Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870
Submitted 11 December 2002 ; accepted in final form 4 April 2003
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ABSTRACT |
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renal ammoniagenesis; metabolic acidosis; posttranscriptional regulation
A protein in rat renal cortical cytosolic extracts was observed to bind
with high affinity and specificity to the 3'-untranslated region
(3'-UTR) of GA mRNA
(23). This protein was
subsequently identified as -crystallin/NADPH:quinone reductase
(
-crystallin) (29). The
protein-binding element within GA mRNA was mapped to a 29-base sequence that
contained a direct repeat of two 8-base AU-rich elements (AREs)
(23). The function of this
sequence was established by characterizing the effect of medium pH on the
half-lives (t1/2) of various chimeric
-globin
(
G) mRNAs that were stably expressed in LLC-PK1-F+
cells (21). The inclusion of a
76-nt GA mRNA segment containing the direct repeat of the two AREs was
sufficient to produce a pH-responsive stabilization of a nonresponsive
G-PEPCK mRNA. Furthermore, when the two AREs within a
G-GA mRNA
were mutated, the pH-responsive stabilization was abolished. The cumulative
data indicate that the two AREs within GA mRNA function as a pH-response
element (pHRE) and that enhanced binding of
-crystallin to this sequence
during acidosis may mediate the stabilization of GA mRNA
(7,
22).
Previous experiments suggest that the adaptive increase in rat renal GDH mRNA is also mediated through stabilization of its mRNA. For example, the increase in GDH mRNA following the acute onset of metabolic acidosis occurs in the same tubular segments (8, 33, 34) and with similar kinetics as observed for GA mRNA (20). For both mRNAs, there is an 8- to 10-h lag between the onset of acidosis and the initial increase in mRNA levels. In addition, when LLC-PK1-F+ cells were transferred to acidic medium (pH 6.9) and treated with actinomycin D to inhibit transcription, an apparent threefold stabilization of endogenous GDH mRNA was observed (20). These data suggest that GDH mRNA may be stabilized by the same mechanism that is used to stabilize GA mRNA. The 3'-UTR of rat GDH mRNA (10) contains four eight-base AREs that are 88% identical to one of the two sequences found in GA mRNA. However, unlike the direct repeat found in GA mRNA, the putative GDH pHREs are distributed throughout the 3'-UTR and, based on previous data, it was uncertain whether an individual ARE could function as a pHRE.
In the present study, direct binding and competition assays were performed
to determine whether purified -crystallin binds to the 3'-UTR of
GDH mRNA and to the individual AREs from GA and GDH mRNAs. The 3'-UTR of
GDH mRNA and a representative GDH ARE were then cloned into
G reporter
constructs and stably expressed in LLC-PK1-F+ cells. To
determine whether the sequences also function as pHREs,
t1/2 analyses were performed. The cumulative data strongly
support the conclusion that individual AREs within the two mRNAs can bind
-crystallin with sufficient affinity to function as a pHRE.
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MATERIALS AND METHODS |
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Purification of -crystallin.
-Crystallin was
purified from a rat kidney cortical cystosolic extract by affinity
chromatography using a biotinylated RNA ligand
(29). The column was
equilibrated, washed, and eluted as described previously except that the bound
proteins were eluted with an additional 2.5 ml of binding buffer containing
500 mM potassium acetate and 50 mM MgCl2. Aliquots of the eluted
fractions were separated on a 10% polyacrylamide gel containing 1% SDS and
stained with 0.1% silver nitrate. Fractions containing the 36-kDa
-crystallin protein were pooled and dialyzed overnight vs. binding
buffer using a Slide-A-Lyzer cassette. The solution was concentrated 5- to
10-fold using a Microcon-30 column from Millipore.
