Characterization of cis-acting element in renal NaPi-2 cotransporter mRNA that determines mRNA stability

Yulia Moz, Justin Silver, and Tally Naveh-Many

Minerva Center for Calcium and Bone Metabolism, Nephrology Services, Hadassah University Hospital, Jerusalem, Israel 91120


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hypophosphatemia leads to an increase in Na+-Pi cotransporter (NaPi-2) mRNA levels. This increase is posttranscriptional and correlates with a more stable transcript mediated by the terminal 698 nt of the NaPi-2 mRNA. A 71-nt binding element was identified with renal proteins from rats fed control and low-Pi (-Pi) diet. The binding of -Pi renal proteins to this transcript was increased compared with control proteins. The functionality of the cis element was demonstrated by an in vitro degradation assay. -Pi renal proteins stabilized transcripts that included the cis element compared with control renal extracts. The full-length NaPi-2 transcript, but not control transcripts, was stabilized by -Pi extracts. Insertion of the binding element into green fluorescent protein (GFP) as a reporter gene decreased chimeric GFP mRNA levels in transfection experiments. Our results suggest that the protein-binding region of the NaPi-2 mRNA functions as a cis-acting instability element. In hypophosphatemia there is increased binding to the cis-acting element and subsequent stabilization of NaPi-2 mRNA.

phosphate; messenger ribonucleic acid half-life; protein-ribonucleic acid interactions


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PI homeostasis is maintained by a well-defined membrane transport system (31). In mammals, renal Pi reabsorption is essential to Pi homeostasis. The renal tubule has an intrinsic ability to adjust the reabsorption rate of Pi according to need and availability of Pi (22). The active reabsorption is mediated by the Na+-dependent Pi transporters (NaPi). NaPi type IIa transporters (NaPi-2) are expressed at the apical brush-border membrane (BBM) of the renal proximal tubules and are responsible for the regulated reabsorption of Pi in response to changes in dietary Pi (16, 20). NaPi type IIb transporters are homologous to NaPi type IIa but are found in a variety of other tissues such as intestine, lung, prostate, and pancreas (4). Disruption of the NaPi-2 gene in mice resulted in increased urinary Pi excretion, an 85% loss in BBM Na+-Pi cotransport, and significant hypophosphatemia (1). Mice homozygous for NaPi-2 gene deletion did not respond to Pi depletion with an adaptive increase in Na+-Pi cotransport or to parathyroid hormone (PTH) with a decrease in transport. Therefore, NaPi-2 is the major regulator of renal Pi homeostasis. There is a rapid adaptive increase in proximal tubule apical BBM Na+-Pi cotransport activity and NaPi-2 protein abundance that is mediated by microtubule-dependent translocation of presynthesized NaPi-2 cotransporter protein to the apical BBM (13). Dexamethasone inhibits Na+-Pi cotransport, and this is associated with a decrease in renal BBM NaPi-2 cotransporter abundance and an increase in glucosylceramide content of the BBM (15). Levi et al. (15) suggested that the increase in BBM glucosylceramide content plays a role in mediating the effect of dexamethasone on Na+-Pi cotransport activity. PTH and a high-Pi diet lead to a decrease in renal proximal tubular Na+-Pi cotransport, which correlates with a decrease in the number of NaPi-2 transporters in the BBM because of their routing to the lysosome and subsequent degradation (6, 7, 23, 24). Several interacting proteins have been identified that may contribute either to its apical distribution or its subapical/lysosomal traffic (5, 22). However, chronic Pi depletion not only increases the transfer of preformed NaPi-2 to the apical BBM but also increases the level of the NaPi-2 mRNA and protein level (9, 30). The adaptive response to Pi depletion also occurs in vitro in opossum kidney cells, where it has been shown to be a posttranscriptional effect (2, 17, 21). In vivo in rats we previously showed (9) by nuclear transcript run-on experiments that the effect of chronic Pi depletion is mainly posttranscriptional, although others also found a transcriptional effect (8). We studied the mechanisms involved in the posttranscriptional effect and found that this was dependent on protein-RNA interactions (19). Cytosolic renal proteins showed increased binding to the NaPi-2 mRNA 5'-untranslated region (UTR), and this was associated with increased translation of NaPi-2 in vitro (19). Renal proteins from rats fed a low-Pi diet (-Pi) stabilized the NaPi-2 transcript in vitro, and this was dependent on the presence of the terminal 698 nt at the 3' end of the mRNA. In the present studies we have defined the region in the NaPi-2 mRNA 698 nt that mediates the binding of -Pi proteins. The functionality of the NaPi-2 mRNA protein-binding sequence was demonstrated in an in vitro degradation assay with renal proteins from control and low-phosphate rats. In addition, the protein binding region was inserted into a green fluorescent protein (GFP) reporter gene that was transfected into human embryonic kidney (HEK)293 cells to study its effect on GFP expression. The sequence was shown to be an instability region in both systems. Therefore, the increase in binding of -Pi renal proteins to this region stabilizes the NaPi-2 mRNA.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Experimental 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 3 wk. This low-phosphate diet resulted in a serum phosphate of 4.1 ± 0.5 mg/dl (control = 9.6 ± 0.9 mg/dl) and a serum calcium of 12.3 ± 0.7 mg/dl (control = 10.6 ± 0.5 mg/dl). After 3 wk, the kidneys were removed under pentobarbital sodium anesthesia and blood samples were taken for measurements of serum calcium and phosphate in a Roche autoanalyzer. Tissues for protein extracts were used immediately as described below.

