH RNA (11). The 3'-UTR is 42% conserved
between human and rat, while the coding region is 78% conserved at the
nt level (11).
We have shown that cytosolic proteins from PTs bind to the 3'-UTR of
the rat PTH mRNA (9). PT proteins from hypocalcemic rats show
increased binding to the PTH mRNA 3'-UTR by mobility shift and UV
cross-linking assays and this protein-RNA binding is decreased with
hypophosphatemic PT proteins. Thus the level of protein-RNA binding
directly correlates with PTH mRNA levels. Since there is no PT cell
line, an in vitro PTH RNA stability assay was utilized. This
assay showed stabilization of the PTH transcript by hypocalcemic PT
proteins and marked instability with PT hypophosphatemic proteins (9).
A PTH transcript that did not include the 3'-UTR was not degraded by PT
proteins in this assay. These studies indicate that there are
instability regions in the PTH mRNA 3'-UTR that are protected by
RNA-binding proteins. We have recently identified two proteins that
bind the PTH mRNA 3'-UTR (12, 13). One protein, AUF1, was
identified by affinity chromatography and was shown to stabilize the
PTH transcript in an in vitro degradation assay with PT
proteins (12). In other RNAs such as proto-oncogenes and cytokines,
that have a short half-life, AUF1 is associated with destabilization of the transcripts (14). A second protein, dynein light chain
(Mr 8000) or LC8 was identified by expression
cloning and was shown to mediate the binding of the PTH mRNA to
microtubules (13). LC8 may have a role in the intracellular
localization of PTH mRNA in the PT cell rather than in the
stability of PTH mRNA.
We have now identified the minimal protein binding sequence in the PTH
mRNA 3'-UTR by binding assays, competition experiments, and
oligonucleotide binding interference analysis. To demonstrate the
functionality of this sequence 63 base pairs of the PTH mRNA 3'-UTR
which included the 26-nt protein-binding element and 5'- and
3'-flanking nt were inserted into the GH gene. Using
in vitro degradation assays with cytosolic extracts from
parathyroid glands we demonstrated that this element altered GH
mRNA stability. The 63-nt element also reduced the stability of a
random transcript of the pCRII polylinker, in contrast to a truncated
element inserted into the polylinker which did not bind PT proteins. In
addition, inserting the 63-nt binding sequence into GH RNA conferred
responsiveness of the chimeric transcript to PT proteins from low
calcium and low phosphate rats, similar to the effects of these
proteins on the full-length native PTH transcript. These results
demonstrate that the protein-binding region in the PTH mRNA 3'-UTR
is sufficient to determine PTH mRNA stability in response to
changes in serum calcium and phosphate.
Animals--
Weanling male Sabra rats were fed a normal calcium
(0.6%), normal phosphate (0.3%) diet; a low calcium (0.02%), normal
phosphate (0.3%) diet; or a low phosphate (0.02%), normal calcium
(0.6%) diet (Teklad, IL) for 3 weeks. At 3 weeks the thyroparathyroid tissue was removed under pentobarbital anesthesia and blood samples were taken for serum calcium and phosphate. The low calcium diet resulted in a serum calcium of 4.6 ± 0.4 mg/dl (control = 11.2 ± 0.3 mg/dl). The low phosphate diet resulted in a serum
phosphate of 4.0 ± 0.6 mg/dl (control = 9.8 ± 0.7 mg/dl) and serum calcium of 12.4 ± 0.8 mg/dl.
Cytoplasmic Protein Purification--
Cytoplasmic
thyroparathyroid proteins (S100) for protein-RNA binding were extracted
by a modification of the method of Dignam et al. (15).
Tissues were removed from the rats and immediately washed in cold
phosphate-buffered saline. The tissue was homogenized in 5 volumes of
buffer A containing 10 mM HEPES, 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM DTT, and
0.5 mM phenylmethylsulfonyl fluoride, and incubated on ice
for 10 min. After centrifugation at 600 × g for 10 min
(4 °C) the supernatant was carefully decanted, mixed with 0.1 volumes of buffer B containing 0.3 M HEPES, 1.4 M KCl, and 0.03 M MgCl2, and
centrifuged (4 °C) at 100,000 × g for 1 h
(Beckman Type TL-100). For RNA degradation assays the S100 fraction was
prepared as previously described (9) by homogenizing the tissue in 2 volumes of 10 mM Tris-HCl, pH 7.4, 0.5 mM DTT,
10 mM KCl, 1.5 mM MgCl2. 0.1 Volume
of the extraction buffer (1.5 mM KCl, 15 mM
MgCl2, 100 mM Tris-HCl, pH 7.4, 5 mM DTT, was added and the homogenate was centrifuged at
14,000 × g for 2 min to pellet the nuclei. The
supernatant was centrifuged at 100,000 × g for 1 h at 4 °C. Cytoplasmic extracts were immediately frozen at
RNA Transcripts and Probes--
Labeled and unlabeled RNA was
transcribed from linearized plasmids using an RNA production kit
(Promega, WI) and the appropriate RNA polymerases. The specific
activity of the RNA probe was 0.5-1.0 × 106 cpm/ng.
