From the Department of Biochemistry and Molecular Biology, Colorado State University, Ft. Collins, Colorado 80523-1870
Received for publication, March 2, 2001, and in revised form, April 9, 2001
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ABSTRACT |
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Increased renal ammoniagenesis and bicarbonate
synthesis from glutamine during chronic metabolic acidosis facilitate
the excretion of acids and partially restore normal acid-base balance.
This adaptation is sustained, in part, by a cell-specific stabilization of the glutaminase mRNA that leads to an increased synthesis
of the mitochondrial glutaminase. A direct repeat of an 8-base AU sequence within the 3'-nontranslated region of the glutaminase mRNA
binds a unique protein with high affinity and specificity. Expression
of various chimeric mRNAs in
LLC-PK1-FBPase+ cells demonstrated
that a single 8-base AU sequence is both necessary and sufficient to
function as a pH response element (pH RE). A biotinylated
oligoribonucleotide containing the direct repeat was used as an
affinity ligand to purify the pH RE-binding protein from a cytosolic
extract of rat renal cortex. The purified binding activity retained the
same specific binding properties as observed with crude extracts and
correlated with the elution of a 36-kDa protein. Microsequencing
by mass spectroscopy and Western blot analysis were used to identify
this protein as In contrast to other tissues, where glutamine metabolism is
largely constitutive, the renal catabolism of glutamine is acutely activated in response to the onset of metabolic acidosis (1, 2). During
normal acid-base balance, very little of the plasma glutamine is
extracted and catabolized within the kidney (3). However, during
metabolic acidosis renal ammoniagenesis and gluconeogenesis are
greatly increased, and the kidney becomes the primary site of glutamine
catabolism (4). This process generates ammonium ions and bicarbonate
ions that facilitate the excretion of acids and partially restore
normal acid-base balance (2). During chronic acidosis, the increased
glutamine catabolism in rat kidney is sustained, in part, by increased
expression of multiple genes that encode the regulatory enzymes and
transport proteins that participate in this adaptive response.
The mitochondrial glutaminase
(GA)1 catalyzes the initial
reaction in the primary pathway of renal glutamine catabolism.
Glutaminase activity is increased 7- to 20-fold within the renal
proximal convoluted tubule during chronic acidosis (5, 6). This
increase results from an increased rate of glutaminase synthesis (7) that correlates with a similar increase in the level of GA mRNA (8,
9). The two forms of GA mRNA that are expressed in rat kidney
result from the use of alternative polyadenylation sites. The levels of
both mRNAs are coordinately affected by changes in acid-base
balance. However, the rate of transcription of the GA gene is not
increased during either acute (9) or chronic acidosis (10). Instead,
the increase in glutaminase activity results from the selective
stabilization of the GA mRNA (11).
The stabilization of the GA mRNA was initially demonstrated by
stable transfection of various Experiments using additional chimeric In the current study, an affinity ligand containing the pH RE was used
to purify a 36-kDa protein from a cytosolic extract of rat renal
cortex. This protein binds to the R-2I RNA with the same specificity as
the pH RE-binding protein that is contained in a crude extract. Through
MS/MS microsequencing and Western blot analysis, the purified protein
was identified as Materials--
Male Sprague-Dawley rats (140-160 g) were
acquired from Charles River Laboratories.
[ Rat Kidney Cytosolic Extract--
Rats were made acidotic by
stomach loading with 20 mmol of NH4Cl/kg of body
mass and then providing 0.28 mM NH4Cl as
the sole source of drinking water. After 18-24 h, the rats were
anesthetized with 1 mg of pentobarbital/kg of body mass and
opened with a midline incision. The kidneys were perfused in
situ with Krebs-Henseleit solution, then removed, decapsulated,
sliced longitudinally, and placed in ice-cold Krebs-Henseleit solution.
