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J Biol Chem, Vol. 274, Issue 36, 25266-25272, September 3, 1999
From the Minerva Center for Calcium and Bone Metabolism, Nephrology
Services, Hadasssah University Hospital, Jerusalem 91120, Israel
Hypophosphatemia leads to an increase in type II
Na+-dependent inorganic phosphate
cotransporter (NaPi-2) mRNA and protein levels in the kidney and
increases renal phosphate reabsorption. Nuclear transcript run-on
experiments showed that the effect of a low phosphate diet was
post-transcriptional. In an in vitro degradation assay,
renal proteins from hypophosphatemic rats stabilized the NaPi-2
transcript 6-fold compared with control rats and this was dependent
upon an intact NaPi-2 3'-untranslated region (UTR). To determine an
effect of hypophosphatemia upon NaPi-2 protein synthesis, the
incorporation of injected [35S]methionine into renal
proteins was studied in vivo. Hypophosphatemia led to
increased [35S]methionine incorporation only into NaPi-2
protein. The effect of hypophosphatemia on translation was studied in
an in vitro translation assay, where hypophosphatemic renal
proteins led to increased translation of NaPi-2 and other transcripts.
NaPi-2 RNA interaction with cytosolic proteins was studied by UV
cross-linking and Northwestern gels. Hypophosphatemic proteins led to
increased binding of renal cytosolic proteins to the 5'-UTR of NaPi-2
mRNA. Therefore, hypophosphatemia increases NaPi-2 gene expression
post-transcriptionally, which correlates with a more stable transcript
mediated by the 3'-UTR, and an increase in NaPi-2 translation involving
protein binding to the 5'-UTR. These findings show that phosphate
regulates gene expression by affecting protein-RNA interactions
in vivo.
Dietary phosphorus is converted in the body to the phosphates, in
which form it exerts its widespread physiological functions, as an
essential component of phospholipids, ATP, DNA, phosphorylated proteins, metabolic intermediaries, body buffers, and bone (1). The
renal tubule has an intrinsic ability to adjust the reabsorption rate
of phosphate according to the need and availability of phosphate to the
body. The renal tubule responds to a decrease in filtered phosphate
load with an increase in transport activity, thereby maintaining renal
phosphate homeostasis. The active reabsorption is mediated by the
Na+-dependent inorganic phosphate
cotransporters (NaPi).1 NaPi
type II are expressed at the apical brush border membranes of the
proximal tubules and are predominantly responsible for the regulated
reabsorption of phosphate in response to changes in dietary phosphate
(2-4). In the rat, NaPi type II is termed NaPi-2. Deletion of the
NaPi-2 gene in mice leads to severe phosphate wasting (5). The activity
of NaPi-2 is increased by a low phosphate diet (6-8) and after
parathyroidectomy (9) (10) and decreased by a high phosphate diet,
parathyroid hormone (9), glucocorticoids (11, 12), and metabolic
acidosis (13, 14). The increase in NaPi-2 is the result of an increase
in Vmax, suggesting an increase in the number of
apical NaPi-2 transporters, by a transporter shuttling mechanism, which
is sensitive to disrupters of microtubule integrity (13, 15). A low
phosphate diet for as little as 2 h led to an increased transfer
of preformed NaPi-2 from the endosome to the apical membrane by a
microtubule-dependent mechanism (16). Chronic
hypophosphatemia increases renal NaPi-2 activity not only by increasing
transfer to the apical membrane (9) but also by increasing the levels
of its mRNA as well as the amount of the cotransporter protein
(10). The adaptive response to extracellular Pi deprivation
also occurs in vitro in opossum kidney cells, where it has
been shown to be a post-transcriptional effect (17, 18). The decrease
in NaPi-2 cotransporter activity caused by PTH is associated with
endocytosis of brush border membrane-associated NaPi-2 protein and is
not accompanied by a decrease in its mRNA levels (19, 20).
We have now studied in vivo in the rat how chronic
hypophosphatemia increases the expression of NaPi-2 mRNA and
protein in the kidney. The results show that the effect on NaPi-2
mRNA is post-transcriptional. An in vitro degradation
assay showed that the NaPi-2 transcript was more stable in the presence
of renal proteins from hypophosphatemic rats than proteins from
controls. RNA stabilization by hypophosphatemic proteins was mediated
by the 3'-UTR of the NaPi-2 transcript. Hypophosphatemic renal
proteins, but not hepatic proteins, showed increased binding to the
NaPi-2 5'-UTR by UV cross-linking and Northwestern gels. In
vivo, the incorporation of injected [35S]methionine
into renal NaPi-2 protein was increased by a low phosphate diet. We
demonstrated that kidney cytosolic proteins from hypophosphatemic rats
led to increased synthesis of NaPi-2 in an in vitro
translation assay, suggesting an increase in translation in addition to
the increased mRNA levels.