For competition experiments unlabeled RNA was transcribed similarly in
the presence of 1 mM each of the four nucleotides. The
unlabeled RNAs were quantified by visualization on a 2% agarose gel.
A linearized plasmid construct containing the full-length PTH cDNA
in Bluescript KS (Invitrogen, CA) was used as previously described (9).
For the 3'-UTR of the PTH cDNA, a polymerase chain reaction product
(9) was subcloned into pCRII (Invitrogen, CA) (12). The transcripts of
100, 63, 50, and 40 nt (Fig. 1A) were transcribed from
polymerase chain reaction products that were subcloned into pGEM-T Easy
vector (Promega, WI) (fragment for the 40 and 30 nt) or pCRII
(Invitrogen, CA) (fragments for the 100, 63, 50, and 38 nt). For the
constructs for transcription of the 100-, 63-, and 50-nt transcripts,
polymerase chain reaction was performed with the same 5'
oligonucleotide: GTCTCTTCCAATGAT. The 3' oligonucleotide for the 100-nt
construct was TTCATGATCATTAAACTTTA, for the 63 nt,
AAGTGGAAATGTGTAATACTTTAA, and for the 50 nt,
TAATACTTTAAAAAGAAGAATATATTG. For the 40-nt transcript polymerase chain
reaction was performed with the 5' oligonucleotide,
AATGATTCCATTTCAATAT, and the 3' oligonucleotide, AATACTTTAAAAGAAG. For
the transcript of 38 nt the corresponding cDNA was excised from the
pCRII 63-nt construct and subcloned into pCRII. The transcript of 30 and 26 nt (Fig. 1A) were prepared from annealed sense and
antisense oligonucleotides that were constructed to include the T3 RNA
polymerase sequence (underlined). For the 30-nt the sense
oligonucleotide was
ATTAACCCTCACTAAAGGGACATTTCAATATATTCTTCTTTTTAAAGTATT, and
the antisense oligonucleotide was AATACTTTAAAAAGAAGAATATATTGAAATG. For
the 26 nt the sense oligonucleotide was
ATTAACCCTCACTAAAGGGACAATATATTCTTCTTTTTAAAGTATTA, and the
antisense oligonucleotide was TAATACTTTAAAAAGAAGAATATATTG. For
the calcium-sensing receptor 3'-UTR, we subcloned the 3'-UTR into
Bluescript II KS (Stratagene, La Jolla, CA) using a fragment obtained
by restriction of the BoPCaRI cDNA in pSPORT (2) with NotI and SmaI, as previously described (13).
RNA Electrophoretic Mobility Shift Assays (REMSA)--
Labeled
RNA transcripts (10,000 cpm) spanning different regions of the PTH
3'-UTR RNA were incubated with S100 thyroparathyroid extracts (10 µg), in a final volume of 20 µl containing 4 µg of tRNA, 10 mM HEPES, 3 mM MgCl2, 40 mM KCl, 5% glycerol, and 1 mM DTT (binding
buffer) for 10 min at at 4 °C. In some experiments RNase T1 (Sigma)
was added for further 10 min at room temperature to a final
concentration of 30 units/µl to digest unprotected RNA. Addition of
heparin (5 mg/ml) did not affect binding and the size of the complex.
For competition experiments unlabeled RNA was added as indicated. The
specificity of binding was further demonstrated by the addition of
unlabeled nonrelated transcripts. The samples were run for 3 h on
a native 4% polyacrylamide gel (polyacrylamide:bisacrylamide, 70:1) in
a cold room. RNA-protein binding was visualized by autoradiography of
the dried gels.
