1 Department of Biochemistry and Molecular Biology, Colorado State University, Ft. Collins 80523-1870; and 2 Department of Natural, Applied, and Environmental Sciences, Front Range Community College, Fort Collins, Colorado 80526
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ABSTRACT |
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Phosphoenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme of renal gluconeogenesis. The 3'-nontranslated region of the PEPCK mRNA contains an instability element that facilitates its rapid turnover and contributes to the regulation of PEPCK gene expression. Such processes are mediated by specific protein-binding elements. Thus RNA gel shift analysis was used to identify proteins in rat renal cortical cytosolic extracts that bind to the 3'-nontranslated region of the PEPCK mRNA. Deletion constructs were then used to map the binding interactions to two adjacent RNA segments (PEPCK-6 and PEPCK-7). However, competition experiments established that only the binding to PEPCK-7 was specific. Functional studies were performed by cloning similar segments in a luciferase reporter construct, pLuc/Zeo. This analysis indicated that both PEPCK-6 and PEPCK-7 segments were necessary to produce a decrease in luciferase activity equivalent to that observed with the full-length 3'-nontranslated region. Thus the PEPCK-7 segment binds a specific protein that may recruit one or more proteins to form a complex that mediates the rapid decay of the PEPCK mRNA.
LLC-PFK1-F+ cells; ribonucleic acid-binding protein; messenger ribonucleic acid turnover
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INTRODUCTION |
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THE CYTOSOLIC ISOFORM of phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the rate-limiting reaction in both hepatic and renal gluconeogenesis. However, this activity is not regulated by allosteric mechanisms or by covalent modifications (9). Instead, it is regulated primarily by mechanisms that affect the level of the PEPCK mRNA and subsequently determine the level of the PEPCK protein. This is accomplished through changes in either the rate of synthesis or the rate of degradation of the PEPCK mRNA. Regulation of PEPCK gene expression occurs primarily at the level of transcription, a process that has been characterized extensively (10). In contrast, the mechanism that mediates the turnover of PEPCK mRNA is poorly understood even though the latter process also plays an important role in the physiological regulation of PEPCK gene expression.
The time required for an mRNA to change from one steady-state level to another is largely proportional to its half-life (11). Thus the rapid induction of an mRNA after activation of transcription is feasible only if the mRNA also has a rapid turnover. Previous studies indicate that the PEPCK mRNA is degraded with a half-life of 1 h in rat kidney cortex (9) and of 4 h in LLC-PK1-F+ cells (14). The turnover of the PEPCK mRNA in liver and hepatoma cells occurs even more rapidly (13). The mRNAs that encode various cytokines and the proteins of immediate early genes are typically degraded with half-lives of <1 h (3). The latter mRNAs usually contain AU-rich elements, including AUUUA or UUAUUUAUU sequences, that function as instability elements. However, very little is known about the sequence of potential elements that may mediate the turnover of an mRNA that occurs at the intermediate rate characteristic of the PEPCK mRNA in kidney cells. To characterize the mechanism of mRNA turnover, it is necessary to initially determine the cis-acting elements present in an mRNA molecule that are involved in protein recognition. The elucidated sequences can then be used to purify and characterize the RNA-binding proteins and to identify related mRNAs whose stability may be determined by the same mechanism.
RNA electrophoretic mobility shift assays were used to detect a protein in the cytosolic fraction of rat hepatoma FTO-2B cells that bound to multiple oligoribonucleotides derived from the 3'-nontranslated region of the PEPCK mRNA (24). The observed binding was not sequence specific, but it may have required a stem-loop structure. The presence of another PEPCK mRNA-binding protein in cytosolic extracts of rat hepatocytes (4, 5) has also been described. This binding occurred primarily within the last 256 nucleotides of the PEPCK mRNA, but additional protein-binding sites were detected in the remainder of the 3'-nontranslated region. A protein with the same electrophoretic motility was observed to bind to the 3'-ends of the histone H1 mRNA and the arylsulfatase A mRNA. More recent studies indicate that the light chain of rat ferritin (12) binds to various RNAs, including the 3'-nontranslated region of the PEPCK mRNA. Thus none of the previously identified PEPCK mRNA-binding proteins exhibit specific binding.
In the present study, a specific binding interaction between the 3'-nontranslated region of the PEPCK mRNA and a cytosolic protein expressed in rat kidney cortex was characterized. The specific binding interaction was mapped to a 50-base segment (PEPCK-7). However, functional studies suggest that an adjacent element (PEPCK-6), which may function as a low-affinity protein binding site, is also required to constitute a functional instability element. Thus binding of a specific protein to the PEPCK-7 segment may recruit one or more proteins to the PEPCK-6 element to form a complex that mediates the rapid decay of the PEPCK mRNA.