Plasmids for RNA transcription. The full-length 2,464-nt NaPi-2 transcript (GenBank accession no. L13257) was prepared with T7 RNA polymerase from linearized plasmid construct containing the cDNA in pSPORT (a gift from J. Biber) as previously described (19). The 698 bp of the NaPi-2 cDNA, spanning the region of 1746-2464 of the NaPi-2 cDNA, were subcloned into Bluescript II KS (Stratagene, La Jolla, CA) as previously described (19). RNAs for the 698, 461, 362, 315, 231, and 144 nt were transcribed from this construct linearized with NotI, MscI, StyI, SphI, AvaII, and BbvII, respectively, with T3 RNA polymerase. The Delta 450 transcript was prepared by restriction of the plasmid containing the 698-bp cDNA with BbvII and SphI that removed 172 bp and religation of the plasmid. The 95-nt transcript was prepared by subcloning a corresponding PCR product prepared with the forward oligonucleotide 5'AGTCTTCCTGGAGGAGCTT3' and a reverse oligo 5'TCTGGACCTGCAGCCTAGA3'. The PCR fragment was ligated into pGEM-T Easy vector (Promega, Madison, WI) and linearized with NcoI for the 95-nt transcript and with BcnI for the 71-nt transcript. RNA was transcribed with SP6 RNA Pol. The transcript for the transferrin receptor (Tfr) contained 250 nt of the Tfr 3'-UTR that included three iron responsive elements (IREs).

In vitro RNA synthesis. Radiolabeled RNA probes for RNA electrophoretic mobility shift assay (REMSA), and in vitro degradation assays were prepared from linearized templates with the appropriate RNA polymerase in a transcription reaction containing 1 µg DNA, 0.5 mM ATP, CTP, and GTP, 8 µM UTP, 2 µM UTP, 500 U/ml RNase inhibitor (Promega) 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 were taken for scintillation counting. The specific activity of the RNA probe was 0.5-1.0 × 106 cpm/ng. For competition experiments RNA was transcribed similarly in the presence of the 4 nt at 1 mM. Unlabeled RNAs were quantified by spectrophotometry at 260 nm/280 nm and visualization on agarose gels.

Protein purification for REMSA and in vitro degradation assays. Kidneys were removed from the rats and immediately washed in cold PBS. For binding assays, kidney protein extracts were prepared as described previously (19) by suspending the tissue in buffer D containing 20 mM HEPES, 20% glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT and homogenizing 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 -80°C in aliquots.