The cortex was dissected free of papilla and medulla and then cut into
small pieces. The cortical tissue was placed in an equal volume of 40 mM Hepes buffer, pH 7.4, containing 100 mM
potassium acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, 10 µM leupeptin, 10 µM antipain, and 5 µg/ml phenylmethylsulfonyl fluoride
and disrupted with a Dounce homogenizer. The homogenate was centrifuged
for 10 min at 1,000 × g to pellet intact cells and
nuclei. The supernatant was centrifuged at 10,000 × g
for 10 min to pellet the mitochondria and then for 90 min at
100,000 × g to pellet membrane-bound organelles and
polyribosomes. The final supernatant was divided into 100-µl aliquots
and stored frozen at In Vitro Transcription of RNA--
The R-2I plasmid (14) was
digested with BssHII and XbaI, and the DNA
fragment containing the T7 promoter and the R-2I template was purified
by electrophoresis on an 8% polyacrylamide gel. The R-2I RNA was
transcribed in a 10-µl reaction mixture containing the following
components: 100 ng of template DNA; 20 µCi of
[
Syntheses of unlabeled RNAs were performed using a 100-µl reaction
volume lacking [ RNA Electrophoretic Mobility Shift Assays--
Either 3 µg of
a rat renal cortical extract or 10-50 ng of purified protein were
preincubated for 10 min at room temperature in a 10-µl reaction
mixture containing 10 mM Hepes, pH 7.4, 25 mM
potassium acetate, 2.5 mM magnesium acetate, 0.5 µg of
yeast tRNA, 0.5% Igepal CA 630, 5% glycerol, 1 mM
dithiothreitol, and 10 units of RNAsin. Where indicated, specific
antibodies were added, and the mixture was incubated for 20 min at room
temperature. Subsequently, 20 fmol of labeled R2-I RNA was added. For
competition experiments, specific or nonspecific competitors (100-fold
excess) were also added. The samples were incubated for 20 min at room temperature and then subjected to electrophoresis for 90 min in a 5%
polyacrylamide gel at 170 V using a 90 mM Tris, 110 mM boric acid, 2 mM EDTA running buffer. The
gels were dried and imaged using a PhosphorImager screen.
Synthesis of Affinity Ligand--
The following oligonucleotide
was synthesized by Macromolecular Resources (Ft. Collins, CO):
5'-TTAGUGUGACUCUUUAAAUAUUAAAAUAAUUACUACUAACUGUUCATTdATTT-3'. The 5'-end contained two deoxythymidines, and the 3'-end contained six
deoxyribonucleotides including two biotinylated thymidines (Glen
Research, Sterling, VA; indicated in bold). The
remainder of the oligonucleotide was composed of a sequence of
ribonucleotides derived from the GA mRNA. The underlined
nucleotides are the direct repeat of the 8-base AU sequence that
constitutes the pH response element of the GA mRNA (15). A
comparison of the affinity ligand and the R-2I and R-2H RNAs is shown
in Fig. 1.
Affinity Purification of the pH RE-binding Protein--
A 1-ml
sample of a rat renal cortical cytosolic extract (~20 mg of total
protein) was dialyzed overnight against 1× binding buffer containing
10 mM Hepes, pH 7.4, 25 mM potassium acetate, 2.5 mM magnesium acetate, and 1 mM
dithiothreitol. The following additions were made to the dialyzed
extract: 40 µl of 10% Igepal CA 630, 1 mg of tRNA, 20 µmol of
dithiothreitol, and 20 µl of RNAsin (40 units/µl). The sample was
then incubated at 4 °C for 10 min. The biotinylated RNA ligand was
centrifuged through a Micro Bio-spin P6 column. Approximately 2 nmol of
the purified RNA were added, and the mixture was incubated at 4 °C
for an additional 20 min. A fast protein liquid chromatography column
was packed with 0.5 ml of avidin agarose (10-fold excess with respect
to the biotinylated RNA) and washed extensively with 1× binding
buffer. The total sample was loaded at a flow rate of 0.1 ml/min, and the column was then washed at 0.05 ml/min with 110 ml of binding buffer
in which the concentrations of the potassium acetate and magnesium
acetate were gradually increased 4-fold. The binding activity was then
eluted with 5 ml of buffer in which the potassium acetate and magnesium
acetate concentrations were linearly increased from 4× to 20×. The
collected fractions (0.5 ml) were dialyzed versus 1×
binding buffer and assayed by RNA gel shift analysis. To assess purity,
samples from the collected fractions containing 10 ng of protein were
separated on a 10% polyacrylamide gel containing 1% SDS and stained
with 0.1% silver nitrate.