UV Cross-linking Assay--
UV cross-linking assays were
performed as previously described (9) using 10 µg of S100
thyroparathyroid extracts and 32P-labeled RNA transcripts
of the full-length or parts of the 3'-UTR of the PTH cDNA in a
buffer containing 10 mM HEPES, 3 mM
MgCl2, 40 mM KCl, 5% glycerol, 1 mM DTT, and 5 mg/ml heparin to eliminate nonspecific
binding. After UV cross-linking, the samples were digested by RNase A,
fractionated by SDS-PAGE, and autoradiographed.
Antisense Oligonucleotides and Binding Interference--
The
antisense oligonucleotides used are shown in Fig. 4A.
Corresponding sense oligonucleotides were also synthesized. For binding
interference experiments antisense oligonucleotides (200 pmol) were
mixed with the radiolabeled RNA, heated to 80 °C for 10 min, and
cooled slowly to 25 °C, before addition of protein extract and
REMSA. In some experiments preincubation of the RNA and
oligonucleotides was performed without heating of the RNA. In other
experiments annealed double-strand DNAs were added instead of the
antisense oligonucleotides.
Construction of the Chimeric GH mRNA containing 63 nt of the
PTH mRNA 3'-UTR for in Vitro Degradation Assay--
The 63-base
pair DNA corresponding to the PTH mRNA 3'-UTR 63-nt transcript
(Fig. 1A) was inserted into the SmaI site of the pS16-GH that contained the S16 ribosomal protein promoter linked to the
GH structural gene lacking GH promoter sequences (16). The
control was the same plasmid without the PTH mRNA 3'-UTR 63-base pair DNA. The GH DNA with and without the PTH sequence was
subcloned into Bluescript KS (Invitrogen, CA) to transcribe RNA.
In Vitro RNA Degradation Assay--
Preparation of S100 PT
protein extracts for the RNA degradation assay and the assay itself
were performed as before (9). 0.2 × 106 cpm
transcripts of PTH, GH, or the chimeric GH/PTH 63-nt RNAs were
incubated with 10-20 µg of PT cytoplasmic extracts and 80 units/ml
RNasin (Promega, WI) and at timed intervals samples were removed and
extracted by Tri-reagent (Molecular Research Center, Cincinnati
OH). The labeled RNAs from each sample were run on formaldehyde-agarose
gels, transferred to Hybond membranes (Amersham Pharmacia Biotech), and
autoradiographed (9). The remaining undegraded transcripts at the
different time points were quantified by densitometry.
To measure the effect of PTH sequences in the pCRII polylinker
transcript, EcoRV-linearized pCRII plasmids containing the 63 or 38 nt of the PTH 3'-UTR as described above, or the unmodified pCRII polylinker were used to transcribe RNA with the Sp6 RNA polymerase. The resulting transcripts contained ~100 nt corresponding to the polylinker plus the additional PTH sequences (Fig.
6A). They were analyzed by in vitro degradation
with PT proteins.
Identification of the Minimal Protein Binding Sequence in the PTH
mRNA 3'-UTR--
PT cytosolic proteins specifically bind the
full-length PTH mRNA transcript and a transcript for the PTH
mRNA 3'-UTR. To identify the protein binding sequence in the PTH
mRNA 3'-UTR we analyzed the binding of PT proteins to smaller RNA
transcripts of the PTH mRNA 3'-UTR (Fig.
1A). Uniformly labeled
transcripts were incubated with PT cytosolic extract and the mixture
resolved on native polyacrylamide gels for REMSA. The free probe
for all transcripts ran as two major bands. These bands may represent
secondary structures of the RNA, because denaturing the RNA at 80 °C
followed by slow renaturation at room temperature resulted in a single
band on polyacrylamide gel. This renatured probe showed the same
binding to PT proteins as the untreated transcript (not shown). The
transcripts used for mapping the minimal protein binding element in the
PTH 3'-UTR are shown in Fig. 1A. Transcripts of 174 and 38 nt that excluded the 60-terminal nt of the 3'-UTR did not bind
proteins. A transcript of 234 nt consisting of the full-length UTR and
transcripts of 100 (Fig. 1B), 63, 50, and 40 nt (not shown)
of the 3'-UTR showed a large protein-RNA complex on REMSA with PT
proteins (Fig. 1B). This complex was reduced to a smaller
complex after RNase T1 digestion of the bound protein RNA samples (Fig.
1B). Similar results were obtained when a transcript for the
full-length PTH mRNA was analyzed (not shown). Transcripts of 30 (not shown) and 26 nt formed only the smaller protein-RNA complexes
with or without treatment with RNase T1 (Fig. 1B) and the
larger RNA-protein complex was not formed. These results show that a
transcript of 40 nt was necessary for the formation of the larger
protein-RNA complex that was obtained when the full-length PTH
mRNA 3'-UTR transcript was analyzed. A 26-nt element was sufficient
for protein binding and formed a complex that was similar to the
complex formed with larger transcripts after treatment with RNase T1.