For RNA degradation, a S-100 cytoplasmic fraction was prepared as before (19) by homogenizing the tissue with a Polytron in 1 vol of (in mM) 10 Tris · HCl pH 7.4, 0.5 DTT, 0.5 PMSF, 10 KCl, and 1.5 MgCl2; 0.1 vol of extraction buffer (in mM: 1.5 KCl, 15 MgCl2, 100 Tris · HCl pH 7.4, 5 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 wk. Protein concentration was determined by optical density densitometry (595-µm wavelength) with a Bradford reagent (Bio-Rad, Hercules, CA).

REMSA. Labeled RNA transcripts (10,000 cpm) spanning different regions of the NaPi RNA were incubated with renal protein extracts, in a final volume of 20 µl containing 4 µg tRNA, 10 mM HEPES, 3 mM MgCl2, 40 mM KCl, 5% glycerol, and 1 mM DTT (binding buffer) for 10 min at 4°C. 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 that did not affect binding. The samples were run for 3 h at 4°C 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.

In vitro cell free degradation assay. In vitro degradation was performed essentially as described previously (18, 19). Radiolabeled RNA transcripts (0.3 × 106 cpm) were incubated with 20-60 µg of cytoplasmic extract and 80 U/ml RNasin to prevent nonspecific RNA degradation, in a total volume of 40 µl at room temperature. At each time point 6 µl was transferred to a tube containing 300 µl of TRI reagent (Molecular Research Center, Cincinnati, OH), and 10 µg of tRNA and RNA was extracted. Samples were run on formaldehyde-agarose gels, transferred to Hybond membranes (Amersham, Little Chalfont, UK), and autoradiographed. The remaining undegraded transcripts at the different time points were quantified by densitometry.

Construction of chimeric GFP constructs containing cDNAs of various regions of NaPi-2 mRNA for transfection experiments. The 461-, 362-, 315-, 144-, 95-, and 71-bp cDNAs corresponding to different regions of the NaPi-2 mRNA terminal 698 nt (Fig. 1) were excised by restriction enzymes and inserted into the multiple cloning site of pEGFP-C1 (Clontech, Palo Alto, CA), at the 3' end of GFP cDNA.


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Fig. 1.   Binding of low-Pi (-Pi) diet renal proteins to Na+-Pi cotransporter type IIa (NaPi-2) mRNA maps to a defined region. A: RNA electrophoretic mobility shift assay (REMSA) of renal proteins from rats fed a -Pi diet and transcripts representing different regions of the terminal 698 nt of the NaPi-2 mRNA, from 1,746 to 2,444 nt. For each transcript the first lane is the free probe, and the subsequent lanes represent increasing amounts of renal proteins that were added. B: diagrammatic representation of the 698-bp fragment of the NaPi-2 cDNA that was used as the template to transcribe RNAs of different lengths. The transcript Delta 461 nt was constructed to exclude the sequence between the 144-nt transcript and the 315-nt transcript. The region that was excluded is shown as a broken line. The transcripts of 95 and 71 nt were generated from constructs that were prepared by ligation of PCR products into pGEM-T easy vector. C: binding of cytosolic proteins from rats fed a control diet (N) or a -Pi diet to a 95- and 231-nt transcript. -Pi renal proteins showed increased binding compared with control renal proteins.