Western Blots--
The proteins contained in 150-µl samples of
the individual column fractions were concentrated by
chloroform/methanol precipitation (20) and washed with methanol. The
protein pellets were then resuspended in Laemmli sample buffer. The
samples were separated by SDS-PAGE using a 10% separating gel,
transferred to a nitrocellulose membrane, and incubated with either a
1:500 dilution of Preparation of Sample for Microsequencing--
The
electrophoresis unit and all glassware were incubated overnight with
2% cleansing concentrate (Bio-Rad), followed by extensive washing with
doubly deionized H2O. The purified protein was
concentrated, subjected to SDS-PAGE, and stained with freshly prepared
Coomassie Blue. The 36-kDa protein band was excised, sealed in an
Eppendorf tube, and sent to the Harvard Microchemistry Facility
(William S. Lane) for analysis. The protein was subjected to in-gel
reduction, carboxyamidomethylation, and tryptic digestion. Multiple
peptide sequences were determined by microcapillary reverse-phase
chromatography coupled to a Finnigan LCQ quadrupole ion trap
mass spectrometer. Interpretation of the resulting MS/MS spectra were
facilitated by software developed in the Harvard Microchemistry
Facility and by data base correlation with the algorithm SEQUEST
(21).
Affinity Purification of pH RE-binding Protein--
A biotinylated
RNA (Fig. 1) was designed as a ligand for
the affinity purification of the pH RE-binding protein. When used as a
probe in an RNA gel shift assay, the affinity ligand bound to the pH
RE-binding protein with an affinity equivalent to that observed with
the R-2I RNA (data not shown). Therefore, a cytosolic extract from rat
kidney cortex was incubated with a sufficient excess of the affinity
ligand to bind all of the pH RE-binding protein. The resulting complex
was then applied to a fast protein liquid chromatography column that
had been packed with avidin-agarose and equilibrated with the binding
buffer. The column was washed extensively and then eluted with binding
buffer in which the concentrations of potassium acetate and magnesium
acetate were increased proportionately. The eluant through the wash
with 4× binding buffer (100 mM potassium acetate and 10 mM magnesium acetate) contained very little R-2I RNA
binding activity. Thus, 0.5-ml fractions were routinely collected only
as the steep gradient of 4× to 20× binding buffer was applied to the
column. An aliquot of each fraction was analyzed by SDS-PAGE and silver
staining. As shown in Fig. 2A,
two proteins were recovered in these fractions. The elution of a 43-kDa
protein peaked in fraction 4, and a 36-kDa protein peaked in fraction
7. When larger volumes of the peak fractions were concentrated by
chloroform/methanol precipitation and subjected to SDS-PAGE, additional
protein bands were observed. However, the additional proteins
constituted less than 5% of the total protein (data not shown).
To measure the R-2I RNA binding activity, the fractions were dialyzed
overnight against 1× binding buffer and then analyzed in an RNA
electrophoretic mobility shift assay. As shown in Fig. 2B,
the amount of R2-I-protein complex formed closely correlates with the
elution profile for the 36-kDa protein. The ratio of binding activity
to the amount of 36-kDa protein quantified by silver staining was
nearly constant across the elution profile (Fig. 2C),
suggesting that this protein is the pH RE-binding protein. To further
support this conclusion, the affinity purification was repeated using a
shallower gradient of the 4× to 20× binding buffer to separate the
proteins into 28 fractions. Analysis by SDS-PAGE and silver staining
(data not shown) indicated that fraction 13 contained only the 43-kDa
protein, whereas fraction 20 contained only the 36-kDa protein. After
dialysis, the two fractions were tested for R2-I RNA binding activity
(Fig. 3). Again, only the fraction
containing the 36-kDa protein formed an RNA-protein complex. The
fraction containing the 43-kDa protein neither bound the R-2I RNA nor
affected the level or mobility of the complex formed with the 36-kDa
protein. Thus, the purified 43-kDa protein does not form a specific
complex with the R-2I RNA.
Specificity of the Purified pH RE-binding Protein--
Competition
experiments were performed to determine the specificity of the binding
interaction between the purified pH RE-binding protein and the R2-I RNA
(Fig. 4). For a control, the same binding experiment was performed using a cytosolic extract of rat renal cortex.
