标题: Characterization of cis-acting element in renal NaPi-2cotransporter mRNA that d [打印本页] 作者: 轻羽 时间: 2009-4-21 13:35 标题: Characterization of cis-acting element in renal NaPi-2cotransporter mRNA that d
作者：YuliaMoz, JustinSilver, TallyNaveh-Many作者单位：Minerva Center for Calcium and Bone Metabolism, NephrologyServices, Hadassah University Hospital, Jerusalem,Israel 91120 # D5 W0 x/ \6 j4 Z& _' p/ S: D
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【摘要】 ) r! ]! E# K! f4 F$ B5 D+ T) R Hypophosphatemia leads to an increasein Na -P i cotransporter (NaPi-2) mRNA levels.This increase is posttranscriptional and correlates with a more stabletranscript mediated by the terminal 698 nt of the NaPi-2 mRNA. A 71-ntbinding element was identified with renal proteins from rats fedcontrol and low-P i ( P i ) diet. The binding of P i 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. P i renal proteins stabilized transcripts that includedthe cis element compared with control renal extracts. Thefull-length NaPi-2 transcript, but not control transcripts, wasstabilized by P i extracts. Insertion of the bindingelement into green fluorescent protein (GFP) as a reporter genedecreased chimeric GFP mRNA levels in transfection experiments.Our results suggest that the protein-binding region of the NaPi-2 mRNAfunctions as a cis -acting instability element. Inhypophosphatemia there is increased binding to the cis -acting element and subsequent stabilization of NaPi-2 mRNA. 8 [/ n2 o) \6 E- z$ J
【关键词】 phosphate messenger ribonucleic acid halflife proteinribonucleic acid interactions H" [1 R1 o3 D# j" J u9 D; c
INTRODUCTION( H F9 }# L! ?, |& g, P1 E- y
# k& Q$ Z$ `/ JP I homeostasis is maintained by a well-defined membrane transportsystem ( 31 ). In mammals, renal P i reabsorptionis essential to P i homeostasis. The renal tubule has anintrinsic ability to adjust the reabsorption rate of P i according to need and availability of P i ( 22 ).The active reabsorption is mediated by the Na -dependentP i transporters (NaPi). NaPi type IIa transporters (NaPi-2)are expressed at the apical brush-border membrane (BBM) of the renalproximal tubules and are responsible for the regulated reabsorption ofP i in response to changes in dietary P i ( 16, 20 ). NaPi type IIb transporters are homologous toNaPi type IIa but are found in a variety of other tissues such asintestine, lung, prostate, and pancreas ( 4 ). Disruption ofthe NaPi-2 gene in mice resulted in increased urinary P i excretion, an 85% loss in BBM Na -P i cotransport, and significant hypophosphatemia ( 1 ). Mice homozygous for NaPi-2 gene deletion did not respond to P i depletion with an adaptive increase in Na -P i cotransport or to parathyroid hormone (PTH) with a decrease intransport. Therefore, NaPi-2 is the major regulator of renal P i homeostasis. There is a rapid adaptive increase inproximal tubule apical BBM Na -P i cotransportactivity and NaPi-2 protein abundance that is mediated bymicrotubule-dependent translocation of presynthesized NaPi-2cotransporter protein to the apical BBM ( 13 ).Dexamethasone inhibits Na -P i cotransport, andthis is associated with a decrease in renal BBM NaPi-2 cotransporterabundance 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 theeffect of dexamethasone on Na -P i cotransportactivity. PTH and a high-P i diet lead to a decrease inrenal proximal tubular Na -P i cotransport,which correlates with a decrease in the number of NaPi-2 transportersin the BBM because of their routing to the lysosome and subsequentdegradation ( 6, 7, 23, 24 ). Several interacting proteinshave been identified that may contribute either to its apicaldistribution or its subapical/lysosomal traffic ( 5, 22 ).However, chronic P i depletion not only increases thetransfer of preformed NaPi-2 to the apical BBM but also increases thelevel of the NaPi-2 mRNA and protein level ( 9, 30 ). The adaptive response to P i depletion also occurs in vitro inopossum kidney cells, where it has been shown to be aposttranscriptional