Transient transfection experiments. The plasmids (1 µg DNA/well/24-well plate) were transiently transfected into HEK293 cells by calcium phosphate precipitation. Twenty-four hours after transfection total RNA was extracted by TRI reagent and analyzed for GFP mRNA levels by Northern blot. GFP protein was measured by immunofluorescence microscopy. Expression of cotransfected cytomegalovirus (CMV)-beta -galactosidase plasmid demonstrated transfection efficiency.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Characterization of binding of renal cytosolic proteins to defined region in NaPi-2 mRNA. To study protein-RNA interaction we performed REMSA with renal proteins from control rats or rats fed a low-Pi diet for 3 wk and transcripts representing different regions of the NaPi-2 mRNA. A transcript that excluded the terminal 698 nt of the NaPi-2 mRNA did not bind -Pi renal proteins by REMSA (not shown). A transcript of 698 nt that contained the terminal coding region and the 3'-UTR (Fig. 1B) formed a shifted complex with renal cytosolic proteins from -Pi rats (Fig. 1A). To define the -Pi protein-binding region in the 698-nt NaPi-2 transcript, we transcribed RNAs representing smaller regions in the mRNA and analyzed them for binding with the -Pi renal proteins. Shortening of the transcript by excluding successively larger regions of the 3'-UTR to transcripts of 461, 362, 315 (Fig. 1, A and B), and 231 nt (Figs. 1B and 2) did not affect binding. All these transcripts bound proteins, resulting in a shift of the free probe that was dose dependent. The larger shifted complexes when more protein is added may represent the binding of additional subunits of the protein RNA complex. Further shortening of the transcript to 144 nt resulted in no binding (Fig. 1, A and B). This suggests that the binding region is between 144 and 231 nt (Fig. 1B). Therefore, we transcribed RNA that deleted the 164 nt between the 151- and 315-nt transcripts (Fig. 1B, Delta 461 nt). This Delta 461-nt transcript did not bind renal proteins (Fig. 1, A and B). These results indicate that the binding region is between 151 and 231 nt in the 698 nt represented in Fig. 1B. Transcripts of 95 (not shown) and 71 nt in this region (Fig. 1A) were sufficient for protein binding (Fig. 1, A and B), defining the minimal protein binding region that corresponds to 1889-1960 of the NaPi-2 cDNA at the junction of the coding region and the 3'-UTR. Renal proteins from control rats showed significantly reduced binding to the 71 (not shown)-, 95-, and 231-nt transcripts (Fig. 1C). The decreased binding may have been in part due to some degradation of the transcript by the renal proteins from control rats. This is evident by the partially degraded excess free probe that remains after binding with control renal proteins but not with -Pi proteins. With -Pi proteins the increased binding utilizes all the probe (Fig. 1C).


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Fig. 2.   Competition experiments for REMSA binding of renal proteins to the NaPi-2 mRNA maps a minimal binding region of 71 nt. Renal proteins from rats fed a -Pi diet were incubated with labeled NaPi-2 RNA 231 nt (left) or 698 nt (right) and increasing amounts of unlabeled 231-, 144-, or 71-nt transcripts as indicated and analyzed by REMSA. The first lane in each panel is the free probe, and the second lane shows the binding in the presence of -Pi proteins without and with increasing amounts of unlabeled transcripts as indicated. Excess transcripts of 231 and 71 nt competed for binding, whereas the 144-nt transcript was a far less effective competitor.

To show specificity of the binding we performed competition experiments. The binding of -Pi proteins to the 231-nt transcript was effectively competed for by excess unlabeled 231-nt transcripts already at 20× competitor excess (Fig. 2). The 144-nt transcript, which did not bind renal proteins (Fig. 1A), was also much less effective as a competitor for the binding to the 231-nt transcript (Fig. 2). In addition, in Fig. 2 it is shown that a 71-nt transcript is sufficient to compete for binding of -Pi renal proteins to the 698-nt NaPi-2 transcript. The competition experiments demonstrate the specificity of protein binding to the NaPi-2 element. There is increased binding of -Pi renal proteins to the NaPi-2 transcript compared with proteins of normal rats. This binding is to a 71-nt region spanning the junction of the coding region and the 3'-UTR of the NaPi-2 mRNA.