Animals--
Weanling male Sabra rats were fed a normal
phosphate (0.3%), normal calcium (0.6%), or a low phosphate (0.02%)
normal calcium (0.6%) diet (Teklad) for 2 weeks. This low phosphate
diet resulted in a serum phosphate of 4.2 ± 0.4 mg/dl
(control = 9.7 ± 1.0 mg/dl) and serum calcium of 12.4 ± 0.7 mg/dl (control = 10.6 ± 0.5 mg/dl). After 2 weeks,
the kidneys, liver, and parathyroid glands were removed under
pentobarbital anesthesia and blood samples were taken for measurements
of serum calcium and phosphate in a Roche autoanalyzer. Tissues for
nuclear run-on assays and protein extracts were used immediately as
described below.
Nuclear Run-on Transcription Assay--
Nuclei were prepared
from kidney cortex of one rat in each experiment and nuclear run-on
transcription assays performed as described previously (21, 22).
Elongated 32P-labeled RNA was extracted by TRI reagent
(Molecular Research Center, Cincinnati, OH) and resuspended in 300 µl
of hybridization buffer (7% SDS, 10% polyethylene glycol 8000, 1.5%
saline/sodium phosphate/EDTA). Aliquots of RNA from treated and
untreated samples were counted in a scintillation counter, and an equal
number of counts from each condition (1-2 × l06 cpm)
was hybridized to linearized cDNAs (5 µg) for, parathyroid (PTH)/parathyroid-hormone related protein receptor, NaPi-2, the calcium
sensing receptor, actin, and pBluescript II KS (Stratagene, La Jolla,
CA), which were immobilized to Hybond filters using a slot
blot apparatus. Hybridization was performed at 65 °C for 72 h.
The filters were washed and autoradiographed. In addition, the filters
were exposed to a bio-imaging plate and quantified by a bio-imaging
analyzer (BAS2065; Fuji Photo Film, Tokyo, Japan).
Plasmid Constructs and Labeling of RNA--
NaPi-2 RNA was
transcribed from pSPORT, which contained the full-length NaPi-2
cDNA (a gift from J. Biber) (Fig. 1A). The plasmid was
linearized using different restriction enzymes and transcripts spanning
different parts of the template were generated using T7 RNA polymerase.
The full-length NaPi-2 transcript was prepared by linearizing the
plasmid with NotI; NaPi-2 RNA without most of the 3'-UTR was
transcribed after linearization with NsiI, RNA for the
1043-nt probe with NcoI, and the RNA for the 5'-UTR with
BspHI (Fig. 1A). For the NaPi-2 3'-UTR RNA, the
NaPi-2 3'-UTR cDNA was recloned by inserting the
SmaI-NotI fragment of the full-length cDNA
into Bluescript II KS (Stratagene, La Jolla, CA) (Fig. 1A). This fragment spans the region of the NaPi-2 cDNA from 1746 to 2464 base pairs. A clone in which the 5' of the cDNA insert was adjacent
to the T3 promoter was used for sense RNA synthesis after linearizing
the plasmid with NotI (Fig.
1B). The full-length RNA for
rat p21 in Bluescript KS (gift of B. Vogelstein) was transcribed with
T3 RNA polymerase after linearization with SalI. Human p27 in Bluescript KS (gift of K. Polyak) was similarly transcribed after
linearization with AccI. Rat cyclin D1 (gift of S. Bianchi) in pCEV 27 was transcribed with SP6 RNA polymerase after linearization with NdeI. Transcription of the PTH cDNA was performed
as described previously (22).
Radiolabeled RNA probes for UV cross-linking were prepared from
linearized templates using the appropriate RNA polymerase in a
transcription reaction containing 1 µg DNA, 0.5 mM each
ATP, CTP, GTP, 8 µM UTP, 2 µM BrUTP or UTP,
500 units/ml RNase inhibitor (Promega, Madison, WI), and
[32P] UTP (800 Ci/mmol, 20 mCi/ml). Samples were
incubated at 37 °C for 1 h, purified on Sephadex G-50 columns,
and aliquots taken for scintillation counting. The specific activity of
the RNA probe was 0.5-1.0 × 106 cpm/ng. For
competition experiments and for RNA translation, unlabeled RNA was
transcribed similarly in the presence of 1 mM each of the
four nucleotides. The RNA was visualized on agarose gels and
transcription levels estimated. To measure the effect of RNA capping on
translation, the RNA was transcribed in the presence of
m7G(5')ppp(5')G cap analog (5 mM) and 0.1 mM GTP, according to the manufacturer's instructions
(Promega, Madison, WI).
Protein Purification--
Different tissues were removed from
the rats and immediately washed in cold phosphate-buffered saline.
Kidney cortex proteins were prepared by rapidly separating the cortex
from the medulla at 4 °C. Parathyroid proteins were prepared from
pooled microdissected parathyroids. The tissues were cut with a
scalpel, suspended in buffer D (23) containing 20 mM HEPES,
20% glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM DTT and homogenized with a Polytron. Total protein was extracted by
repeated freezing and thawing of the samples and centrifugation for 15 min at 12,000 × g. Protein extracts were immediately
frozen at UV Cross-linking Assay--
1 ng of RNA probe (0.5-1.0 × 106 cpm) was incubated with different amounts (15-60 µg)
of cytoplasmic protein extracts in a final volume of 20 µl containing
10 mM HEPES, 3 mM MgCl2, 5 mM DTT, 40 mM KCl, and 5% glycerol (binding
buffer). After 30 min at room temperature, heparin was added to a final
concentration of 5 mg/ml, to eliminate nonspecific binding, and the
samples irradiated at 2.5 J/cm2 with a UV light source of
312 nm. RNase A-XII (Sigma) was then added for 15 min at 37 °C to a
final concentration of 1 mg/ml to digest unprotected RNA. The samples
were heated for 5 min at 65 °C after addition of 5 µl of Laemmli
sample buffer. The samples were then loaded on a 10%
SDS-polyacrylamide electrophoresis gel. RNA-protein binding was
visualized by autoradiography. A molecular weight marker (Bio-Rad) was
also run on the gel for size estimation of the protein-RNA bands. In
some experiments, proteinase K (200 µg/ml) was added. For competition
experiments, unlabeled RNA was added.