effect ( 2, 17, 21 ). In vivo in rats wepreviously showed ( 9 ) by nuclear transcript run-onexperiments that the effect of chronic P i depletion ismainly posttranscriptional, although others also found atranscriptional effect ( 8 ). We studied the mechanismsinvolved in the posttranscriptional effect and found that this wasdependent on protein-RNA interactions ( 19 ). Cytosolicrenal proteins showed increased binding to the NaPi-2 mRNA5'-untranslated region (UTR), and this was associated with increasedtranslation of NaPi-2 in vitro ( 19 ). Renal proteins fromrats fed a low-P i diet ( P i ) stabilized theNaPi-2 transcript in vitro, and this was dependent on the presence ofthe terminal 698 nt at the 3' end of the mRNA. In the present studieswe have defined the region in the NaPi-2 mRNA 698 nt that mediates the binding of P i proteins. The functionality of the NaPi-2mRNA protein-binding sequence was demonstrated in an in vitrodegradation assay with renal proteins from control and low-phosphaterats. In addition, the protein binding region was inserted into a green fluorescent protein (GFP) reporter gene that was transfected into humanembryonic 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 P i renal proteinsto this region stabilizes the NaPi-2 mRNA.$ Z- O0 R6 h9 E* ^, D, o+ q( ~4 G
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EXPERIMENTAL PROCEDURES4 C v* y: A) r9 i2 P
E2 i) K/ d8 |1 ^- C- w- ^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 aserum 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 underpentobarbital sodium anesthesia and blood samples were taken formeasurements of serum calcium and phosphate in a Roche autoanalyzer.Tissues for protein extracts were used immediately as described below.5 M, ^4 x. n y% l
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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 plasmidconstruct containing the cDNA in pSPORT (a gift from J. Biber) aspreviously described ( 19 ). The 698 bp of theNaPi-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 Not I, Msc I, Sty I, Sph I, Ava II, and Bbv II, respectively,with T3 RNA polymerase. The 450 transcript was prepared byrestriction of the plasmid containing the 698-bp cDNA with Bbv II and Sph I that removed 172 bp and religationof the plasmid. The 95-nt transcript was prepared by subcloning acorresponding PCR product prepared with the forward oligonucleotide5'AGTCTTCCTGGAGGAGCTT3' and a reverse oligo 5'TCTGGACCTGCAGCCTAGA3'.The PCR fragment was ligated into pGEM-T Easy vector (Promega, Madison,WI) and linearized with Nco I for the 95-nt transcript andwith Bcn I for the 71-nt transcript. RNA was transcribed withSP6 RNA Pol. The transcript for the transferrin receptor (Tfr)contained 250 nt of the Tfr 3'-UTR that included three iron responsiveelements (IREs). ) [* Y. S g( `4 T+ M R& S4 i& u: `; X& i& dIn vitro RNA synthesis. Radiolabeled RNA probes for RNA electrophoretic mobility shift assay(REMSA), and in vitro degradation assays were prepared from linearizedtemplates with the appropriate RNA polymerase in a transcriptionreaction containing 1 µg DNA, 0.5 mM ATP, CTP, and GTP, 8 µM UTP, 2 µM UTP, 500 U/ml RNase inhibitor (Promega) and [ 32 P]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 forscintillation counting. The specific activity of the RNA probe was0.5-1.0 × 10 6 cpm/ng. For competitionexperiments RNA was transcribed similarly in the presence of the 4 ntat 1 mM. Unlabeled RNAs were quantified by spectrophotometry at 260 nm/280 nm and visualization on agarose gels.( Z, D, E5 \% u, ~8 ]
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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 describedpreviously ( 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. Totalprotein was extracted by repeated freezing and thawing of the samplesand centrifugation for 15 min at 12,000 g. Protein extractswere immediately frozen at 80°C in aliquots.3 o1 F( `& s7 f5 G