Synthesis of RNAs. Rat liver GDH cDNA (10) was digested with XmnI and EcoRI to obtain a 1,178-nt fragment that contains the entire 3'-UTR. This fragment was then cloned into pBluescriptII-SK() (pBSSK) that had been previously digested with Asp718, blunted with Klenow polymerase, and then cut with EcoRI to yield the pGDH14 plasmid (prepared by R. Gallien). The synthesis of the plasmid that encodes the R2-I RNA segment of GA mRNA was described previously (23). Transcription vectors containing only a single GA ARE and the individual GDH AREs were constructed by annealing complimentary oligonucleotides that were synthesized by Macromolecular Resources (Ft. Collins, CO). The sequences of the coding strands of the oligonucleotides are the following:
The italicized letters designate the AREs. The resulting double-stranded DNAs encode the ARE sequences and form Asp718 and XbaI overhangs. The annealed oligonucleotides were inserted into pBSSK that had been restricted with Asp718 and XbaI.
A DNA template containing a T7 promoter was obtained by digesting the pGDH14 plasmid with BssHII, which flanks the multicloning site of pBSSK. Generating the other templates required additional digestion with XbaI and SacI. XbaI cleaves DNA immediately after the ARE sequence, and SacI cleaves the similarly sized nontemplate DNA into two pieces so that the template and promoter-less DNA fragments can be separated on an 8% polyacrylamide gel. The templates were extracted from the gel using the crush and soak method (26). The eluted DNA was precipitated with 2 vol of 100% ethanol, washed with 200 µl 70% ethanol, and resuspended in 11 µl diethylpyrocarbonate-treated water. In vitro transcription reactions using T7 RNA polymerase to synthesize 32P-labeled and unlabeled RNAs were performed as described previously (23). The concentrations of the 32P-labeled and unlabeled RNAs were determined by scintillation counting and by measuring the absorbance at 260 nm and using specific extinction coefficients calculated from the nucleotide composition, respectively.
RNA electrophoretic mobility shift assay. This assay was performed
as reported previously (23).
Briefly, 1050 ng of purified -crystallin were incubated for 10
min at room temperature in a 10-µl reaction containing 10 mM HEPES, pH 7.4,
25 mM potassium acetate, 2.5 mM magnesium acetate, 2 µg yeast tRNA, 0.5%
Nonidet P-40, 5% glycerol, 1 mM dithiothreitol, and 10 U of RNasin.
Approximately 20 fmol of labeled RNA were then added, and the sample was
incubated at room temperature for 20 min. For the competition studies, a 30-,
100-, or 300-fold excess of an unlabeled RNA was added along with the labeled
RNA. To compare the binding of the GDH14 and GA(R2-I) RNAs, the samples
were also incubated for 10 min with 15 U of RNase T1 to reduce the length of
the GDH14 RNA. The samples were subjected to electrophoresis for
2
h at 170 V on a 5% polyacrylamide gel using a 90 mM Tris, 110 mM boric acid, 2
mM EDTA running buffer. Gels were then dried and exposed to a PhosphorImager
screen.
G expression vectors. Various
G constructs were
synthesized to contain either the 3'-UTR of the GDH cDNA or an
individual ARE. A 930-nt segment containing the 3'-UTR of GDH cDNA was
PCR-amplified from pGDH14 using primers that add SpeI and
XbaI sites to the 5'- and 3'-ends, respectively. The PCR
product was cloned into the SrfI site of pCR-Script-SK(+), and the
SpeI/XbaI fragment was excised from the plasmid and inserted
into the XbaI site within the multicloning site of p
G
(17). The p
G-GA(R2-I)
and p
G-GDH4 vectors were constructed by annealing complimentary
oligonucleotides (Macromolecular Resources, Ft. Collins, CO) that encode the
GA(R2-I) or the GDH4 sequence and form SpeI and XbaI
overhangs and inserting them into the XbaI site of p
G. The
coding sequences of the GA(R2-I) and GDH4 oligonucleotides are
5'-CTAGTTCTTTAAATATTAAAATAATTCTAAT-3' and
5'-CTAGTAGACATTATTTATATAAGAATGAGT-3', respectively. The
italicized letters in the oligonucleotide sequences designate the AREs. The
p
G-GDH4-PEPCK vector was constructed by K. Propst. Complimentary
oligonucleotides (Macromolecular Resources) that encode the GDH4 sequence and
form XbaI and NheI overhangs were annealed and inserted into
the XbaI and NheI sites of pGEM4Z-PEPCK
(24). The sequence of the
coding strand of the GDH4 oligonucleotide is
5'-CTAGATATCAGACATTATTTATATAAGAATGAGG-3',
where the bold italicized letters designate an EcoRV site that was
used to detect the presence of the inserted sequence, and the italicized
lightface letters designate the ARE. The pGEM4Z-GDH4-PEPCK vector was digested
with XbaI and SpeI to obtain the GDH4-PEPCK sequence that
was inserted into XbaI/SpeI-digested p
G to produce
p
G-GDH4-PEPCK.