In Vitro Cell Free Degradation Assay--
In
vitro cell free degradation was performed essentially as
described (22, 25). Radiolabeled RNA transcripts (0.3 × 106 cpm) were incubated with 20-60 µg of cytoplasmic
extract and 80 units/ml RNasin to prevent nonspecific RNA degradation,
in a total volume of 40 µl at room temperature. At each time point, 6 µl were transferred to a tube containing 300 µl of TRI reagent (Molecular Research Center, Cincinnati, OH) and 10 µg of tRNA and RNA
extracted. Samples were run on formaldehyde-agarose gels, transferred
to Hybond membranes (Amersham Pharmacia Biotech), and immediately
autoradiographed. The remaining undegraded transcripts at the different
time points were quantified by densitometry.
In Vitro Translation Assay--
A RNA transcript (1 µg) for
the full-length NaPi-2 mRNA (2464 nt) or for 1043 nt were
translated using a rabbit reticulocyte lysate system (Promega, Madison,
WI) according to the manufacturer's instructions using
[35S]methionine (1000 Ci/mmol, Amersham Pharmacia
Biotech). Translation was conducted in the presence of proteins (30 µg). The translation product was analyzed on SDS-PAGE and
autoradiographed. In some experiments, trace amounts (200,000 cpm,
equivalent to 0.2 ng) of the transcript were added to the translation
reaction for estimation of RNA recovery. At the end of the experiment,
RNA was extracted from a sample of the translation reaction, run on an
RNA gel, and autoradiographed to measure the amount of remaining transcript.
[35S]Methionine Incorporation into Renal NaPi-2
Protein--
[35S]Methionine (250 µCi/100 g, body
weight) was injected ip to rats fed a control or low phosphate diet.
After 20 min, proteins were extracted from kidney cortex and equal
amounts of radioactivity were incubated overnight at 4 °C with
rabbit anti-rat NaPi-2 antibody (gift of M. Levi) and
immunoprecipitated with anti-rabbit IgG immunoprecipitation reagent
(Sigma). A small amount of each sample before immunoprecipitation was
run together with the immunoprecipitated samples on SDS-PAGE and autoradiographed.
Statistical Analysis--
Results were analyzed by one-way
analysis of variance with the post hoc Bonferroni
multiple comparisons test to determine the significance of differences
between means. Probability values under 5% were determined as
significance. The results are presented as the mean ± S.E.
Hypophosphatemia Increases NaPi-2 mRNA Levels
Post-transcriptionally--
Dietary phosphate restriction is
associated with up-regulation of NaPi-2 mRNA and protein. To
determine if the increase in NaPi-2 mRNA levels by hypophosphatemia
in vivo was transcriptional or post-transcriptional, nuclear
transcript run-on experiments were performed. Weanling rats were fed a
low phosphate (0.02%) or a normal phosphate (0.3%) diet. The low
phosphate diet, given for 14 days, led to hypophosphatemia,
hypercalcemia and increased serum 1,25-(OH)2D3
together with decreased serum PTH levels and a 3-fold increase in
NaPi-2 mRNA and protein (10). Nuclear transcription run-on
experiments showed that the transcription of NaPi-2 and of other
control genes expressed in the kidney were the same in nuclei from
control and hypophosphatemic rats (Fig.
2). This result is representative of
three repeat experiments. These results indicate that the effect of
hypophosphatemia to increase NaPi-2 mRNA and protein levels is
post-transcriptional. We then performed further studies to determine a
mechanism for the in vivo effect.