The complexes produced with both protein samples exhibited identical
electrophoretic mobilities. A 100-fold excess of various unlabeled RNAs
was added to compare specificity. The mut1, mut2, and mut3 competitors
(14) are R2-I RNAs in which the first, second, and both of the AU-rich
regions, respectively, were mutated. The (AUUU)5A RNA
contains five tandem repeats of an AUUUA sequence. This sequence binds
a variety of AUUUA-binding proteins (22). The pBS RNA was
transcribed from the multicloning site of pBlueScript SK(+) and was
included as a nonspecific competitor. Finally, the R2-H RNA is a
76-nucleotide sequence that includes all of R2-I plus flanking
sequences from the GA mRNA (Fig. 1A). Formation of the
protein-R2-I complex with either the purified protein or the crude
extract was almost completely inhibited by the R2-H RNA. In addition,
both complexes were slightly competed by the (AUUU)5A and
mut2 RNAs, but not any of the other RNAs. Thus, the purified pH
RE-binding protein exhibits a specificity identical to that observed
with the crude extract.
MS/MS Microsequencing--
The purified and concentrated pH
RE-binding protein was subjected to SDS-PAGE and stained with Coomassie
Blue. The 36-kDa protein band was cut from the gel, digested with
trypsin, and sequenced by mass spectroscopy. A total of 13 peptides
were identified. The peptides were classified into two groups. Class I
peptides include eight sequences that are identical to tryptic peptides derived from the 36-kDa mouse
The identities of the two proteins contained in the purified
preparation of the pH RE-binding protein were confirmed by Western blot
analysis (Fig. 5). Antisera produced
against the full-length
Immunoblocking assays were performed to confirm that
RNA-binding proteins play an important role in the turnover of
mRNAs (24). For example, mRNAs that encode various cytokines (25), transcription factors (26), and other immediate-early gene
products (27) generally turn over with half-lives of less than 1 h. Specific AU-rich elements within the coding sequence or the
3'-nontranslated region of the mRNAs function as instability elements (22). They recruit proteins that enhance 3'-deadenylation and
the loss of poly(A)-binding proteins, leading to the rapid exonucleolytic degradation of the mRNA. Alternatively, the turnover of an mRNA may be initiated by a site-specific endonucleolytic cleavage that generates sites for rapid exonucleolytic degradation (28,
29). Selective stabilization of the latter class of mRNAs is
mediated by sequence-specific binding of unique proteins that inhibit
the endonucleolytic cleavage.
In the current study, an affinity ligand was used to purify from rat
kidney cortex the protein that binds to the pH RE of the GA mRNA.
The purified protein formed a complex with the R-2I RNA that retained
the same electrophoretic mobility and specificity as observed with the
crude cytosolic extract. In both cases, the observed binding was
strongly competed by an oligonucleotide (R-2H) that contained both AU
elements. When characterized as independent binding sites, the initial
8-nucleotide element of the direct repeat was shown to have greater
affinity for the pH RE-binding protein.2 These data
are consistent with the observation that the mut2 oligonucleotide, which retains the initial 8-base element, functions as
a weak competitor. The (AUUU)5A oligonucleotide was
previously shown to be a weak competitor (15). None of the other tested oligonucleotides was able to compete the specific binding observed with
either the crude extract or the purified pH RE-binding protein.
The preparation of purified pH RE-binding protein contained two
proteins that were identified by MS/MS sequencing and Western blot
analysis. One of the proteins was the TIAR, a well known RNA-binding
protein that contains three RNA recognition motifs (30). TIAR may
participate in Fas-mediated apoptotic cell death (18, 31). It also
binds to the AU-rich element that mediates the translational regulation
of tumor necrosis factor The second protein identified in the purified preparation of the pH
RE-binding protein was The onset of metabolic acidosis may activate a signal transduction
pathway that results in increased expression and/or covalent modification of the pH RE-binding protein. Identification of
-crystallin/NADPH:quinone reductase. The purified
protein contained eight tryptic peptides that were identical to
sequences found in mouse
-crystallin and three peptides that
differed by only a single amino acid. The observed differences may
represent substitutions found in the rat homolog. A second protein
purified by this protocol was identified as T-cell-restricted
intracellular antigen-related protein (TIAR). However, the
purified TIAR neither bound nor affected the binding of
-crystallin/NADPH:quinone reductase to the pH RE. Furthermore, specific antibodies to
-crystallin, but not TIAR, blocked the formation of the complex between the pH RE and either the crude cytosolic extract or the purified protein. Thus,
-crystallin/NADPH:quinone reductase is a pH response element-binding protein.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-globin (
G) constructs (12) into
LLC-PK1-FBPase+ cells, a pH-responsive
porcine proximal tubule-like cell line (13). Expression of p
G
produced a high level of a very stable mRNA
(t1/2 > 30 h) that was not affected by
transfer of the cells to acidic medium (pH 6.9, 10 mM
HCO
G-GA, which
encodes an mRNA containing an additional 956 bases from the
3'-nontranslated region that is common to both GA mRNAs, was
expressed at significantly lower levels. The decreased expression
resulted from the more rapid turnover (t1/2 = 4.6 h) of the
G-GA mRNA. Transfer of the latter cells to
acidic medium resulted in a pronounced stabilization (6-fold) and a
gradual induction of the
G-GA mRNA. These studies indicated that
the 3'-nontranslated region of the GA mRNA contains a pH response element (pH RE).