Renal cytosolic proteins from -Pi stabilize NaPi-2 transcript in an in vitro degradation assay at the protein binding region. We previously showed (19) that the increase in renal NaPi-2 mRNA levels in hypophosphatemic rats can be reproduced by an in vitro degradation assay. In this assay labeled transcripts for the NaPi-2 mRNA are incubated with cytosolic renal proteins from control or -Pi rats. The amounts of transcripts remaining with time are determined, and this has been shown to represent the degradatory processes that occur in vivo. We showed that the full-length 2,464-nt NaPi-2 transcript and the terminal 698 nt were stabilized by -Pi proteins, correlating with mRNA levels in vivo. However, a transcript of the 5' 1,043 nt that does not include the 698-nt binding region was not stabilized in this assay (19). We have now performed the in vitro degradation assay with the shorter transcripts that we have now shown are relevant for binding. We analyzed the transcripts of 461, 231, and 95 nt (Fig. 1B) that showed increased binding to the -Pi renal proteins compared with control proteins. These three transcripts were stabilized in five different in vitro degradation assay experiments (Fig. 3A). The half-time (t1/2) for the degradation of the 95-nt transcript was ~2 h with renal proteins from control rats and ~5 h with proteins from -Pi rats. The t1/2 for both the 231- and the 461-nt transcripts was <0.5 h with control proteins and >2 h with -Pi proteins. Despite the differences in degradation time between the shorter transcript of 95 nt (2 h) and the longer transcripts (0.5 h) in this assay, all of the transcripts were stabilized by the -Pi proteins compared with proteins from control rats. In contrast, a nonrelevant transcript for Tfr showed the same degradation rate with both control and -Pi renal proteins (Fig. 3B) similar to the 5' 1,043-nt NaPi-2 transcript (19). These results indicate that the protein binding region of the NaPi-2 mRNA is also the target region that determines NaPi-2 mRNA stability in response to a -Pi diet. We also studied renal proteins from rats fed a low-calcium diet for 10 days, in which serum phosphate was increased, serum calcium decreased, and PTH levels markedly increased with no change in NaPi-2 mRNA levels (9). The in vitro degradation time course of the 461-nt NaPi-2 transcript with proteins from control and low-calcium rat kidneys was the same (Fig. 3D). This result demonstrates that the in vitro studies accurately reflect the in vivo changes in NaPi-2 mRNA and underlines the stabilizing effect of -Pi renal proteins in this assay.


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Fig. 3.   Renal cytosolic proteins from -Pi rats increase NaPi-2 mRNA stability and not that of a control RNA, the transferrin receptor (Tfr), in an in vitro degradation assay. NaPi-2 RNA of 95 nt (A), 231 nt (B), and 461 nt and Tfr RNA (C) were incubated with cytosolic proteins from rats fed control or -Pi diets. At different time periods RNA was extracted and analyzed by gel electrophoresis. The 461-nt NaPi-2 transcript and the Tfr transcript were incubated with protein extract and analyzed together (C). The NaPi-2 transcripts, and not the control Tfr transcript, were stabilized by -Pi renal proteins. The 461-nt transcript was also studied with renal cytosolic proteins from control rats and rats fed a low-calcium diet (D), in which NaPi-2 mRNA levels are unchanged (9). There was no change in the in vitro degradation rates.

Protein-binding region decreases levels of reporter gene mRNA and protein in transfected cells. The correlation between binding and stabilization of the NaPi-2 transcript by -Pi renal proteins suggests that the protein binding protects the NaPi-2 transcript in hypophosphatemia, resulting in increased NaPi-2 mRNA and protein levels in vivo. To study the instability effect of the protein-binding region, we inserted different cDNAs of the NaPi-2 transcript at the 3' end of the GFP cDNA in an expression vector driven by a CMV promoter. The wild-type and chimeric constructs were transiently transfected into HEK293 cells. These cells have the same protein-binding pattern to the NaPi-2 transcript as does protein from rat kidney tissue (not shown). At 24 h GFP mRNA levels were analyzed by Northern blots and GFP protein levels by immunofluorescence. Chimeric transcripts containing 461, 362, and 95 nt decreased GFP mRNA and protein levels (Fig. 4), with the effects of 461 > 95 > 362 nt. These transcripts all contained the NaPi-2 mRNA protein-binding region (Fig. 1). Figure 4B shows a representative gel for GFP mRNA levels performed in triplicate for each chimeric construct. Quantification of four separate experiments showed that the GFP mRNA levels were decreased with the NaPi-2 461-, 95-, and 362-nt inserts by 70-90%. Insertion of the 144 nt that did not bind the -Pi renal proteins did not affect GFP mRNA. Correction after cotransfection with a beta -gal expression plasmid confirmed that the results were not due to differences in transfection efficiency (not shown). The chimeric GFP cDNAs all used the same CMV promoter, suggesting that the differences in expression are not a transcriptional effect. Furthermore, when actinomycin D was added to cells transfected with the GFP plasmids containing such insertions, there was no difference in GFP mRNA decay (not shown). Therefore, the effect of the protein-binding element of the NaPi-2 mRNA to decrease GFP levels is posttranscriptional at the level of mRNA stability. The amount of GFP protein in the transfected cells correlated with GFP mRNA levels (Fig. 4C). However, the amount of GFP protein expression as shown by immunofluorescence provides a qualitative rather than a quantitative representation of the amount of GFP protein. The measurement of GFP mRNA in the Northern blot is the direct indication of the effect of the NaPi-2 inserts on transcript levels and stability. The transfection experiments together with the in vitro degradation and binding experiments suggest that the junctional region between the 3' end of the coding region and the 5' end of the 3'-UTR is important for binding and regulation of NaPi-2 RNA stability.