Cytosolic Proteins from Hypophosphatemic Rats Stabilize the NaPi-2
mRNA Transcript in an in Vitro Degradation Assay--
To determine
a role for renal cytosolic proteins in the post-transcriptional
regulation of NaPi-2 by hypophosphatemia, NaPi-2 mRNA stability was
studied in an in vitro degradation assay. The labeled
full-length NaPi-2 transcript was incubated with kidney cortex
cytosolic proteins. At timed intervals, samples were taken and the RNA
extracted and run on a formaldehyde gel to determine the level of
intact radiolabeled transcript remaining. With kidney proteins from
control rats, the full-length transcript was rapidly degraded at 10 min
and, in contrast, with kidney proteins of the low phosphate rats, the
RNA was still stable after 60 min (Fig. 3A). When RNA probes of 1043 nt (Fig. 3B) and a probe of 2065 nt (data not shown), which
did not include the 3'-UTR, were incubated with either control or low
phosphate renal proteins, there was no difference in RNA degradation
(Fig. 3B). However, when a transcript for the isolated
3'-UTR of NaPi-2 RNA was used in the degradation assay, the 3'-UTR
transcript was stabilized by renal proteins from hypophosphatemic rats
(Fig. 3C) similar to the full-length NaPi-2 transcript. This
indicates that the 3'-UTR is necessary for the stabilization by low
phosphate proteins. When no protein was added, there was no degradation
of the transcript (Fig. 3, A and B) demonstrating
that the degrading factors are in the protein extract. Quantification
of the results for the degradation of either a full-length or a 1043-nt
RNA are shown graphically in Fig. 3D. These results show
that in this in vitro degradation assay proteins from
kidneys of hypophosphatemic rats stabilized the NaPi-2 mRNA, in
correlation with the increase in mRNA levels and stability found
in vivo. The degradation of the probe of 1043 nt by
hypophosphatemic proteins was the same as the degradation of this probe
or the full-length probe by control proteins. This indicates that the
3'-UTR is necessary for hypophosphatemic renal proteins to stabilize
NaPi-2 mRNA, and that without the 3'-UTR the RNA is not
stabilized.
Protein-RNA Binding to the NaPi-2 5'-UTR mRNA Is Increased by
Hypophosphatemia--
Post-transcriptional regulation is often
mediated by proteins, which interact with sequences in the mRNA. To
study whether protein RNA binding is involved in the
post-transcriptional regulation of NaPi-2, after a low phosphate diet,
we performed protein-RNA binding assays. A radiolabeled riboprobe for
the NaPi-2 cDNA was prepared, incubated with rat kidney cortex
proteins, cross-linked by UV light, digested with RNase A, and run on a
SDS-polyacrylamide gels. In these UV cross-linking assays, specific
protein-RNA bands were present which were not seen when no protein was
added to the reaction mix (Fig.
4A) or when the samples were
treated with proteinase K (data not shown), indicating that protein
binding had protected the radiolabeled NaPi-2 mRNA sequences. The
use of transcripts spanning different lengths of the NaPi-2 cDNA
enabled us to determine which region of the NaPi-2 mRNA bound the
cytoplasmic proteins. A radiolabeled RNA probe representing only the
5'-UTR showed 2 prominent protein-RNA bands of approximately 40 and 55 kDa and a faint band at 35 kDa. This binding was increased when the RNA
was incubated with renal proteins from rats fed a low phosphate diet
(Fig. 4A). A transcript for part (1043 nt) of the NaPi-2
mRNA spanning the 5'-UTR and part of the coding region of the
mRNA and also a probe for the full-length RNA, both bound cytoplasmic proteins with the same pattern consisting of two major bands at 35 and 40 kDa (Fig. 4A). The 40-kDa protein RNA
band was present with all three probes and was increased by low
phosphate. The longer transcripts (1043 nt and the full-length) showed
an intense band at 35 kDa which was less intense with the 5'-UTR (Fig.
4A) which may be due to different secondary structures in the RNA. These results indicate that the 5'-UTR was the region necessary for protein binding. Hypophosphatemic renal protein extracts
showed increased binding to full-length NaPi-2 mRNA as compared
with controls (Fig. 4A). This increase in binding of kidney
proteins of hypophosphatemic rats was also observed with the shorter
RNA probes spanning 1043 nt or just the 5'-UTR of the NaPi-2 mRNA
(Fig. 4A).
A probe for the 3'-UTR of the NaPi-2 mRNA showed no specific
binding of proteins to the RNA (data not shown), indicating that the
isolated 3'-UTR did not bind renal proteins. It is noteworthy that the
3'-UTR was necessary for the effect of hypophosphatemic proteins on the
in vitro degradation of the NaPi-2 transcript (Fig. 3), but
in its isolated form it did not show binding.
The same binding pattern to the NaPi-2 mRNA full-length and 5'-UTR
was also demonstrated by UV cross-linking in proteins from parathyroid and liver (Fig. 4B). However, proteins from the
parathyroid and liver of the same hypophosphatemic rats bound NaPi-2
mRNA at the same level as controls, indicating that the
increased binding of hypophosphatemia was unique to the kidney (Fig.
4B).
Competition experiments with excess unlabeled NaPi-2 mRNA indicated
the specificity of the binding of proteins to the NaPi-2 mRNA.
Excess unlabeled RNA representing the first 1043 nt of NaPi-2 mRNA
or its 5'-UTR competed for binding of the full-length NaPi-2 mRNA
probe, confirming that the 5'-UTR was the protein binding site (Fig.
5A). Other mRNA
transcripts of cDNAs such as PTH and the cell cycle inhibitors p27
(Fig. 5B) and p21 and cyclin D1 (data not shown) also
competed for binding to the NaPi-2 mRNA, indicating that the
proteins which bound the NaPi-2 mRNA may also recognize sequences
in these mRNAs. In addition, PTH mRNA 5'-UTR bound renal
cytoplasmic proteins (data not shown). It is of interest that the
translation in vitro of all these mRNAs is regulated by
renal hypophosphatemic proteins (see later).