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For RNA degradation, a S-100 cytoplasmic fraction was prepared asbefore ( 19 ) by homogenizing the tissue with a Polytron in1 vol of (in mM) 10 Tris · HCl pH 7.4, 0.5 DTT, 0.5 PMSF, 10 KCl, and 1.5 MgCl 2; 0.1 vol of extraction buffer (in mM:1.5 KCl, 15 MgCl 2, 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 at100,000 g for 1 h at 4°C. Cytoplasmic extracts wereimmediately frozen at 80°C in aliquots, which were stable only upto 2 wk. Protein concentration was determined by optical densitydensitometry (595-µm wavelength) with a Bradford reagent (Bio-Rad,Hercules, CA).. P) m1 Q+ ?! u% h
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REMSA. Labeled RNA transcripts (10,000 cpm) spanning different regions of theNaPi RNA were incubated with renal protein extracts, in a final volumeof 20 µl containing 4 µg tRNA, 10 mM HEPES, 3 mM MgCl 2, 40 mM KCl, 5% glycerol, and 1 mM DTT (binding buffer) for 10 min at4°C. Addition of heparin (5 mg/ml) did not affect binding and thesize of the complex. For competition experiments unlabeled RNA wasadded as indicated. The specificity of binding was further demonstratedby the addition of unlabeled nonrelated transcripts that did not affectbinding. 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. , z+ C* r9 U+ N: I3 ]3 e+ S & p9 d& @. A9 u* w9 Q8 {% k2 TIn vitro cell free degradation assay. In vitro degradation was performed essentially as described previously( 18, 19 ). Radiolabeled RNA transcripts (0.3 × 10 6 cpm) were incubated with 20-60 µg of cytoplasmicextract and 80 U/ml RNasin to prevent nonspecific RNA degradation, in atotal volume of 40 µl at room temperature. At each time point 6 µlwas 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), andautoradiographed. The remaining undegraded transcripts at the differenttime points were quantified by densitometry.! u: m+ K7 w y" p' G
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Construction of chimeric GFP constructs containing cDNAs ofvarious regions of NaPi-2 mRNA for transfection experiments. The 461-, 362-, 315-, 144-, 95-, and 71-bp cDNAs corresponding todifferent regions of the NaPi-2 mRNA terminal 698 nt (Fig. 1 ) were excised by restriction enzymesand inserted into the multiple cloning site of pEGFP-C1 (Clontech, PaloAlto, CA), at the 3' end of GFP cDNA.- E8 X/ T7 \! d: t9 x2 ~5 Y" H
! c; n, I. h7 x! S# K1 \ U+ wFig. 1. Binding of low-P i ( P i ) diet renalproteins to Na -P i cotransporter type IIa(NaPi-2) mRNA maps to a defined region. A : RNAelectrophoretic mobility shift assay (REMSA) of renal proteins fromrats fed a P i diet and transcripts representing differentregions 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 thesubsequent lanes represent increasing amounts of renal proteins thatwere added. B : diagrammatic representation of the 698-bpfragment of the NaPi-2 cDNA that was used as the template to transcribeRNAs of different lengths. The transcript 461 nt was constructed toexclude the sequence between the 144-nt transcript and the 315-nttranscript. The region that was excluded is shown as a broken line. Thetranscripts of 95 and 71 nt were generated from constructs that wereprepared by ligation of PCR products into pGEM-T easy vector. C : binding of cytosolic proteins from rats fed a controldiet (N) or a P i diet to a 95- and 231-nt transcript. P i renal proteins showed increased binding compared withcontrol renal proteins.6 ~* b3 ?. x* p, n: ^
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Transient transfection experiments. The plasmids (1 µg DNA/well/24-well plate) were transientlytransfected into HEK293 cells by calcium phosphate precipitation. Twenty-four hours after transfection total RNA was extracted by TRIreagent and analyzed for GFP mRNA levels by Northern blot. GFP proteinwas measured by immunofluorescence microscopy. Expression ofcotransfected cytomegalovirus (CMV)- -galactosidase plasmid demonstrated transfection efficiency./ n/ r# _- E1 r& [$ i