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Fig. 4.   The NaPi-2 RNA protein-binding region decreases green fluorescent protein (GFP) mRNA and protein levels in HEK293 cells. HEK293 cells were transiently transfected with wild-type GFP or chimeric GFP-NaPi-2 cDNA constructs containing NaPi-2 mRNA inserts of 461 nt and different transcripts spanning the 461 nt (A). Twenty-four hours after transfection the levels of GFP mRNA were determined by Northern blot (B) and the expression of GFP protein by immunofluorescence (C). Transcripts of 461, 95, and 362 nt decreased GFP mRNA and protein levels, but not the transcript of 144 nt, which also did not bind renal proteins (Fig. 1).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Renal phosphate homeostasis involves the coordinate regulation of Pi reabsorption by both intrinsic renal sensing as well as the response to hormonal signals such as PTH (14). The regulated Pi reabsorption is through the NaPi-2 cotransporter whose gene is Npt2. The regulation of the NaPi-2-mediated Na+Pi cotransport by dietary Pi occurs primarily at the posttranscriptional level (21, 26), despite the identification of several cis-acting elements in the promoter region (8, 27, 28). We previously showed (19) that -Pi cytoplasmic renal proteins stabilized the NaPi-2 transcript in an in vitro degradation assay, which was dependent on an intact NaPi-2 3'-end. We also showed that there was increased translation of NaPi-2 in vitro in reticulocyte lysate assays and in vivo in pulse chase experiments with [35S]methionine-injected rats. This increased translation correlated with increased binding of -Pi renal proteins to the 5'-UTR by UV cross-linking gels. Therefore, the -Pi diet regulates NaPi-2 gene expression by affecting protein-RNA interactions. In addition, Levi et al. (14) showed that at short time intervals (2 h) when rats were transferred from a high to a -Pi diet there was an increase in NaPi-2 protein with no change in mRNA levels, indicating an effect at the level of translation or protein stability. In the chronic -Pi model there is an increase in mRNA levels that our results suggest is posttranscriptional (19). We have now defined the region in the NaPi-2 mRNA at the junction of the coding region and the 3'-UTR that binds -Pi renal proteins by REMSA. UV cross-linking gels do not detect the binding to this region (19). REMSA is a more sensitive assay for RNA-protein binding and more closely represents the interactions in vivo because it utilizes nondenaturing conditions. The binding of renal proteins to the defined NaPi-2 region was increased by -Pi renal proteins. A transcript for this region was stabilized by -Pi renal proteins in an in vitro degradation assay, similar to the full-length NaPi-2 transcript. The stabilizing effect of -Pi renal proteins correlates with NaPi-2 mRNA levels in vivo. Therefore, this defined region in the NaPi-2 transcript is a cis-acting instability element. Under normal conditions there is limited binding of cytosolic proteins to this cis-acting element, and this determines the steady-state levels of NaPi-2 mRNA and NaPi-2 protein. In hypophosphatemia there is increased binding to this element, which protects the RNA from degradation resulting in an increase in NaPi-2 mRNA levels.