The binding of proteins to the NaPi-2 mRNA was also demonstrated by
Northwestern blots (Fig. 6). This method
determines the size of the proteins that bind NaPi-2 mRNA without
the bound radiolabeled RNA sequences that are present in the UV
cross-linking assay. Kidney cortex proteins from control and rats fed a
low phosphate diet were run on SDS-polyacrylamide gels and transferred
to nitrocellulose membranes which were hybridized with a RNA probe for
NaPi-2 mRNA. The binding was increased with renal hypophosphatemic
proteins (Fig. 6) but not with hepatic hypophosphatemic proteins (data not shown). These proteins were similar in size to those demonstrated by UV cross-linking. It is noteworthy that, in some experiments, an
additional band of ~55 kDa was present (Figs. 4A and 5),
while in other instances this larger band was very faint (Fig.
4A).
To determine a role for the differences in NaPi-2 5'-UTR RNA-protein
binding of renal proteins from hypophosphatemic rats, we analyzed their
effect on NaPi-2 RNA translation.
Increased Incorporation of [35S]Methionine into
NaPi-2 Protein in the Kidneys of Hypophosphatemic Rats--
To
demonstrate that hypophosphatemia led to an increase in NaPi-2 protein
synthesis, we compared the in vivo rates of NaPi-2 synthesis. Rats fed a normal or a low phosphate diet were injected intraperitoneally with L-[35S]methionine.
After 15 min, the kidneys were removed and kidney cortex protein
extracts prepared. Incorporation of radioactivity into renal protein
was estimated by precipitation with trichloroacetic acid. NaPi-2 was
isolated from the extracts by antibody precipitation. Equal amounts of
radioactivity were added to an excess of anti rat NaPi-2 serum and
incubated overnight at 4 °C. After precipitation the samples were
analyzed by SDS-PAGE. Radioactivity of the gels was visualized by
autoradiography. The size of the immunoprecipitated NaPi-2 was ~45
kDa, which is the size of monomeric NaPi-2 protein (26). The additional
bands present at ~65 and 180 kDa may represent alternative forms of
NaPi-2 (27). There was an increase in incorporation of the labeled
methionine in the immuno-precipitated NaPi-2 protein from kidney in
extracts of hypophosphatemic rats compared with control (Fig.
7), and not in the total labeled proteins
before immunoprecipitation (Fig. 7). Immunoprecipitation enriched the samples for NaPi-2 protein as compared with the sample of the total
protein. These results suggest that the increase in NaPi-2 protein in
rats fed a low phosphate diet was due to increased synthesis and was
specific for NaPi-2 protein and not for other renal proteins (Fig. 7).
The increase in NaPi-2 protein may be a result of the increase in
NaPi-2 mRNA or there could be an additional increase in
translation. In vivo we were unable to separate the increase
in NaPi-2 protein due to the increase in NaPi-2 mRNA levels from an
additional translational effect. To answer this question an in
vitro translation assay was used.
Cytosolic Proteins from Hypophosphatemic Rats Increase NaPi-2
mRNA Translation in an in Vitro Translation Assay--
A
functional role for the increased binding of kidney proteins to the
NaPi-2 5'-UTR mRNA was provided by an in vitro
translation assay. Kidney proteins from control and hypophosphatemic
rats were added with NaPi-2 mRNA transcripts to an in
vitro rabbit reticulocyte lysate translation system in the
presence of [35S]methionine. At the end of the reaction,
the 35S-labeled translation products were run on a
SDS-polyacrylamide gel and the labeled NaPi-2 protein synthesized was
visualized by autoradiography. There was a marked increase in
translation of NaPi-2 mRNA in the presence of proteins from kidneys
of rats fed the low phosphate diet (Fig.
8A). This increase was evident when the full-length NaPi-2 transcript and also when a transcript of
1043 nucleotides were used for translation (Fig. 8A). The
translation of other mRNAs such as PTH, cyclin D1 (Fig.
8B), p21 (Fig. 8C), and also p27 (data not shown)
was also increased in the presence of proteins from kidneys of
hypophosphatemic rats. It is noteworthy that, in addition to the
increase in translation of these RNAs, they all competed for binding of
proteins to the NaPi-2 mRNA (Fig. 5B).
To determine whether RNA capping affected the increased translation in
the presence of renal proteins from low phosphate rats, we compared the
translation of both capped and uncapped transcripts for NaPi-2 and
cyclin D1. The in vitro translation of both the capped and
uncapped transcripts was increased with low phosphate proteins,
indicating that capping had no additional effect in this assay (data
not shown).
The increase in in vitro translation with hypophosphatemic
renal proteins may have been due to differences in degradation of the
RNA transcripts in the presence of the proteins (Fig. 3). To exclude
this possibility, we added radiolabeled NaPi-2 transcript in trace
amounts, together with the unlabeled NaPi-2 transcript and renal
proteins to the reticulocyte assay. At the end of the experiment, RNA
was extracted from a part of the reaction and run on a gel and the
amount of labeled NaPi-2 transcript was visualized by autoradiography.