Therefore, additional nucleotides in the 5' of this element were
necessary for formation of the larger complex that was formed in the
absence of RNase T1. PT protein binding to these smaller transcripts
(100, 63, and 40 nt) was increased by hypocalcemia and decreased by
hypophosphatemia (Fig. 2), as with the
full-length PTH transcript in our previous results (9). These results
demonstrate that the regulation of binding is preserved with the
smaller transcripts. Further experiments were performed to confirm the
minimal binding element.
To demonstrate the specificity of the binding of PT proteins to the
protein-binding element we used competitor RNAs from overlapping regions of the 3'-UTR. Fig. 3 shows a
representative REMSA that demonstrates the binding of PT cytosolic
proteins to the PTH mRNA 3'-UTR. Addition of excess unlabeled RNA
transcript for the 3'-UTR that did not include the terminal
60-nucleotide of the 3'-UTR (Fig. 3, transcript A) did not compete for
binding of PT proteins to the PTH 3'-UTR. However, excess transcript of
the 26-nt element or of 63 nt that included the 26-nt binding element
(Fig. 3, transcripts B and C) both competed for protein binding to the
3'-UTR. Excess of an unrelated transcript for the 3'-UTR of the
calcium-sensing receptor mRNA (Fig. 3, transcript D), which is also
expressed in the PT, did not compete for binding. Addition of heparin
had no effect on complex formation (not shown). These results
demonstrate the specificity of the protein-RNA interaction and indicate
that the 26-nt transcript was sufficient to compete for the binding of
PT proteins to the PTH mRNA 3'-UTR.
To further characterize the protein binding element in the PTH mRNA
3'-UTR we designed short single stranded antisense DNA oligonucleotides
complimentary to portions of the 3'-UTR and analyzed their effect on
protein-RNA binding by REMSA and UV cross-linking analysis. Fig.
4A shows the sequence of the
100 terminal nt of the PTH mRNA 3'-UTR including the 26 nt of the
proposed protein-binding element and the antisense oligonucleotides
used for the binding interference experiments. A representative REMSA
for the binding of cytosolic PT proteins to the 3'-UTR transcript that
had been preincubated with different antisense oligonucleotides is
shown in Fig. 4B. The antisense oligonucleotides were
annealed to the labeled 3'-UTR transcript that had been heated to
80 °C to unfold secondary structures in the RNA. PT protein extracts
were then added and protein binding analyzed. Corresponding
double-stranded DNAs were used as controls. Fig. 4B shows
the 3'-UTR transcript after RNase T1 treatment and the protein-RNA
complexes formed after addition of PT cytosolic extracts followed by
RNase treatment. Preincubation of the PTH mRNA 3'-UTR transcript
with antisense oligonucleotides 1, 2, and 6 (Fig. 4A) that
did not span the protein binding sequences had no effect on protein
binding (Fig. 4B). Preincubation with oligonucleotides
spanning the 26-nt element or part of this sequence with or without
3'-flanking sequences (oligonucleotides 3, 4, and 5 in Fig.
4A) abolished the binding of PT proteins to the 3'-UTR (Fig.
4B). Corresponding sense or double-stranded DNAs had no
effect on protein-RNA complex formation (not shown). The effect of
antisense oligonucleotides on PT protein binding to the PTH RNA 3'-UTR
was also analyzed by UV cross-linking experiments. In this assay
RNA-binding proteins from cytosolic extracts are cross-linked to
labeled transcript in solution and complexes resolved by denaturing
SDS-PAGE. Fig. 4C shows that 3 cross-linked protein-RNA
complexes of ~110, 60, and 50 kDa were formed when a transcript for
the PTH mRNA 3'-UTR was analyzed with PT protein extracts, as in
our previous reports (9). When the transcript was denatured at 80 °C
and preincubated with the antisense oligonucleotides the same
inhibitory effect of the oligonucleotides corresponding to the binding
region on protein binding was observed (Fig. 4C), similar to
the REMSA (Fig. 4B). When the same UV cross-linking experiment was performed without denaturing the RNA by heating to
80 °C there was no effect of preincubation of the RNA with any of
the oligonucleotides (Fig. 4C). In addition, when there was
no preheating of the RNA, the relevant oligonucleotides did not
interfere with binding also by REMSA (not shown). Together, these
results indicate that the protein-binding recognition site of the
3'-UTR includes the element of 26 nt and that this region plays a role
in protein-RNA binding. Moreover, the interference of binding only
after unfolding the RNA at 80 °C suggests that protein binding to
this region is dependent on secondary structures in the RNA.