To demonstrate the destabilizing properties of the defined cis-acting element, we inserted the cDNA coding for the element into the 3'-end of a GFP reporter gene and studied its effect on GFP mRNA and protein levels by transient transfections in HEK293 cells. These cells have the same protein binding pattern to the NaPi-2 transcript as does protein from rat kidney tissue (not shown), indicating that these RNA binding proteins are preserved. Therefore, the HEK293 cells were used to study the correlations between binding and degradation. The expression of the chimeric GFP-NaPi-2 cis-element was reduced compared with the wild-type GFP, confirming that the cis-acting element is a destabilizing element.

The NaPi-2 cis-acting element is not homologous to any other reported cis-acting element. Defined elements in many mRNAs have been characterized that function in RNA stability, translation, and localization (3). The paradigm for such elements is the IRE, which is present in a number of mRNAs that code for proteins that are regulated by iron (12, 29). PTH gene expression is also regulated at the posttranscriptional level by Pi, but in the opposite direction from NaPi-2 (10). In the parathyroid low Pi results in posttranscriptional decrease in PTH mRNA levels, which is dependent on a 26-nt cis-acting element in the PTH mRNA 3'-UTR (11). A -Pi diet leads to less binding of parathyroid cytosolic proteins to this element and a more rapid degradation of the PTH mRNA. A low-calcium diet leads to increased binding of parathyroid cytosolic proteins to this element and a more stable PTH transcript. One of the cytosolic proteins that bind the defined cis element in the parathyroid is AU-rich binding protein (AUF1), which has been shown to stabilize the PTH transcript in the in vitro degradation assay (25). The cytosolic proteins that bind to the NaPi-2 transcript are as yet unknown. The definition and functional characterization of the NaPi-2 cis-acting element may help in the isolation of renal cytosolic proteins that respond to -Pi diet and bind to and stabilize the NaPi-2 mRNA.


    ACKNOWLEDGEMENTS

This work was supported in part by grants from the Israel Academy of Sciences, the US-Israel Binational Foundation (BSF), the Hadassah Research Fund for Women's Health (to T. Naveh-Many), and the Minerva Foundation.


    FOOTNOTES

Address for reprint requests and other correspondence: T. Naveh-Many, Minerva Center for Calcium and Bone Metabolism, Nephrology Services, Hadassah Hebrew Univ. Hospital, PO Box 12000, Jerusalem, Israel 91120 (E-mail: tally{at}huji.ac.il).

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.

First published December 10, 2002;10.1152/ajprenal.00332.2002

Received 16 September 2002; accepted in final form 3 December 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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13.   Levi, M, Arar M, Kaissling B, Murer H, and Biber J. Role of microtubules in the rapid regulation of renal phosphate transport in response to acute alterations in dietary phosphate content. J Clin Invest 99: 1302-1312, 1997[Abstract/Free Full Text].

14.   Levi, M, Lotscher M, Sorribas V, Custer M, Arar M, Kaissling B, Murer H, and Biber J. Cellular mechanisms of acute and chronic adaptation of rat renal Pi transporter to alterations in dietary Pi. Am J Physiol Renal Fluid Electrolyte Physiol 267: F900-F908, 1994[Abstract/Free Full Text].

15.   Levi, M, Shayman JA, Abe A, Gross SK, McCluer RH, Biber J, Murer H, Lotscher M, and Cronin RE. Dexamethasone modulates rat renal brush border membrane phosphate transporter mRNA and protein abundance and glycosphingolipid composition. J Clin Invest 96: 207-216, 1995[ISI][Medline].

16.   Magagnin, S, Werner A, Markovich D, Sorribas V, Stange G, Biber J, and Murer H. Expression cloning of human and rat renal cortex Na/Pi cotransport. Proc Natl Acad Sci USA 90: 5979-5983, 1993[Abstract].

17.   Markovich, D, Verri T, Sorribas V, Forgo J, Biber J, and Murer H. Regulation of opossum kidney (OK) cell Na/Pi cotransport by Pi deprivation involves mRNA stability. Pflügers Arch 430: 459-463, 1995[ISI][Medline].