The result showed that the same amount of transcript was present at the
end of the translation assay in the presence of normal and low
phosphate renal proteins (data not shown). This indicated that there
was an increase in in vitro translation in this assay and
not an effect on RNA degradation under the conditions of this
experiment. In the degradation assay (Fig. 3), there was a
decrease in NaPi-2 RNA after incubation with normal phosphate
renal proteins; this is because the protein preparation for the
degradation assay is designed to retain its catalytic functions.
Addition of proteins from other tissues such as liver (Fig.
8C) and parathyroid (data not shown) of these same rats did
not show any difference in translation levels, indicating that the effect was specific for proteins from kidneys. The binding of hypophosphatemic proteins from these other tissues to NaPi-2 RNA was
also not increased (Fig. 4B). The increased translation
in vitro of NaPi-2 mRNA and other mRNAs by renal
hypophosphatemic proteins correlated with the increased binding of
these proteins to the NaPi-2 mRNA 5'-UTR. These effects were
specific for renal proteins and not found with hepatic proteins. The
results suggest that the increased binding has a functional role in the
increased synthesis of NaPi-2 protein.
The renal adaptation to hypophosphatemia involves an increase in
the proximal tubular reabsorption of phosphate that is mediated by
NaPi-2 (3, 13). The increase in NaPi-2 activity involves an increase in
the levels of NaPi-2 mRNA, NaPi-2 protein, and translocation of the
protein to the luminal membrane. In opossum kidney cells, it has been
shown that a low phosphate increases NaPi-2 mRNA levels
post-transcriptionally (17, 18). To understand how a low phosphate
regulates cellular processes that increases NaPi-2 in vivo,
we have used a model of dietary induced chronic hypophosphatemia. We
show in vivo that the increase in the mRNA levels is
post-transcriptional and have performed experiments to elucidate the
mechanisms that contribute to this effect. An in vitro
degradation assay showed that the proteins from the kidneys of
hypophosphatemic rats, when incubated with a NaPi-2 transcript, led to
a more stable transcript, which is dependent upon the presence of the
3'-UTR.
The parathyroid is another organ that responds to phosphate (21,
28-30). The parathyroid has a different NaPi cotransporter named
PiT-1, which is homologous to the type III NaPi cotransporters, and is
increased in rats fed a low phosphate diet (31). Interestingly, hypophosphatemia decreases PTH mRNA levels post-transcriptionally, and this is also mediated by cytosolic proteins interacting with the
3'-UTR of the PTH mRNA (22). In the parathyroid this interaction results in a rapid degradation of the PTH transcript. It is intriguing that the effect of phosphate in these two organs and on the different mRNAs is post-transcriptional and mediated by protein-RNA
interactions at the 3'-UTR.
UV cross-linking gels showed no binding of renal proteins to an
isolated NaPi-2 RNA 3'-UTR. However, the 3'-UTR was essential for
stabilization of the NaPi-2 transcript by hypophosphatemic renal
proteins. Moreover, the isolated 3'-UTR was stabilized by low phosphate
renal proteins in the in vitro degradation assay. This
suggests that the inhibition of degradation with the low phosphate
proteins is not due to proteins binding to the 3'-UTR and protecting it
from degradation, but rather to an effect on the degrading proteins
(Fig. 9). However, our results do not
exclude the possibility that in the full-length NaPi-2 RNA, proteins
may bind to sequences other than the 3'-UTR and contribute to RNA stabilization by low phosphate.
At the 5'-UTR, hypophosphatemia led to an increased binding of renal
cytosolic proteins as demonstrated by both UV cross-linking and
Northwestern gels. The binding studies showed that hypophosphatemia increased binding to the NaPi-2 RNA 5'-UTR, only with renal cytosolic proteins, and not with proteins from other organs, such as the liver
and parathyroid that also express these proteins. A number of other
mRNAs tested competed for binding of renal proteins to the NaPi-2
5'-UTR by UV cross-linking. This suggests that other mRNAs have
sites that recognize the same proteins, which are regulated by
hypophosphatemia in the kidney. The binding of proteins to the 5'-UTR
often indicates a translational role. One possibility is that these
proteins are factors that are involved in the initiation of RNA translation.
To measure NaPi-2 synthesis, [35S]methionine was injected
into control and hypophosphatemic rats and the renal proteins
synthesized analyzed. There was more [35S]methionine
incorporation into NaPi-2 in the hypophosphatemic rat kidneys. This
effect in vivo was specific for NaPi-2 protein, and there
was no general increase in [35S]methionine incorporation
into other renal proteins, suggesting that there was more NaPi-2
synthesized. A short labeling time was used in this in vivo
experiment, and therefore the amount of 35S-labeled NaPi-2
protein detected represented newly synthesized NaPi-2. However, in this
in vivo experiment, we were unable to control the ratio of
[35S]methionine to unlabeled methionine from
intracellular proteolysis, which may dilute the labeled amino acid. The
effect of hypophosphatemia on NaPi-2 translation was therefore studied
using an in vitro translation assay. In the presence of
hypophosphatemic cytosolic proteins, there was an increase in NaPi-2
translation compared with controls. There was increased in
vitro translation also with a shorter transcript that did not
include the 3'-UTR and part of the coding region, indicating that these
excluded regions were not important to this regulation. Proteins from
other tissues of these same rats showed no effect on NaPi-2
translation. Of interest, the translation of a number of other
transcripts was also increased, indicating that the hypophosphatemic
proteins in vitro affected other mRNAs as well. The
increase in translation correlates with the ability of these mRNAs
to compete for binding of kidney protein to NaPi-2 mRNA. The
increased translation of all these mRNAs by hypophosphatemic
proteins was specific to the kidney. These observations suggest that
hypophosphatemia enhances the renal translational machinery and this
may affect other mRNAs in addition to NaPi-2. However, in
vivo, the incorporation of [35S]methionine did not
show a generalized increase in translation with hypophosphatemic
proteins but only an increase in NaPi-2 protein (Fig. 7). Our results
do not exclude the possibility that the translation of some specific
proteins were increased. It is known that the activity of other renal
transporters, such as the sodium-dependent glucose and
sulfate transporters, are not increased by hypophosphatemia (32-34).