Cell culture. LLC-PK1-F+ cells were obtained from Gerhard Gstraunthaler and cultured as described previously (16). Cells were grown in a 50:50 mixture of DMEM and Ham's F-12 containing 5 mM glucose and 10% fetal bovine serum at 37°C in a 5% CO2-95% air atmosphere. Normal medium (pH 7.4) contains 25 mM sodium bicarbonate, whereas acidic medium (pH 6.9) contains 10 mM sodium bicarbonate supplemented with 15 mM sodium chloride to maintain an equivalent osmolarity and sodium ion concentration. LLC-PK1-F+ cell lines that stably express the chimeric mRNAs were produced by transfection of 3-day postsplit cells with calcium phosphate-precipitated DNA (3) and selection with medium containing 0.8 mg/ml G418. The medium was changed every 2 days. After 1421 days, three 10-cm plates containing multiple colonies were combined. Following the next split, the cells were grown in normal medium containing 0.2 mg/ml G418.
Analysis of t1/2. The various transfected
LLC-PK1-F+ cell lines were generally split 1:10 and
grown for 710 days in pH 7.4 medium containing 0.2 mg/ml G418. They
were then maintained in pH 7.4 medium without G418 for 24 h and subsequently
treated for 12 h with normal or acidic medium. At time 0, 65 µM
56 dichloro-1--ribofuranosylbenzimidazole (DRB), a specific
inhibitor of RNA polymerase II transcription
(11), dissolved in 95% ethanol
was added to each plate. An equivalent concentration of 95% ethanol was added
to control plates. The final concentration of ethanol never exceeded 0.5%. At
0, 3, 6, and 9 h post-DRB treatment, total cellular RNA was isolated using the
method of Chomczynski and Sacchi
(6). The RNA concentration was
determined by measuring the absorbance of the RNA at 260 nm.
Northern blot analysis. A 507-bp fragment of rabbit G cDNA
was excised by restricting pRSV-
G
(15) with HindIII and
BglII. A 2.0-kb fragment of the 18S ribosomal RNA cDNA from
Acanthamoeba castellanii was excised by restricting pAr2 with
HindIII and EcoRI
(9). The fragments were
separated on 1% agarose gels, excised, and purified using a GENECLEAN kit. A
synthesis of oligolabeled cDNA probes and Northern blot analysis was performed
as described previously (17).
The blots were exposed to a PhosphorImager screen and quantified using
Molecular Dynamics software. The level of the chimeric
G mRNA was
divided by the level of the corresponding 18S rRNA to correct for errors in
sample loading. The log of normalized data was then plotted vs. the time of
treatment with DRB.
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RESULTS |
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To determine whether -crystallin can bind to individual eight-base
ARE sequences, it was necessary to synthesize short RNAs of similar lengths
that contain a single ARE. As a control, RNAs containing the individual AREs
from GA mRNA were synthesized. GA(R2-IA) RNA contained the UUUAAAUA element
(bases 25962603), and GA(R2-IB) RNA contained the UUAAAAUA element
(bases 26042611) in the context of the surrounding sequence of the
3'-UTR of GA mRNA (28).
Purified
-crystallin binds to the two RNAs to form complexes that have
mobilities identical to those observed with GA(R2-I) RNA
(Fig. 2). Thus it is unlikely
that multiple copies of
-crystallin bind to GA(R2-I) RNA even though it
contains two potential binding elements.
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Experiments were also performed to determine whether -crystallin
binds to the individual eight-base AREs from the 3'-UTR of GDH mRNA. The
sequences and locations of the AREs within the 3'-UTR of GDH are
UUUAAGUA (GDH1; bases 19731980); CUAAAAUA (GDH2; bases
23132320); UUCAAAUA (GDH3; bases 25372544); and UUUAUAUA (GDH4;
bases 27492756) (10).