Sequence analysis of the 26-nt element in the PTH mRNA revealed
high conservation of the rat element in the PTH mRNA 3'-UTR to
mouse (23 of 26 nt), human (19 of 26 nt), and canine (19 of 26 nt),
with human and canine being identical (Table
I). Such conservation of this sequence
that lies outside of the coding region, among different species,
suggests a functional role for this element.
Functionality of the Protein-binding Region of the PTH mRNA
3'-UTR Inserted into Heterologous RNAs--
To demonstrate that the
protein-binding region has a role in determining mRNA stability and
response to calcium and phosphate, we inserted the fragment for the 63 nt of the PTH mRNA 3'-UTR, which contains the 26-nt element (Fig.
1A), into the structural gene of human growth hormone (GH).
This is shown schematically in Fig.
5A. We used these constructs
to study whether this inserted 63-nt sequence affected GH mRNA
stability in an in vitro degradation assay with PT proteins
of rats fed normal, low calcium, or low phosphate diets.
The in vitro degradation assay reflects the
post-transcriptional regulation of PTH mRNA stability in
vivo in response to changes in calcium and phosphate. A low
calcium diet increases PTH mRNA levels in vivo and PT
proteins from these rats stabilized the full-length PTH transcript
using the in vitro degradation assay (Fig. 5B, upper
panel, and Ref. 9). A low phosphate diet decreases PTH mRNA
levels and led to rapid degradation of the PTH transcript by PT
proteins from these rats (Fig. 5B, upper panel, and Ref. 9).
We then analyzed the effect of the PTH mRNA 63-nt protein binding
sequence on GH mRNA degradation in the presence of these PT
proteins. With PT proteins from rats fed a normal diet, the chimeric GH
transcript containing 63 nt of the PTH 3'-UTR was degraded more rapidly
than the native GH transcript (t1/2 = 130 min: >180
min) (Fig. 5B, middle and bottom panels, normal diet). This suggests that this element is an instability element. We
then studied the effect of PT proteins from hypocalcemic or hypophosphatemic rats on degradation of the GH transcripts. When the
transcript for the native GH mRNA was analyzed with PT proteins from the different diets, there was no effect on native GH degradation (Fig. 5B, middle panel). In contrast, the chimeric GH
transcript was stabilized by PT proteins from low calcium rats and more
rapidly degraded with PT proteins of low phosphate rats
(t1/2 > 180 min:30 min) (Fig. 5, B, bottom
panel, and C), similar to the full-length PTH
transcript (Fig. 5B, top panel). Therefore the protein
binding sequences in the PTH mRNA 3'-UTR were sufficient to confer
responsiveness to changes in PT proteins induced by dietary calcium and
phosphate to another mRNA that itself was not regulated by calcium
and phosphate.
To determine the specificity of the effect of the protein-binding
region, the protein-binding segment of 63 nt was inserted into a random
sequence, the pCRII polylinker. In addition a shorter PTH mRNA
3'-UTR RNA of 38 nt, that itself did not bind PT proteins (Fig.
1A) was also inserted at the same site into the pCRII
polylinker (Fig. 6A). The
stability of the polylinker and chimeric RNAs was determined in the
in vitro degradation assay with PT proteins. The PTH
mRNA 63 nt was recognized and cleaved more rapidly by the PT
extract (t1/2 = 10 ± 2 min, n = 3) than the RNA without the PTH mRNA insert
(t1/2 = 35 ± 5 min, n = 3)
(Fig. 6). Insertion of the shorter PTH RNA 38 nt did not destabilize
the chimeric transcript (t1/2 = 40 ± 5 min,
n = 3). These results suggest that the PTH 63-nt RNA
destabilized the random RNA sequence of pCRII. This effect was similar
to the effect of the 63 nt when it was inserted into a larger
transcript, GH RNA, representing a cellular mRNA. The destabilizing
effect was dependent on an intact protein-binding transcript, because a
shorter transcript that disrupted protein binding did not have the same
effect.
We have identified by gel shift binding, competition experiments,
and the use of single-strand antisense oligonucleotides the minimal
sequence of 26 nt for protein binding in the PTH mRNA 3'-UTR.