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Characterization of binding of renal cytosolic proteins to definedregion in NaPi-2 mRNA. To study protein-RNA interaction we performed REMSA with renal proteinsfrom control rats or rats fed a low-Pi diet for 3 wk and transcriptsrepresenting different regions of the NaPi-2 mRNA. A transcript thatexcluded the terminal 698 nt of the NaPi-2 mRNA did not bind P i renal proteins by REMSA (not shown). A transcript of698 nt that contained the terminal coding region and the 3'-UTR (Fig. 1 B ) formed a shifted complex with renal cytosolic proteins from P i rats (Fig. 1 A ). To define the P i protein-binding region in the 698-nt NaPi-2transcript, we transcribed RNAs representing smaller regions in themRNA and analyzed them for binding with the P i renalproteins. Shortening of the transcript by excluding successively largerregions of the 3'-UTR to transcripts of 461, 362, 315 (Fig. 1, A and B ), and 231 nt (Figs. 1 B and 2 ) did not affect binding. All thesetranscripts bound proteins, resulting in a shift of the free probe thatwas dose dependent. The larger shifted complexes when more protein isadded may represent the binding of additional subunits of the proteinRNA complex. Further shortening of the transcript to 144 nt resulted inno binding (Fig. 1, A and B ). This suggests thatthe binding region is between 144 and 231 nt (Fig. 1 B ).Therefore, we transcribed RNA that deleted the 164 nt between the 151- and 315-nt transcripts (Fig. 1 B, 461 nt). This 461-nttranscript 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. 1 B.Transcripts of 95 (not shown) and 71 nt in this region (Fig. 1 A ) were sufficient for protein binding (Fig. 1, A and B ), defining the minimal protein bindingregion that corresponds to 1889-1960 of the NaPi-2 cDNA at thejunction of the coding region and the 3'-UTR. Renal proteins fromcontrol rats showed significantly reduced binding to the 71 (notshown)-, 95-, and 231-nt transcripts (Fig. 1 C ). Thedecreased binding may have been in part due to some degradation of thetranscript by the renal proteins from control rats. This is evident bythe partially degraded excess free probe that remains after binding with control renal proteins but not with P i proteins.With P i proteins the increased binding utilizes all theprobe (Fig. 1 C ).! @5 Y, s: D" i9 D
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Fig. 2. Competitionexperiments for REMSA binding of renal proteins to the NaPi-2 mRNA mapsa minimal binding region of 71 nt. Renal proteins from rats fed a P i diet were incubated with labeled NaPi-2 RNA 231 nt( left ) or 698 nt ( right ) and increasing amountsof unlabeled 231-, 144-, or 71-nt transcripts as indicated and analyzedby REMSA. The first lane in each panel is the free probe, and thesecond lane shows the binding in the presence of P i proteins without and with increasing amounts of unlabeled transcriptsas indicated. Excess transcripts of 231 and 71 nt competed for binding,whereas the 144-nt transcript was a far less effective competitor. % |2 n, q! e: H1 _6 z6 y+ p. E. L0 L) \4 D% o
To show specificity of the binding we performed competitionexperiments. The binding of P i proteins to the 231-nttranscript was effectively competed for by excess unlabeled 231-nttranscripts already at 20× competitor excess (Fig. 2 ). The 144-nttranscript, which did not bind renal proteins (Fig. 1 A ), wasalso much less effective as a competitor for the binding to the 231-nttranscript (Fig. 2 ). In addition, in Fig. 2 it is shown that a 71-nttranscript is sufficient to compete for binding of P i renal proteins to the 698-nt NaPi-2 transcript. The competitionexperiments demonstrate the specificity of protein binding to theNaPi-2 element. There is increased binding of P i renalproteins to the NaPi-2 transcript compared with proteins of normalrats. This binding is to a 71-nt region spanning the junction of thecoding region and the 3'-UTR of the NaPi-2 mRNA. * ]5 I/ H3 y/ O. c: `% K7 r p! O3 e5 R8 v