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MATERIALS AND METHODS |
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Materials.
[-32P]UTP (specific activity 3,000 Ci/mmol) was
purchased from ICN Biochemicals or Amersham. Restriction enzymes, T4
DNA ligase, T7 RNA polymerase, and RNase T1 were acquired from
Boehringer Mannheim and New England Biolabs. RNasin and the
Dual-Luciferase Reporter Assay System were obtained from Promega. The
PCR site-directed mutagenesis kit and the PCR-Script Amp cloning kit
were purchased from Stratagene. All other biochemicals were purchased
from Sigma.
Construction of transcription vectors.
Various plasmids (Fig. 1) were
synthesized to utilize T7 RNA polymerase to transcribe the RNAs
necessary to map the binding site of proteins that interact with the
3'-nontranslated region of the PEPCK mRNA. The PEPCK/pPCR-Script
plasmid contains 593 bp of the 3'-nontranslated region of the PEPCK
cDNA that extends from 2,003 to 2,595 bp. It was synthesized by using
the oligonucleotides 5'-ATCAGCTAGCTGTAATCCCG-3' (forward)
and 5'-TTCCAACTAGTGGTCAGGATACC-3' (reverse) as
primers to PCR amplify the desired sequence from the GP plasmid
(24). The reaction introduced Nhe I and
Spe I restriction sites at the 5'- and 3'-ends,
respectively. The Nhe I and Spe I sites in the
respective oligonucleotides are underlined, and the mutated bases are
shown in bold letters. The resulting PCR fragment was subsequently
cloned into the Srf I site of pPCR-Script Amp SK(+).
Restriction analysis was used to identify a PEPCK/pPCR-Script plasmid
in which the 5'-end of the PEPCK sequence was positioned near the
Kpn I site of the multicloning site. The desired plasmid was
restricted with Asp718 and Nhe I, and the
resulting vector fragment was religated to produce the PEPCK-1 plasmid.
The deletion eliminated the intervening muticloning sites between the
T7 RNA polymerase promoter and the sequence that encodes the
3'-nontranslated region of the PEPCK mRNA.
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Generation of DNA templates.
The pPCR-Script Amp SK(+) and pBlueScript II SK() vectors contain a
BssH II site immediately upstream of the T7 promoter. Thus
DNA templates are normally obtained by restricting either expression
vector with BssH II and a second restriction enzyme. However, for many of the constructs described above, it was not possible to restrict with BssH II because of the presence of
this site within the 3'-nontranslated region of the PEPCK cDNA. Thus a
Pvu II site that is located just upstream of the
BssH II site had to be used in the construction of some of
the DNA templates. The PEPCK-1 template was a Pvu
II/Spe I fragment of the PEPCK-1 plasmid. The PEPCK-2
template was obtained by restricting the PEPCK-1 plasmid with
BssH II. The PEPCK-3 and PEPCK-4 templates were
Pvu II/Spe I and Pvu
II/Hind III fragments that were excised from the PEPCK-3 and
PEPCK-4 plasmids, respectively. The PEPCK-5, PEPCK-6, and PEPCK-7
templates were obtained by restricting the respective plasmids with
BssH II and either Xba I (PEPCK-6) or Spe I (PEPCK-5 and PEPCK-7).
In vitro transcription.
Transcription reactions were performed using a slight modification of
the standard method (23). A 10-µl reaction mixture containing 10 ng/µl DNA template, 40 µCi of
[-32P]UTP, 0.2 mM ATP, CTP, and GTP, 50 µM unlabeled
UTP, 20 units of RNasin, and 10 mM dithiothreitol was incubated at
37°C for 1 h. Next, 1 unit of RNase-free DNase was added, and
the reaction mixture was incubated at 37°C for an additional 15 min.
The final reaction volume was adjusted to 50 µl with diethyl
pyrocarbonate (DEPC)-treated water, and the mixture was then applied to
a Micro Bio Spin P-30 column (Bio-Rad Labs). This process eliminates
>99% of the free nucleotides. The RNA transcripts were further
purified by adding 15 µl of 2 M sodium acetate, pH 4.0, 85 µl
DEPC-treated water, 75 µl water-saturated phenol, and 75 µl
chloroform-isoamyl alcohol (24:1). The mixture was vortexed, allowed to
stand for 5 min, and then centrifuged at 14,000 g for 2 min
at 4°C. The aqueous upper layer was removed, and 150 µl of
isopropanol was added. The RNA was kept at
20°C for 20 min and then
was centrifuged at 21,000 g at 4°C for 15 min. The pellet
was washed in 80% ethanol, air-dried, and resuspended in 20-30
µl of DEPC-treated water. Synthesis of unlabeled RNA was performed
using a 100-µl reaction mixture lacking [
-32P]UTP
but containing 0.2 mM of each ribonucleotide. All transcripts were
stored at
70°C and were used within 4 days. The radiolabeled products were quantified by scintillation counting, and the volume was
adjusted to yield the desired concentration. The concentration of the
cold RNA transcripts was determined by measuring the absorbance at 260 nm and using specific extinction coefficients calculated from the
nucleotide composition.