Unlike gel shifts, UV cross-linking identified 40 nt as the smallest
binding transcript and not the 26- or 30-nt transcripts. The 26 and 30 nt are part of the 40-nt binding transcript. It is not readily evident
why the 26-nt element was not sufficient for protein binding by UV
cross-linking. However, protein-RNA binding analysis by REMSA utilizes
a native PAGE after incubating protein and RNA and therefore may be
more physiological than denaturing gels of UV cross-linked RNA-protein
complexes. Single-strand antisense oligonucleotides spanning the 26-nt
core binding element, or parts of it, interfered with the binding of PT
proteins to the 3'-UTR both by REMSA and UV cross-linking. The
antisense data together with the competition experiments with the 26-nt
element show that the 26-nt element is important for binding. It is
possible that the larger sequence of 40 nt may represent the required
sequence for protein-RNA interaction, with the 26 nt representing the
core element.
We have previously shown that PT proteins from rats fed a low calcium
diet, where there is a marked increase in PTH mRNA levels, have
increased binding to the PTH mRNA full-length and 3'-UTR transcripts. In contrast, PT proteins from rats fed a low phosphate diet where there is decrease in PTH mRNA levels, have decreased binding (9). We have now shown that protein binding to shorter transcripts (100, 63, and 40 nt) was also regulated by the different diets by UV cross-linking (Fig. 2). The binding of PT proteins to the
26-nt transcript by REMSA was similarly regulated by calcium and
phosphate (not shown). These results demonstrate that the smaller
transcripts retain the properties of the previously characterized full-length transcript (9).
The rat PTH mRNA 3'-UTR is 234 nt long. Sequence analysis of the
PTH mRNA 3'-UTR of different species revealed a preservation of the
26-nt core protein-binding element in rat, murine, human, and canine
3'-UTRs. In particular, there is a stretch of 14 nt within the element
that is present in all four species. In the 26-nt element, the identity
among the species varies between 73 and 89%. For instance, the human
and rat are 73% identical in the 26-nt element, and only 42%
identical in their 3'-UTR (11). The canine and rat are 73% identical
in their 26-nt element and 50% identical in their 3'-UTR (17). The
human and canine are 100% identical in the 26 nt of the element and
only 70% identical in their 3'-UTR. Comparison of the 26-nt sequence
in rat and mouse showed 89% identity, however, their 3'-UTRs show a
comparable degree of identity. All in all, this analysis suggests that
the binding element may represent a functional unit that has been evolutionarily conserved, but sequencing of the 3'-UTR in many other
species is needed to establish this conclusion.
One of the PT cytosolic proteins involved in the stabilization of the
PTH transcript was identified by affinity chromatography to the PTH
mRNA 3'-UTR as AUF1 (12). Gel shift assays showed that recombinant
AUF1 bound the full-length, 3'-UTR (12) and shorter transcripts
including the 26-nt core
element.2 There is no PT cell
line and therefore PTH mRNA stability was measured by an in
vitro degradation assay with rat PT proteins (9). Recombinant AUF1
stabilized the PTH transcript in the in vitro degradation
assay with PT proteins (12).
To study functionality of the protein-binding region, we used the 63-nt
sequence to create a chimeric transcript. We inserted a 63-base pair
fragment of the PTH 3'-UTR into the structural gene of human
GH. The RNA transcribed from this chimeric gene includes the
26-nt core element within the 63 nt of the PTH-binding region. We then
studied the effect of the protein-binding region to confer PTH-like
responsiveness to calcium and phosphate on a heterologous gene. There
is no PT cell line and therefore, to study the effect of the PTH 3'-UTR
element on RNA decay, we performed in vitro degradation
assays with cytosolic proteins from PT glands. With PT proteins, the
chimeric transcript was less stable than the native GH transcript
indicating that in this assay, with PT proteins the 63-nt element was
an instability element. Furthermore, the chimeric GH mRNA
transcript containing the 63 nt of the PTH mRNA responded to PT
proteins from low calcium and phosphate similar to the PTH mRNA.
Therefore, the 63-nt protein-binding region of the PTH mRNA 3'-UTR
is both necessary and sufficient to regulate RNA stability and to
confer responsiveness to changes in PT proteins by calcium and phosphate.