Renal cytosolic proteins from P i stabilize NaPi-2 transcript in an in vitro degradation assay at theprotein binding region. We previously showed ( 19 ) that the increase in renalNaPi-2 mRNA levels in hypophosphatemic rats can be reproduced by an invitro degradation assay. In this assay labeled transcripts for theNaPi-2 mRNA are incubated with cytosolic renal proteins from control or P i rats. The amounts of transcripts remaining with timeare determined, and this has been shown to represent the degradatoryprocesses that occur in vivo. We showed that the full-length 2,464-ntNaPi-2 transcript and the terminal 698 nt were stabilized by P i proteins, correlating with mRNA levels in vivo.However, a transcript of the 5' 1,043 nt that does not include the698-nt binding region was not stabilized in this assay ( 19 ). We have now performed the in vitro degradation assaywith the shorter transcripts that we have now shown are relevant for binding. We analyzed the transcripts of 461, 231, and 95 nt (Fig. 1 B ) that showed increased binding to the P i renal proteins compared with control proteins. These three transcriptswere stabilized in five different in vitro degradation assayexperiments (Fig. 3 A ). Thehalf-time ( t 1/2 ) for the degradation of the95-nt transcript was ~2 h with renal proteins from control rats and~5 h with proteins from P i rats. The t 1/2 for both the 231- and the 461-nttranscripts was 2 h with P i proteins. Despite the differences in degradation timebetween the shorter transcript of 95 nt (2 h) and the longertranscripts (0.5 h) in this assay, all of the transcripts werestabilized by the P i proteins compared with proteins fromcontrol rats. In contrast, a nonrelevant transcript for Tfr showed thesame degradation rate with both control and P i renalproteins (Fig. 3 B ) similar to the 5' 1,043-nt NaPi-2 transcript ( 19 ). These results indicate that the proteinbinding region of the NaPi-2 mRNA is also the target region thatdetermines NaPi-2 mRNA stability in response to a P i diet. We also studied renal proteins from rats fed a low-calcium dietfor 10 days, in which serum phosphate was increased, serum calciumdecreased, and PTH levels markedly increased with no change in NaPi-2mRNA levels ( 9 ). The in vitro degradation time course ofthe 461-nt NaPi-2 transcript with proteins from control and low-calciumrat kidneys was the same (Fig. 3 D ). This result demonstratesthat the in vitro studies accurately reflect the in vivo changes in NaPi-2 mRNA and underlines the stabilizing effect of P i renal proteins in this assay. 9 v( P/ Q0 R% q( S 4 r9 B7 a( u- I3 lFig. 3. Renal cytosolic proteins from P i ratsincrease NaPi-2 mRNA stability and not that of a control RNA, thetransferrin receptor (Tfr), in an in vitro degradation assay. NaPi-2RNA of 95 nt ( A ), 231 nt ( B ), and 461 nt and TfrRNA ( C ) were incubated with cytosolic proteins from rats fedcontrol or P i diets. At different time periods RNA wasextracted and analyzed by gel electrophoresis. The 461-nt NaPi-2transcript and the Tfr transcript were incubated with protein extractand analyzed together ( C ). The NaPi-2 transcripts, and notthe control Tfr transcript, were stabilized by P i renalproteins. The 461-nt transcript was also studied with renal cytosolicproteins 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 degradationrates.+ `6 y- x) `, o" G" o P1 g: d# k
, h' `, Q2 J( I, V5 wProtein-binding region decreases levels of reporter gene mRNA andprotein in transfected cells. The correlation between binding and stabilization of the NaPi-2transcript by P i 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 theinstability effect of the protein-binding region, we inserted differentcDNAs of the NaPi-2 transcript at the 3' end of the GFP cDNA in anexpression vector driven by a CMV promoter. The wild-type and chimericconstructs were transiently transfected into HEK293 cells. These cellshave the same protein-binding pattern to the NaPi-2 transcript as doesprotein from rat kidney tissue (not shown). At 24 h GFP mRNAlevels were analyzed by