Purified
-crystallin binds to each of the RNAs containing a single ARE
from the GDH 3'-UTR (Fig.
2) but to a lesser extent than observed for GA(R2-I) RNA or the
individual GA AREs. Of the four GDH AREs, GDH2 and GDH4 RNAs demonstrate the
highest apparent affinity for
-crystallin. Thus the individual GDH AREs
can function as binding elements for
-crystallin. An additional, more
slowly migrating band was reproducibly observed only with GDH4 RNA. Then
composition or the significance of this band is unkown.
Competition studies were performed to assess the relative affinity of
-crystallin binding to individual GA and GDH AREs. Purified
-crystallin was bound to 32P-labeled GA(R2-I) RNA, and
increasing amounts of unlabeled RNAs were added as competitors. Competition
studies demonstrated that a 100-fold excess of unlabeled GA(R2-IA) RNA or
GA(R2-IB) RNA was required to produce a level of competition similar to that
observed with a 30-fold excess of GA(R2-I) RNA
(Fig. 3). Thus the two
individual elements exhibit similar affinities for
-crystallin.
Furthermore, the relative affinity for the two individual elements is
approximately one-third of that for the RNA that contains the direct repeat of
the individual binding sites.
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RNAs containing individual GDH AREs were also tested as competitors of labeled GA(R2-I) RNA (Fig. 4). GDH2 and GDH4 RNAs were more effective competitors than GDH3 and GDH1 RNAs. A 300-fold excess of GDH2 or GDH4 RNA competes slightly less effectively than a 100-fold excess of GA(R2-I) RNA. In contrast, GDH1 and GDH3 RNAs were weaker competitors. Thus the competition pattern observed with individual GDH elements confirms the results of the direct binding studies. The lower bands observed when GA(R2-IA) and GA(R2-IB) RNAs (Fig. 2) and GDH2 RNA (Fig. 3) were added as competitors represent undissociated dimers of 32P-labeled GA(R2-I) RNA.
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Functional studies were performed to determine whether the 3'-UTR of
GDH mRNA is sufficient to produce a pH-responsive stabilization of a reporter
mRNA. This was accomplished by measuring the half-lives of various chimeric
G mRNAs expressed in LLC-PK1-F+ cells treated with
either normal (pH 7.4, 25 mM
) or
acidic (pH 6.9, 10 mM
) medium. The
p
G plasmid contains a promoter derived from the Rous sarcoma virus long
terminal repeat, a transcription start site, the coding region from rabbit
G genomic DNA containing three exons and two introns, a multicloning
site, and a 3'-UTR and polyadenylation site from bovine growth hormone
(bGH) cDNA (17).
As a control, pG was stably expressed in the split of
LLC-PK1-F+ cells used in the present experiments.
Neither the level nor the t1/2 (>30 h) of
G mRNA
was affected by growing the cells in either normal or acidic medium (data not
shown). As a second control, the LLC-PK1-F+ cells were
also stably transfected with the p
G-GA plasmid that contains 955-bases
from the 3'-UTR of GA mRNA
(17). The p
G-GA vector
produced an unstable mRNA that had a t1/2 of
12 h in
cells maintained in normal medium. However,
G-GA mRNA exhibited a
pH-responsive stabilization (t1/2 >30 h) when the cells
were transferred to acidic medium (data not shown). These observations confirm
the previous conclusion that the 3'-UTR of GA mRNA contains an
instability element and imparts a pH-responsive stabilization to
G mRNA
(17).
Functional studies were performed using the pG-GDH vector that
encodes the 930-base 3'-UTR of GDH mRNA including all four of the
eight-base AREs (Fig. 5). The
resulting
G-GDH mRNA had a t1/2 of
13 h in
LLC-PK1-F+ cells that were grown in normal medium. When
the cells were transferred to acidic medium, the t1/2 of
G-GDH mRNA was increased to >30 h. Thus this mRNA also exhibits a
pH-responsive stabilization. This observation indicates that the 3'-UTR
of GDH mRNA contains one or more pHREs that impart selective stabilization to
GDH mRNA in response to treatment with an acidic medium.