G constructs indicated that a
340-base segment of the GA mRNA, termed R-2, retained most of the
functional characteristics of the 3'-nontranslated region (14). Mapping
studies, using RNA electrophoretic mobility shift assays, demonstrated
that the specific binding of a unique protein mapped to the
29-nucleotide R-2I RNA that contained a direct repeat of an
8-base AU sequence. Site-directed mutation of the direct repeat of the
8-base AU sequence completely abolished the pH-responsive stabilization
of the
G-GA mRNA (15). A
G reporter construct that contained
the 3'-nontranslated region of the phosphoenolpyruvate
carboxykinase mRNA, p
G-phosphoenolpyruvate carboxykinase, was
designed to further test the function of the AU element. When expressed
in LLC-PK1-FBPase+ cells, the half-life
of the
G-phosphoenolpyruvate carboxykinase mRNA was only
slightly stabilized (1.3-fold) by growth in acidic medium. However,
insertion of short segments of GA cDNA containing either the direct
repeat or a single 8-base AU sequence was sufficient to impart a 5-fold
pH-responsive stabilization to the chimeric mRNA. Thus, either the
direct repeat or a single copy of the 8-base AU sequence is both
necessary and sufficient to function as a pH RE. The apparent binding
to the pH RE is increased 3-fold in cytosolic extracts prepared from
LLC-PK1-FBPase+ cells that were grown in
acidic medium (16). Extracts prepared from the renal cortex of rats
that were made acutely acidotic also exhibit a similar increase in
binding to the direct repeat of the pH RE. The time course for the
increase in binding correlates with the temporal increase in GA
mRNA. Thus, the protein that binds to the pH RE may mediate the
pH-responsive stabilization of the GA mRNA.
-crystallin/NADPH:quinone reductase. Furthermore,
antiserum specific for
-crystallin blocks the formation of the
complex formed between the R2-I RNA and either the purified protein or
the crude cytosolic extract. Thus,
-crystallin/NADPH:quinone reductase is a pH RE-binding protein.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]UTP (specific activity, 800 Ci/mmol),
horseradish peroxidase-conjugated secondary antibody, and ECL-Plus kits
were obtained from Amersham Pharmacia Biotech. Restriction enzymes,
RNase T1, T7 polymerase, and yeast tRNA were obtained from Roche
Molecular Biochemicals and New England Biolabs. Chemicals for
acrylamide gels, Micro Bio-spin chromatography columns, and protein
standards were purchased from Bio-Rad. Immobilon NC membrane and
Microcon columns were obtained from Millipore. RNasin was obtained from
Promega. GelBond PAG films were purchased from Intermountain
Scientific. DNA preparation kits were from Promega or Qiagen.
Slide-a-lyzer cassettes were obtained from Pierce. Other chemicals were
acquired from Sigma. Anti-sera against
-crystallin/NADPH:quinone reductase (17) were kindly provided by Dr.
J. Samuel Zigler, Jr. (National Eye Institute). A monoclonal antibody
against the T-cell-restricted intracellular antigen-related protein,
TIAR (18), was kindly provided by Dr. Nancy Kedersha (Harvard Medical School).
70 °C. The protein concentration of the
cytosolic extract was determined by a Bradford assay (19) using bovine
serum albumin as the standard.
-32P]UTP; 0.5 mM ATP, CTP, and GTP; 50 µM unlabeled UTP; 20 units of RNAsin; and 10 mM
dithiothreitol. After the mixture was incubated at 37 °C for
1 h, 1.0 unit of RNase-free DNase was added, and the reaction
mixture was incubated at 37 °C for 15 min. The RNA was then purified
by chromatography on a Micro Bio-spin column, and its concentration was
determined by scintillation counting.