Northern blots and GFP protein levels byimmunofluorescence. Chimeric transcripts containing 461, 362, and 95 ntdecreased GFP mRNA and protein levels (Fig. 4 362 nt. These transcripts all contained the NaPi-2 mRNAprotein-binding region (Fig. 1 ). Figure 4 B shows arepresentative gel for GFP mRNA levels performed in triplicate for eachchimeric construct. Quantification of four separate experiments showedthat the GFP mRNA levels were decreased with the NaPi-2 461-, 95-, and362-nt inserts by 70-90%. Insertion of the 144 nt that did notbind the P i renal proteins did not affect GFP mRNA.Correction after cotransfection with a -gal expression plasmidconfirmed that the results were not due to differences in transfectionefficiency (not shown). The chimeric GFP cDNAs all used the same CMVpromoter, suggesting that the differences in expression are not atranscriptional effect. Furthermore, when actinomycin D was added tocells transfected with the GFP plasmids containing such insertions,there was no difference in GFP mRNA decay (not shown). Therefore, theeffect of the protein-binding element of the NaPi-2 mRNA to decrease GFP levels is posttranscriptional at the level of mRNA stability. Theamount of GFP protein in the transfected cells correlated with GFP mRNAlevels (Fig. 4 C ). However, the amount of GFP protein expression as shown by immunofluorescence provides a qualitative ratherthan a quantitative representation of the amount of GFP protein. Themeasurement of GFP mRNA in the Northern blot is the direct indicationof the effect of the NaPi-2 inserts on transcript levels and stability.The transfection experiments together with the in vitro degradation andbinding experiments suggest that the junctional region between the 3'end of the coding region and the 5' end of the 3'-UTR is important forbinding and regulation of NaPi-2 RNA stability.+ K4 k5 W! ]/ s8 h/ G9 T) A
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Fig. 4. The NaPi-2 RNA protein-binding region decreases greenfluorescent protein (GFP) mRNA and protein levels in HEK293 cells.HEK293 cells were transiently transfected with wild-type GFP orchimeric GFP-NaPi-2 cDNA constructs containing NaPi-2 mRNA inserts of461 nt and different transcripts spanning the 461 nt ( A ).Twenty-four hours after transfection the levels of GFP mRNA weredetermined by Northern blot ( B ) and the expression of GFPprotein by immunofluorescence ( C ). Transcripts of 461, 95, and 362 nt decreased GFP mRNA and protein levels, but not thetranscript of 144 nt, which also did not bind renal proteins (Fig. 1 ). 4 \2 }0 r8 I9 T4 i9 w- y# Q. K# p. _2 C+ o& b; Z
DISCUSSION 9 N3 ~; u7 i% H2 i$ r% u! A3 E 2 V+ h+ K) x N2 K3 XRenal phosphate homeostasis involves the coordinate regulation ofP i reabsorption by both intrinsic renal sensing as well asthe response to hormonal signals such as PTH ( 14 ). Theregulated P i reabsorption is through the NaPi-2cotransporter whose gene is Npt2. The regulation of the NaPi-2-mediatedNa P i cotransport by dietary P i 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 P i cytoplasmic renalproteins stabilized the NaPi-2 transcript in an in vitro degradationassay, which was dependent on an intact NaPi-2 3'-end. We also showedthat there was increased translation of NaPi-2 in vitro in reticulocytelysate assays and in vivo in pulse chase experiments with[ 35 S]methionine-injected rats. This increased translationcorrelated with increased binding of P i renal proteins tothe 5'-UTR by UV cross-linking gels. Therefore, the P i diet regulates NaPi-2 gene expression by affecting protein-RNAinteractions. In addition, Levi et al. ( 14 ) showed that atshort time intervals (2 h) when rats were transferred from a high to a P i diet there was an increase in NaPi-2 protein with nochange in mRNA levels, indicating an effect at the level of translationor protein stability. In the chronic P i model there is anincrease in mRNA levels that our results suggest is posttranscriptional( 19 ). We have now defined the region