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To test the function of shorter segments, the 29-base R2-I fragment of GA
cDNA encoding the direct repeat of the two eight-base AREs of GA mRNA was
cloned into pG. This construct lacks the 3'-UTR of PEPCK mRNA that
was included in previous studies
(21). Therefore, it was used
to determine whether the GA sequence can function as both a destabilizing
element and a pHRE. When LLC-PK1-F+ cells were stably
transfected with p
G-GA(R2-I) and grown in normal medium,
G-GA(R2-I) mRNA decayed with a t1/2 of 12 h
(Fig. 6). When the same cells
were transferred to acidic medium, the chimeric RNA was degraded with a
t1/2 of >30 h. This indicates that the sequence
contained within the GA(R2-I) region of GA 3'-UTR is sufficient to
function as an instability element and a pHRE.
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The direct binding and competition analyses indicated that the fourth ARE
was one of two sites within GDH mRNA that binds -crystallin with the
greatest affinity. Therefore, the GDH4 sequence was cloned into p
G to
test whether a single GDH element was sufficient to function as a pHRE. The
t1/2 of
G-GDH4 mRNA in
LLC-PK1-F+ cells maintained in either normal or acidic
medium was >30 h (data not shown). Thus the 35-base GDH4 sequence does not
significantly destabilize
G mRNA. Given the inherent stability of this
construct, it could not be used to determine whether the GDH4 sequence can
function as a pH-responsive stabilizing element.
To assess whether the GDH4 sequence can function as a pHRE, the
3'-UTR of PEPCK cDNA was cloned downstream of the GDH4 element to
produce the pG-GDH4-PEPCK plasmid. Insertion of the PEPCK sequence
destabilizes the resulting
G-GDH4-PEPCK mRNA
(Fig. 7). The
t1/2 of this mRNA in LLC-PK1-F+
cells grown in pH 7.4 medium was 16 h. However, when the cells were treated
with pH 6.9 medium, the t1/2 was increased to >30 h.
Therefore, the GDH4 sequence can function as a pHRE but not as a destabilizing
element.
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DISCUSSION |
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The 5'- and 3'-ends of most eukaryotic mRNAs are protected from
exonuclease degradation by the binding of eIF4E and poly(A) binding proteins
(PABP) to the 7-methyl-guanosine cap and the poly(A) tail, respectively. The
two ends are then linked together by eIF4G that acts as a bridging protein by
binding to eIF4E and PABP (13,
14). Deadenylation is the
initial step in the degradation of most mRNAs
(4,
31). In yeast, deadenylation
is usually followed by decapping and 5' 3' exonucleolytic
degradation. However, in mammals the prominent pathway involves initial
deadenylation followed by 3'
5' degradation. Various AREs,
including the canonical AUUUA sequence
(4), function as instability
elements by binding proteins, such as TTP
(2), that recruit the 3'
5' poly(A)-specific deadenylase DAN/PARN
(13) and a complex of 3'
5' exoribonucleases that has been termed the exosome
(30). The deadenylation and
subsequent degradation of an mRNA can be averted by the alternative
recruitment of a stabilizing ARE-binding protein such as HuR
(5).
On the basis of previous experiments, it was hypothesized that the pHRE of
GA mRNA also functions as the recognition site for a sequence-specific
endonuclease (7). During normal
acid-base balance, the weak interaction of -crystallin with the pHRE may
allow for recruitment of the endonuclease that initiates the rapid degradation
of GA mRNA. The onset of acidosis leads to an enhanced interaction of
-crystallin with the pHRE
(22) that may be mediated by a
kinase that is upstream of the p38 stress-activated protein kinase
(12). The enhanced binding of
-crystallin may stabilize GA mRNA by blocking the recruitment of the
sequence-specific endonuclease. However, more recent pulse-chase experiments
indicate that the degradation of the chimeric
G-GA mRNA in
LLC-PK1-F+ cells is preceded by deadenylation and occurs
without apparent endonucleoytic cleavage (Schroeder JM and Curthoys NP,
unpublished observations). Thus the pHRE may function as a site that
alternatively binds the stabilizing protein,
-crystallin, or an
ARE-binding protein that recruits a deadenylase and the exosome.