This suggests that, in the kidney in vivo, there is a
mechanism that restricts the increase in translational activity after
hypophosphatemia to NaPi-2.
Hypophosphatemia, therefore, increases NaPi-2 translation and the
binding of renal cytosolic proteins to NaPi-2 5'-UTR, effects that are
specific to the kidney. In addition, hypophosphatemia increases the
NaPi-2 mRNA stability by an effect on its 3'-UTR. These mechanisms
are of relevance to the understanding of both the post-transcriptional
increase in NaPi-2 mRNA and the possible increase in translation of
NaPi-2 protein due to hypophosphatemia. Further understanding of these
mechanisms will involve characterization of the binding proteins and
definition of the RNA sequences on the NaPi-2 mRNA with which they interact.
Regulation of transcript stability and translation by RNA-protein
interaction is a well defined paradigm (35). RNA stability is
determined by stabilizing and degrading factors which act on specific
sequences in the mRNA, often in the 3'-UTR (36). Translation initiation is generally the rate-limiting step in the overall process
of translation and is influenced by elements in the 5'-UTR of the
mRNA (37). An increasing number of trans-acting factors, mainly
proteins associated with mRNA, are now being recognized as
modulators of translation. Translation control can be very specific,
aimed at translation of only part of the mRNAs in the cell or even
a single one, and it can effect a wider population of mRNAs. For
NaPi-2, we show that hypophosphatemia increases its RNA stability and
translation. We propose a model where a low phosphate induces a change
in renal cytosolic proteins, which result in less degradation (Fig. 9).
This could be due to an inhibitory factor interacting with a
ribonuclease rather than with the 3'-UTR, because there was no binding
to the isolated 3'-UTR by UV cross-linking. Protein-RNA interactions at
the 5'-UTR regulate NaPi-2 translation. This in vivo model
can now be used to identify protein-RNA interactions that regulate
NaPi-2 gene expression, which is central to renal phosphate homeostasis.
We thank Miriam Offner for excellent technical assistance.
*
This work was supported in part by a grant from the Israel
Academy of Sciences (to T. N.-M.) 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.
The abbreviations used are:
NaPi, Na+-dependent inorganic phosphate;
UTR, untranslated region;
PAGE, polyacrylamide gel electrophoresis;
PTH, parathyroid hormone;
DTT, dithiothreitol.
Protein-RNA Interactions Determine the Stability of the Renal
NaPi-2 Cotransporter mRNA and Its Translation in Hypophosphatemic
Rats*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The rat NaPi-2 cDNA constructs and the
restriction enzymes used for in vitro synthesis of RNA
transcripts. A, the full-length NaPi-2 cDNA. T7 RNA
polymerase was used to transcribe the full-length cDNA after
linearization of the plasmid with NotI; a 2065-nt transcript
excluding the 3'-UTR with NsiI; a 1043-nt transcript, which
included the 5'-UTR and only part of the translated region, and
excluded the 3'-UTR, with NcoI; and a 53-nt transcript
consisting only of the 5'-UTR, with BspHI. B,
subcloning of the NaPi-2 3'-UTR into Bluescript KS. The 3'-UTR was
transcribed with T3 RNA polymerase after linearization of the plasmid
with NotI. The transcript length (nt) is denoted under the
restriction sites.
80 °C in aliquots. For RNA degradation a S-100
cytoplasmic fraction was prepared as before (24) by homogenizing the
tissue with a Polytron in 1 volume of 10 mM Tris/HCl, pH
7.4, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl
fluoride, 10 mM KCl, and 1.5 mM
MgCl2. 0.1 volumes of extraction buffer (1.5 mM
KCl, 15 mM MgCl2, 100 mM Tris/HCl,
pH 7.4, 5 mM DTT) were 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 80 °C in aliquots, which were stable only up to 2 weeks. In
some experiments, these proteins were used for binding and in
vitro translation. Protein concentration was determined by optical
density densitometry (595 µm wave length) using a Bradford reagent
(Bio-Rad).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
NaPi-2 run-on transcription rates are the
same for the nuclei of control and hypophosphatemic kidneys.
Nuclear transcript run-ons for PTH receptor (PTH-R), NaPi-2,
the calcium sensing receptor (CaSR), Bluescript KS plasmid,
and actin, for rats fed a control or low phosphate (Low P) diet for 14 days after weaning.