To document a correlation between protein-binding and RNA stability,
the 63-nt protein-binding region or a shorter sequence of 38 nt that
did not bind PT proteins were inserted into a random transcript of
pCRII polylinker. The stability of these transcripts and the transcript
of the original polylinker were then studied in the in vitro
degradation assay with PT proteins from normal rats. The chimeric
transcript was destabilized by the 63-nt insert and not by the
truncated insert. This indicates that the 63-nt binding element also
functions as an instability element in a random sequence. Moreover, the
lack of effect of the truncated transcript indicates that a sequence,
which binds PT proteins is necessary for the degradatory function in
another RNA. A random transcript of a polylinker had been used by
others to show that the ribonuclease cleavage site in the Increasing evidence demonstrates that mRNA decay is an actively
regulated process that determines gene expression. This process involves trans-acting protein factors that interact with
specific cis-elements in a mRNA and under different
physiological conditions leads to rapid decay or stability. Defined
elements in mRNAs bind specific RNA-binding proteins and have been
shown to mediate in addition to RNA stability, subcellular localization
and RNA translation (19, 20). The information encoded by such elements
in the RNA can be packaged as primary sequences and secondary or
tertiary structures or a combination of both. cis-Elements
that determine mRNA stability or instability have been determined
in a number of mRNAs. The primary sequence of some of these
cis-acting elements is highly conserved among species (21).
A well defined example is the adenosine uridine-rich element. Repeats
of this AUUUA pentamer in the 3'-UTR of mRNAs of various cytokines
targeted them for rapid decay by their interaction with cytoplasmic
trans-factors (22, 23). In the brains of Alzheimer's
patients there are increased levels of
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C in aliquots. Protein concentration was determined by O.D.
densitometry (595 µm wavelength) using a Bradford reagent (Bio-Rad).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Different PTH mRNA 3'-UTR transcripts
used for binding assays revealed the minimal sequence for protein
binding by REMSA. A, the full-length PTH mRNA
3'-UTR (234 nt) and smaller transcripts which were tested for binding
by REMSA. The minimal sequence for binding (26 nt) is shown in a
lighter shade. The binding of PT proteins by each transcript
is indicated on the right. B, REMSA for the binding of
parathyroid cytosolic proteins to the 234- (full-length), 100-, and
26-nt transcripts of the 3'-UTR. For each transcript, the free probe is
shown (single vertical line) and after digestion with RNase
T1 (second lane in each panel). The protein
complex formed with each probe in the presence of proteins is shown
without RNase T1 (arrow) and after RNase T1 (double
vertical lines). All the free probes, except the 26-nt probe, ran
as two bands (see "Results").
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Fig. 2.
Parathyroid protein-binding to the shorter
transcripts of the PTH mRNA 3'-UTR is increased in rats fed a low
calcium diet and decreased in rats fed a low phosphate diet. UV
cross-linking of PT proteins from normal (N), low calcium
( Ca), and low phosphate (
P) rats to
transcripts of 100-, 63-, and 40-nt of the PTH 3'-UTR. Molecular
mass markers in kDa are indicated on the right.
Calcium and phosphate regulated binding to all 3 transcripts.
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Fig. 3.
A 26-nt transcript is sufficient to compete
for the binding of PT cytosolic proteins with the full-length PTH
mRNA 3'-UTR. Competition experiment for the binding of PT
proteins to the 3'-UTR by REMSA. PT proteins were incubated with the
full-length transcript either without (lane 2) or with ×50
or ×100 of transcript A, the 3'-UTR without the terminal 60 nt; ×25
or ×50 of transcript B, the 26-nt binding element; ×25 or ×50 of
transcript C, the 63 nt including the binding element; and ×50 or
×100 of transcript D, the calcium sensing receptor 3'-UTR. After
binding the samples were treated with RNase T1 and then separated on
native PAGE. Lane 1 shows the labeled transcript after RNase
T1 without added protein. There was competition by transcripts
B and C, which include the binding element, but
not by A and D.
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Fig. 4.
Antisense oligonucleotides corresponding to
the protein-binding element prevent binding of PT proteins to the PTH
mRNA 3'-UTR. A, the nt sequence corresponding to
the terminal 100 nt of the 3'-UTR (as shown in Fig. 1A) and
the single stranded antisense oligonucleotides used for binding
interference are shown. The 26-nt protein-binding element is emphasized
in bold. B, REMSA for the binding of PT proteins to the
3'-UTR without and with antisense oligonucleotides. All the samples
were treated with RNase T1. Lane 1 shows the digested free
probe in the absence of protein. Lane 2, shows the
protein-RNA complexes formed in the presence of protein. For
lanes 3-8, the RNA transcripts were preincubated at
80 °C with the different antisense oligonucleotides (1-6) depicted
in Fig. 3A and then protein binding was analyzed by REMSA.