The revised hypothesis is also supported by the analysis of the turnover of
the chimeric G-GA(R2-I) mRNA that contains the direct repeat of the
eight-base AREs of GA mRNA (Fig.
6). Insertion of this segment was sufficient to destabilize
G mRNA in LLC-PK1-F+ cells maintained in normal
medium (pH 7.4) and produce a pH-responsive stabilization when the cells were
transferred to acidic medium (pH 6.9). Furthermore, the magnitude of both
effects was similar to that observed in the control experiment that utilized
G-GA mRNA. Therefore, the 29-base R2-I segment functions as both a
destabilizing element and a pHRE. This may occur through the alternate
recruitment of a destabilizing ARE-binding protein and
-crystallin.
The binding of -crystallin to the individual AREs of the GA
3'-UTR was previously studied using a crude rat renal cytosolic extract
and GA(R2-I) RNAs in which the eight-base AREs were mutated to include five
guanine and cytosine residues
(23). The RNAs containing a
single mutated ARE exhibited a large reduction in apparent binding affinity
for
-crystallin compared with wild-type GA(R2-I) RNA. When both of the
AREs were mutated, binding was abolished. In contrast, both the direct binding
studies (Fig. 2) and the
competition analysis (Fig. 3)
performed with GA(R2-IA) and GA(R2-IB) RNAs, which contain a single ARE in the
context of the surrounding sequence from the 3'-UTR of GA mRNA, indicate
that a single eight-base element binds
-crystallin with only slightly
less affinity than the complete R2-I sequence. Thus the decreased binding
affinity observed with the single-site mutations may have been caused by the
increase in the GC content of the mRNAs and not the missing ARE.
Previous studies suggested that the increase in renal GDH mRNA during
acidosis is also mediated by a cell-specific stabilization
(20). The 3'-UTR of GDH
mRNA contains four well-spaced eight-base segments that are 88% identical to
one of the AREs in the GA mRNA
(9). The reported experiments
establish that the 3'-UTR of GDH mRNA binds -crystallin and
contains both an instability element and a pHRE. Furthermore, the direct
binding studies and the competition analysis indicate that all four putative
AREs bind
-crystallin with varying affinities, all of which were less
than that observed for the individual elements from GA mRNA. The GDH2 and GDH4
sequences showed a greater binding affinity than the GDH1 and GDH3 elements.
The putative element with GDH4 RNA contains only A and U residues, whereas the
GDH2 element contains a single C residue at the 5'-end of the eight-base
sequence. In contrast, the GDH3 and GDH1 elements contain C and G
substitutions at the number 3 and 6 positions, respectively.
Therefore, substitutions that interrupt the stretch of AU nucleotides may have
a greater negative impact on the binding of
-crystallin.
The greater affinity of -crystallin for the direct repeat of the AREs
within GA mRNA compared with the individual AREs within GDH mRNA is consistent
with the observed changes in the levels of the two enzymes that occur during
acidosis. The GA activity is increased 8- to 20-fold within the renal proximal
convoluted tubule during chronic acidosis
(8,
34), whereas the GDH activity
within the same cells is increased only three-fold
(33). Therefore, the
preferential binding of a limiting amount of
-crystallin could
contribute to the greater fold-stabilization of GA mRNA.
Functional studies using pG-PEPCK mRNA indicated that the GDH4 ARE
can function as a pHRE. However, either the multiple AREs or an alternative
element within the 3'-UTR is needed to impart a significant
destabilization to GDH mRNA. Identification of the destabilizing element will
require the synthesis and characterization of additional constructs that
individually assess the function of the three additional AREs or that mutate
the individual sites within the pGDH14 plasmid.
The combined binding and functional studies establish that individual AREs
within GA and GDH mRNAs bind -crystallin with different affinities and
that a single ARE from GDH mRNA is sufficient to mediate a pH-responsive
stabilization. A recent analysis using cDNA microarrays indicated that the
onset of metabolic acidosis leads to increased expression of a large number of
genes within the renal cortex
(32). Thus it will be
interesting to determine how many of these genes encode an eight-base sequence
in their 3'-UTR that is similar to pHRE sequences of GA or GDH
mRNAs.
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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