18.   Moallem, E, Silver J, Kilav R, and Naveh-Many T. RNA protein binding and post-transcriptional regulation of PTH gene expression by calcium and phosphate. J Biol Chem 273: 5253-5259, 1998[Abstract/Free Full Text].

19.   Moz, Y, Silver J, and Naveh-Many T. Protein-RNA interactions determine the stability of the renal NaPi-2 cotransporter mRNA and its translation in hypophosphatemic rats. J Biol Chem 274: 25266-25272, 1999[Abstract/Free Full Text].

20.   Murer, H. Homer Smith Award. Cellular mechanisms in proximal tubular Pi reabsorption: some answers and more questions. J Am Soc Nephrol 2: 1649-1665, 1992[Abstract].

21.   Murer, H, Forster I, Hernando N, Lambert G, Traebert M, and Biber J. Posttranscriptional regulation of the proximal tubule NaPi-II transporter in response to PTH and dietary Pi. Am J Physiol Renal Physiol 277: F676-F684, 1999[Abstract/Free Full Text].

22.   Murer, H, Hernando N, Forster L, and Biber J. Molecular mechanisms in proximal tubular and small intestinal phosphate reabsorption. Mol Membr Biol 18: 3-11, 2001[ISI][Medline].

23.   Pfister, MF, Lederer E, Forgo J, Ziegler U, Lotscher M, Quabius ES, Biber J, and Murer H. Parathyroid hormone-dependent degradation of type II Na+/Pi cotransporters. J Biol Chem 272: 20125-20130, 1997[Abstract/Free Full Text].

24.   Pfister, MF, Ruf I, Stange G, Ziegler U, Lederer E, Biber J, and Murer H. Parathyroid hormone leads to the lysosomal degradation of the renal type II Na/Pi cotransporter. Proc Natl Acad Sci USA 95: 1909-1914, 1998[Abstract/Free Full Text].

25.   Sela-Brown, A, Silver J, Brewer G, and Naveh-Many T. Identification of AUF1 as a parathyroid hormone mRNA 3'-untranslated region binding protein that determines parathyroid hormone mRNA stability. J Biol Chem 275: 7424-7429, 2000[Abstract/Free Full Text].

26.   Soumounou, Y, Gauthier C, and Tenenhouse HS. Murine and human type I Na-phosphate cotransporter genes: structure and promoter activity. Am J Physiol Renal Physiol 281: F1082-F1091, 2001[Abstract/Free Full Text].

27.   Taketani, Y, Miyamoto K, Chikamori M, Tanaka K, Yamamoto H, Tatsumi S, Morita K, and Takeda E. Characterization of the 5' flanking region of the human NPT-1 Na+/phosphate cotransporter gene. Biochim Biophys Acta 1396: 267-272, 1998[ISI][Medline].

28.   Taketani, Y, Segawa H, Chikamori M, Morita K, Tanaka K, Kido S, Yamamoto H, Iemori Y, Tatsumi S, Tsugawa N, Okano T, Kobayashi T, Miyamoto K, and Takeda E. Regulation of type II renal Na+-dependent inorganic phosphate transporters by 1,25-dihydroxyvitamin D3. Identification of a vitamin D-responsive element in the human NAPi-3 gene. J Biol Chem 273: 14575-14581, 1998[Abstract/Free Full Text].

29.   Theil, EC, and Eisenstein RS. Combinatorial mRNA regulation: iron regulatory proteins and iso-iron-responsive elements (Iso-IREs). J Biol Chem 275: 40659-40662, 2000[Free Full Text].

30.   Werner, A, Kempson SA, Biber J, and Murer H. Increase of Na/Pi-cotransport encoding mRNA in response to low Pi diet in rat kidney cortex. J Biol Chem 269: 6637-6639, 1994[Abstract/Free Full Text].

31.   Werner, A, and Kinne RK. Evolution of the Na-Pi cotransport systems. Am J Physiol Regul Integr Comp Physiol 280: R301-R312, 2001[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 284(4):F663-F670
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