-32P]UTP but containing a 0.5 mM concentration of each ribonucleotide. The appropriate
templates were prepared as described previously (14, 15). The
concentrations of the unlabeled RNA samples were determined by
measuring their absorbance at 260 nm and using specific extinction
coefficients calculated from the nucleotide composition of the
individual transcripts.
-crystallin/NADPH:quinone reductase antiserum or a
1:2000 dilution of TIAR monoclonal antibody. The membrane was then
incubated with a 1:1500 dilution of horseradish peroxidase-conjugated
secondary antibody. Images were developed with the ECL-Plus kit and
visualized on a Storm system (Molecular Dynamics).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Comparison of the RNAs used for gel shift
assays and as a ligand for affinity purification. All three RNAs
contain the pH RE from the GA mRNA that is a direct repeat of two
8-nucleotide AU sequences. In total, the R2-I and R2-H RNAs contained
29 and 72 nucleotides, respectively. The affinity ligand contained 44 ribonucleotides and the indicated 5'- and 3'-deoxyribonucleotides (in
italics) including two biotinylated thymidines (labeled
B).
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Fig. 2.
Analyses of samples that were collected from
the avidin-agarose column and then dialyzed overnight against 1×
binding buffer. A, aliquots of fractions
4-9 were separated by 10% SDS-PAGE and silver-stained.
B, aliquots of the same fractions were analyzed for binding
activity using an RNA gel shift assay containing 25 fmol of
32P-labeled R2-I RNA. C, specific binding
activity was estimated as the ratio of the digitized intensities of the
protein-RNA complex observed in B to those of the 36-kDa
protein from A.
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Fig. 3.
Comparison of the binding activities observed
with fractions that contained either the 36- or 43-kDa proteins.
Samples containing ~10 ng of protein from each fraction were
incubated with 25 fmol of 32P-labeled R2-I RNA and then
separated on a native polyacrylamide gel.
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Fig. 4.
Comparison of the binding specificity of the
pH RE binding activity of a crude extract (A) and the
purified protein (B). In both experiments, 25 fmol of 32P-labeled R2-I RNA was incubated either in the
absence (lane 1) or presence (lanes 2-8) of
either 3 µg of a cytosolic extract of rat kidney cortex or 10 ng of
purified pH RE-binding protein. Samples in lanes 3-8 also
contained a 100-fold excess of the indicated competitor.
pBS, pBlueScript.
-crystallin/NADPH:quinone reductase protein. Three other Class I peptides differ from tryptic peptides of
mouse
-crystallin by a single amino acid substitution and may
represent sequences from the rat homolog. The two Class II peptides
correspond to tryptic peptides contained in the 43-kDa mouse
RNA-binding protein, TIAR.
-crystallin/NADPH:quinone reductase purified
from a guinea pig lens (17) reacted with only the single 36-kDa protein
when tested versus a crude cytosolic extract of rat renal
cortex (data not shown). When used versus a purified sample
containing both the 43- and 36-kDa proteins (Fig. 5A), the
anti-
-crystallin antibody again bound only to the 36-kDa protein
(Fig. 5B). Similarly, a monoclonal antibody (18), which is
specific for TIAR, bound to only the 43-kDa protein. Thus, a slight
contamination of the isolated 36-kDa protein band with the larger
protein accounts for the two Class II peptides that were identified by
microsequencing.
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Fig. 5.
Western blot analysis of the two peptides
contained in the purified preparation of the pH RE-binding protein.
A, the fractions from an avidin-agarose column that
contained pH RE binding activity were pooled, and an aliquot was
separated by 10% SDS-PAGE and silver-stained. B, an
identical sample was used for Western blot analysis with either
antiserum against -crystallin/NADPH:quinone reductase or a
monoclonal antibody versus TIAR.