View larger version (33K):
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Fig. 3.
Renal cytosolic proteins from
hypophosphatemic rats increase NaPi-2 RNA stability in an in
vitro degradation assay. A, gel
electrophoresis of full-length labeled NaPi-2 transcript incubated with
cytosolic proteins from rats fed control or a low phosphate diet
(low P) for different time periods. B,
a 1043-nt transcript incubated with the same proteins and analyzed as
in A. C, degradation of a transcript for the
3'-UTR with cytosolic proteins from control and low phosphate rats.
D, time-response curves of in vitro degradation
assays for the full-length NaPi-2 RNA after incubation with renal
cytosolic proteins of control ( 
) and low phosphate (
), and the
1043-nt RNA with control (
) and low phosphate (
) proteins. Each
point represents the mean ± S.E. of five different experiments.
The NaPi-2 transcript is degraded very rapidly by proteins from control
rats or when the 3'-UTR is not present. When the 3'-UTR is intact,
hypophosphatemic proteins stabilize the transcript. This indicates that
the 3'-UTR is necessary for the stabilization of NaPi-2 by low
phosphate renal proteins.
View larger version (72K):
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Fig. 4.
A low phosphate diet increases binding of
renal proteins and not hepatic proteins to the 5'-UTR of NaPi-2 by UV
cross-linking. A, the 32P-labeled NaPi-2
5'-UTR, the 1043-nt RNA, and the full-length NaPi-2 RNA were UV
cross-linked with no proteins ( ) or with renal proteins from control
(N) or low phosphate (P) rats, run on SDS-PAGE,
and visualized by autoradiography. There is protein binding to the
three probes, and the binding is increased with renal cortical proteins
from the low phosphate rats. B, UV cross-linking of kidney,
parathyroid (PT), and liver of control (N) and
low phosphate (P) rat proteins to the NaPi-2 1043-nt
transcript. There was increased binding with low phosphate proteins
only from the kidney and not from other tissues to the NaPi-2 RNA. The
arrows indicate the RNA-binding proteins, and the size of
molecular weight markers is shown on the right.
View larger version (64K):
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Fig. 5.
Competition experiments for UV cross-linking
of renal proteins to the NaPi-2 RNA probe with unlabeled RNA.
A, renal proteins were incubated with labeled NaPi-2 1043-nt
transcript and increasing amounts of unlabeled 1043-nt transcript or
unlabeled NaPi-2 5'-UTR transcript, and analyzed by UV cross-linking.
B, as in A but with unlabeled PTH and p27
transcripts. The arrows indicate the RNA-binding proteins,
and the size of molecular weight markers is shown on the
right.
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Fig. 6.
A low phosphate diet increases binding of
renal proteins to the NaPi-2 RNA by Northwestern gels. Renal
cortical proteins from control (N) and low phosphate
( P) rats were run on a SDS-PAGE and transferred to
membranes which were hybridized to NaPi-2 32P-riboprobe for
Northwestern analysis. There was increased binding to the low phosphate
proteins at three main bands.
View larger version (62K):
[in a new window]
Fig. 7.
More NaPi-2 protein is synthesized by
measuring [35S]methionine incorporation in
vivo in the kidney. [35S]Methionine was
injected intraperitoneally to control rats (N) and rats fed
a low phosphate ( P) diet, and after 20 min renal cortical
proteins were extracted. Equal amounts of radioactivity were
immunoprecipitated with NaPi-2 antibody and run a SDS-PAGE
(IP). In addition, a sample before immunoprecipitation from
control and low phosphate proteins was run on a SDS-PAGE
(Total protein). The gels were autoradiographed.
There was an increase in labeled NaPi-2 (arrow) in low
phosphate kidneys but no generalized increase in protein
synthesis.
View larger version (35K):
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Fig. 8.
Renal proteins from hypophosphatemic rats
increase the translation in vitro of NaPi-2 RNA.
A, in vitro translation of NaPi-2 1043-nt and
full-length NaPi-2 in the presence of [35S]methionine and
renal proteins from control (N) and low phosphate
( P) rats. The translation products were rum on SDS-PAGE
and autoradiographed. The 1043-nt translation product was ~40 kDa and
the full-length product ~60 kDa, which are the predicted sizes from
these transcripts. There is increased translation by low phosphate
proteins of both transcripts. B, translation of NaPi-2
1043-nt probe, PTH, and cyclin D1 RNAs as in A. The
translation of all transcripts was increased. C, translation
of NaPi-2 and p21 RNAs as in A in the presence of kidney and
liver proteins. The translation of both transcripts was increased by
low phosphate proteins from the kidney and not the liver.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (11K):
[in a new window]
Fig. 9.
A model of the regulation of renal NaPi-2
mRNA stability and translation by a low phosphorus diet. Low
phosphate leads to an increase in binding of renal cytosolic proteins
( 
) to the NaPi-2 5'-UTR and increased NaPi-2 translation. At the
3'-UTR, a low Pi results in an inhibition of degradation of
the RNA by ribonucleases (shaded crescents) and
increased mRNA stability.
ACKNOWLEDGEMENT
FOOTNOTES
ABBREVIATIONS
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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