in the NaPi-2 mRNA atthe junction of the coding region and the 3'-UTR that binds P i renal proteins by REMSA. UV cross-linking gels do notdetect the binding to this region ( 19 ). REMSA is a moresensitive assay for RNA-protein binding and more closely represents theinteractions in vivo because it utilizes nondenaturing conditions. Thebinding of renal proteins to the defined NaPi-2 region was increased by P i renal proteins. A transcript for this region wasstabilized by P i renal proteins in an in vitrodegradation assay, similar to the full-length NaPi-2 transcript. Thestabilizing effect of P i renal proteins correlates withNaPi-2 mRNA levels in vivo. Therefore, this defined region in theNaPi-2 transcript is a cis -acting instability element. Undernormal conditions there is limited binding of cytosolic proteins tothis cis -acting element, and this determines thesteady-state levels of NaPi-2 mRNA and NaPi-2 protein. Inhypophosphatemia there is increased binding to this element, whichprotects the RNA from degradation resulting in an increase in NaPi-2mRNA levels.* a& A( {4 {! p9 B
+ p+ @; S# U9 ^To demonstrate the destabilizing properties of the defined cis -acting element, we inserted the cDNA coding for theelement 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-2transcript 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 bindingand 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.* l( l# v9 Y/ T9 G+ I6 ?, W0 _% e
4 t) j% `& l/ w: i, z3 pThe NaPi-2 cis -acting element is not homologous to any otherreported cis -acting element. Defined elements in many mRNAshave been characterized that function in RNA stability, translation, and localization ( 3 ). The paradigm for such elements isthe IRE, which is present in a number of mRNAs that code for proteins that are regulated by iron ( 12, 29 ). PTH gene expressionis also regulated at the posttranscriptional level by P i,but in the opposite direction from NaPi-2 ( 10 ). In theparathyroid low P i results in posttranscriptional decreasein PTH mRNA levels, which is dependent on a 26-nt cis -actingelement in the PTH mRNA 3'-UTR ( 11 ). A P i diet leads to less binding of parathyroid cytosolic proteins to thiselement and a more rapid degradation of the PTH mRNA. A low-calciumdiet leads to increased binding of parathyroid cytosolic proteins tothis element and a more stable PTH transcript. One of the cytosolicproteins that bind the defined cis element in theparathyroid is AU-rich binding protein (AUF1), which has been shown tostabilize the PTH transcript in the in vitro degradation assay( 25 ). The cytosolic proteins that bind to the NaPi-2transcript are as yet unknown. The definition and functionalcharacterization of the NaPi-2 cis -acting element may helpin the isolation of renal cytosolic proteins that respond to P i diet and bind to and stabilize the NaPi-2 mRNA. / u/ M* L Z) e/ a 6 v$ F, h0 o. u6 i9 M" b* `ACKNOWLEDGEMENTS & G- x: x+ I; o: i0 e* J! V* r6 u* L/ S2 @; v
This work was supported in part by grants from the Israel Academyof Sciences, the US-Israel Binational Foundation (BSF), the HadassahResearch Fund for Women's Health (to T. Naveh-Many), and the Minerva Foundation.! t% W$ D7 O3 y& h) b" W0 q1 F) c5 u
【参考文献】 ! j ]* L3 Z6 y! D4 `' m 1. Beck, L,Karaplis AC,Amizuka N,Hewson AS,Ozawa H,andTenenhouse HS. Targeted inactivation of Npt2 in mice leads to severe renal phosphate wasting, hypercalciuria, and skeletal abnormalities. Proc Natl Acad Sci USA 95:5372-5377,1998 . Z m; [; k/ N: f
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- Y0 X# {- Z, O4 D2. Biber, J,Forgo J,andMurer H. Modulation of Na -Pi cotransport in opossum kidney cells by extracellular phosphate. Am J Physiol Cell Physiol 255:C155-C161,1988 .8 B: D1 N3 j0 }) J
$ W2 o) Y% H1 }, @9 { 9 n3 W- F- u9 h6 t8 e8 s% k ) ^2 `1 L6 K4 C8 H7 J& W& x3 O3. Guhaniyogi, J,andBrewer G. Regulation of mRNA stability in mammalian cells. Gene 265:11-23,2001 .' M6 P8 ]6 S7 A s' r
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