-crystallin/NADPH:quinone reductase is a pH RE-binding protein (Fig. 6A). Preincubation of a
cytosolic extract of rat renal cortex with increasing amounts of
anti-
-crystallin antiserum completely blocked the formation of the
specific R2-I RNA-protein complex. The same inhibition pattern was
observed when the gel shifts were performed with the purified pH
RE-binding protein (data not shown). The observed inhibition was
specific, because anti-glutaminase antiserum (23) had no effect on the
complex formation (Fig. 6A). The affinity ligand used to
purify the pH RE-binding protein was slightly longer than the R2-I RNA
probe. Thus, labeled R-2H RNA, which contains all of the
ribonucleotides present in the RNA affinity ligand, was synthesized and
used as a probe for gel shift analysis (Fig. 6B). Because of
the larger size of this probe, the resulting complex was digested with
RNase T1 before electrophoresis. Again, the resulting complex is
completely blocked by preincubation with anti-
-crystallin antiserum
but is not affected by pretreatment with antibody specific for TIAR.
Thus,
-crystallin/NADPH:quinone reductase binds with high affinity
and specificity to the pH RE.
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Fig. 6.
Immunoblocking analysis of the pH RE-binding
protein. A, samples containing either zero (lane
1) or 3 µg (lanes 2-6) of a crude extract were
preincubated with increasing amounts (0.25, 0.5, or 1 µg) of
-crystallin antiserum or 1 µg of glutaminase antiserum. Then 25 fmol of 32P-labeled R2-I RNA was added, and the samples
were separated on a native polyacrylamide gel. B, samples
containing either zero (lane 1) or 5 ng of purified protein
(lanes 2-4) were preincubated with either 1 µg of
-crystallin antiserum or 4 µg of TIAR monoclonal antibody. Then 10 fmol of 32P-labeled R2-H RNA was added, and the samples
were digested with RNase T1 and separated on a native polyacrylamide
gel.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mRNA (32). An in vitro
analysis of its binding preference revealed that TIAR also has an
affinity for RNAs that contain short stretches of uridylate residues
(30). This broad specificity may account for the purification of TIAR
by the AU-rich pH RE affinity ligand. However, the purified TIAR failed
to bind to either the R-2I or the longer R-2H RNA. In addition,
specific antibodies to TIAR did not block formation of specific
complexes that are formed by incubating either RNA with a cytosolic
extract of rat kidney cortex. Thus, it is unlikely that TIAR functions
as a pH RE-binding protein.
-crystallin/NADPH:quinone reductase.
-crystallin constitutes 10% of the total protein present in the lens of hystricomorph rodents (33) and camelids (34). In these species,
the
-crystallin gene contains an alternative promoter that accounts
for its lens-specific overexpression (17, 35, 36). Similar to other
lens crystallins with a limited phylogenetic distribution,
-crystallin also has a catalytic activity and is expressed at
enzymatic levels in various tissues of different species (17, 37, 38).
-crystallin possesses a novel NADPH-dependent quinone
oxidoreductase activity that reduces various quinones through the
sequential transfer of single electrons (33).
-Crystallin/NADPH:quinone reductase was not previously known to
function as an RNA-binding protein. However, the ability of bovine
-crystallin to bind to different forms of DNAs had been quantified
through an ELISA assay (39). It preferentially binds to double-stranded
Z-DNA and to single-stranded DNA but has a 5-7-fold lower affinity for
double-stranded B-DNA. The sequence specificity of the observed
interactions was not examined. However, the binding of
-crystallin
to single-stranded DNA was effectively competed by NADPH. Thus, the
NADPH binding site of
-crystallin may constitute a portion of its
DNA binding site. The observation that antiserum versus
mouse
-crystallin reacts specifically with the 36-kDa protein
contained in the purified pH RE-binding protein and blocks formation of
the specific complex formed with the pH RE indicates that the
-crystallin/NADPH:quinone reductase also has a high affinity binding
site for the AU-rich pH RE.
-crystallin/NADPH:quinone reductase as a pH RE-binding protein will
facilitate the characterization of the process by which this binding
activity is enhanced during acidosis and the mechanism by which this
interaction leads to the cell-specific stabilization of the GA mRNA.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant DK-37124.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 970-491-5566;
Fax: 970-491-0494; E-mail: ncurth@lamar.colostate.edu.
Published, JBC Papers in Press, April 9, 2001, DOI 10.1074/jbc.M101941200
2 J. Schroeder and N. P. Curthoys, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
GA, glutaminase;
G,
-globin;
pH RE, pH response element;
MS, mass
spectrometry;
TIAR, T-cell-restricted intracellular antigen-related
protein;
PAGE, polyacrylamide gel electrophoresis.
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