Preincubation with the antisense oligonucleotides 3-5, which
correspond to the protein-binding element or parts of it, prevented
protein binding to the PTH mRNA 3'-UTR. C, UV
cross-linking analysis for the binding of PT proteins to the 3'-UTR
without and with antisense oligonucleotides. The assay was performed
without (first lane) or after preincubation with antisense
oligonucleotides 1-6, as for Fig. 3B either after unfolding
at 80 °C (above) or without heating to 80 °C (below). Molecular
mass markers (kDa) are shown on the right. Preincubation
with the antisense oligonucleotides 3-5, which correspond to the
protein-binding element or parts of it, prevented protein binding to
the PTH mRNA 3'-UTR only if the RNA was denatured at
80 °C.
The 26-nt protein-binding element in the PTH mRNA 3'-UTR is
conserved among species
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Fig. 5.
Insertion of the 63-nt protein-binding region
of the PTH mRNA 3'-UTR into GH RNA conferred responsiveness to GH
mRNA by PT proteins from rats fed low calcium or low phosphate
diets in an in vitro degradation assay.
A, schematic representation of the GH mRNA (above) and
the chimeric GH mRNA containing the PTH 3'-UTR 63-nt element
inserted at the end of the GH coding region (below). B,
representative gels of in vitro degradation assays for
labeled transcripts for PTH (top), GH (middle),
and GH + 63 nt of the PTH 3'-UTR (bottom) with PT proteins
from rats fed a normal, low calcium, or low phosphate diet. At timed
intervals after protein and RNA incubation samples were removed for RNA
analysis. C, time response curves of transcripts for GH + 63 nt of the PTH 3'-UTR after incubation with PT cytosolic proteins as in
A (bottom panel). Each point represents the
mean ± S.E. of three different experiments. The PTH 63-nt insert
into the GH mRNA resulted in stabilization of the transcript with
low calcium PT proteins and destabilization with low phosphate PT
proteins, similar to the native PTH transcript. The PT proteins from
the different diets did not affect the native GH transcript.
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Fig. 6.
Insertion of the 63-nt protein-binding region
of the PTH mRNA 3'-UTR into a random pCRII RNA resulted in
decreased RNA stability that was dependent upon protein binding.
A, cDNA fragments corresponding to 63 or 38 nt of the PTH mRNA 3'-UTR, as in Fig. 1A, were
inserted into a polylinker of pCRII. Chimeric transcripts and a
transcript of the polylinker were analyzed by in vitro
degradation with PT proteins from normal rats. B, the last
3 lanes show the different transcripts at 60 min without
added PT protein. The chimeric 63-nt transcript was less stable than
the native and the chimeric 38-nt transcript.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-globin
3'-UTR could be conferred to a heterologous RNA (18). Therefore, the
effect of the 63 nt on RNA stability was dependent upon an intact
protein-binding region both with the native PTH RNA (9) as well as in
the heterologous RNAs.
-amyloid protein and often
the amyloid precursor protein mRNA as well (21). A 29-base element
in the 3'-UTR has been defined that is bound by
trans-factors and determines the amyloid precursor protein
mRNA decay (21). The iron response element is another well defined
cis-element. This element in the ferritin 5'-UTR controls
translation of this mRNA and in the transferrin receptor mRNA
it is present in multiple iterations where it regulates mRNA
stability (24, 25). We have now identified a novel
cis-element in the PTH mRNA 3'-UTR that determines PTH
mRNA stability in response to changes in serum calcium and phosphate.
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ACKNOWLEDGEMENTS |
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We thank Oded Meuhas for the GH plasmid and Miriam Offner for excellent technical assistance.
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FOOTNOTES |
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* This work was supported in part by grants from the Israel Science Foundation and the U.S.-Israel Binational Science Foundation (to T.N-M.), Horovitz Foundation, and the Minerva Foundation.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: Nephrology
Services, Hadassah University Hospital, P.O. Box 12000, Jerusalem, Israel 91120. Tel.: 972-2-6436778; Fax: 972-2-6421234; E-mail: tally@cc.huji.ac.il.
Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.M005471200
2 R. Kilav, J. Silver, and T. Naveh-Many, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: PTH, parathyroid hormone; PT, parathyroid; UTR, untranslated region; nt, nucleotide(s); DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; REMSA, RNA electrophoretic mobility shift assay; GH, growth hormone.
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REFERENCES |
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