RNA electrophoretic mobility shift assay. This assay was performed as described previously with some modifications (1). An aliquot of rat renal cortical extract containing 3-5 µg of protein was 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% Nonidet P-40, 1 mM dithiothreitol, 10% glycerol, and 10 units of RNasin. Next, 10-40 fmol of 32P-labeled RNA and the indicated amounts of specific or nonspecific competitor RNAs were added simultaneously. The reaction mixture was incubated at room temperature for 20 min. Next, 15 units of RNase T1 were added to reactions containing transcripts of 50 bases or longer, and the sample was incubated at room temperature for 15 min. Samples were then loaded on a 5% polyacrylamide gel and subjected to electrophoresis.
Rat renal cortical cytoplasmic extracts.
Rats were anesthetized with 1 mg/kg body wt of pentobarbital sodium,
and was opened with a midline incision. The kidneys were perfused in
situ with a Krebs-Henseleit solution (KHS). They were removed
immediately, decapsulated, sliced longitudinally, and placed in
ice-cold KHS. The cortex was dissected from the papilla and medulla,
cut into small pieces, weighed, and placed in an equal volume of 40 mM
HEPES, pH 7.4, containing 100 mM potassium acetate, 10 mM magnesium
acetate, 1 mM dithiothreitol, 10 µM leupeptin, 10 µM antipain, and
10 µg/ml phenylmethylsulfonyl fluoride. The tissue was homogenized
using a Dounce homogenizer. An aliquot of homogenate was examined
microscopically for released nuclei. The sample was then centrifuged at
1,000 g for 10 min at 4°C to pellet intact cells and
nuclei. The supernatant was centrifuged at 10,000 g for 10 min at 4°C to pellet mitochondria and then at 100,000 g
for 2 h at 4°C to pellet membrane-bound organelles and
polyribosomes. The final supernatant was separated into aliquots and
was frozen at 70°C. Protein concentration was determined by the
Lowry assay using BSA as the standard (21).
Construction of luciferase vectors.
The pLuc/Zeo plasmid was designed to facilitate the rapid detection and
mapping of mRNA instability elements (O. F. Laterza, C. Cole, W. Liu, L. Taylor, and N. P. Curthoys, unpublished observation). It
contains the cytomegalovirus (CMV) promoter, the entire coding region
of the firefly luciferase gene, a segment of the 3'-nontranslated region of the Simina virus 40 containing a single intron, a
multicloning site containing nine unique restriction sites, and the
3'-nontranslated region and polyadenylation site of the bovine growth
hormone gene. pLuc-G/Zeo was produced by isolating a 507-bp
Hind III/Bgl II fragment from pRSV-
-globin
(6) and inserting it into the multicloning site in
pLuc/Zeo. The pLuc/Zeo plasmid contains two Hind III sites. Thus a partial digest was performed to generate the vector fragment that was restricted only at the Hind III site within the
multicloning site.
Luciferase assay. LLC-PK1-F+ cells (7) were grown on 10-cm plates using a 50:50 mixture of DMEM and Ham's F-12 medium containing 5 mM glucose. At 3 days postsplitting in six-well plates, the cells were transiently cotransfected by calcium phosphate precipitation (2) of 1.0 µg of the various pLuc/Zeo plasmids and 0.2 µg of pRL-TK (Promega). Approximately 24 h later, the transfection media were removed, and fresh media were added. The cells were cultured for an additional 24 h and washed two times with 2 ml of PBS, and cell extracts were prepared and assayed using the reagents contained in the Dual-Luciferase Reporter Assay System (Promega). The firefly luciferase activities obtained from the various pLuc/Zeo plasmids were standardized vs. the corresponding Renilla luciferase activities to correct for differences in transfection efficiency. In a single experiment, each transfection was performed in triplicate. For comparative purposes, the mean of the standardized luciferase activities measured with the parent construct was normalized to a value of one. This arbitrary unit was selected so that a decrease in the stability of a chimeric mRNA is indicated by a number less than one. Statistical analysis of the data was performed using a Student's t-test.
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RESULTS |
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Mapping of protein binding sites.
RNA gel shift analysis was performed to detect the presence of proteins
in cytosolic extracts of rat kidney cortex that bind to the
3'-nontranslated region of the PEPCK mRNA. The PEPCK-1 RNA that
contains 593 nucleotides of the 3'-nontranslated region of the PEPCK
mRNA was used as the initial probe. When the 32P-labeled
PEPCK-1 RNA was incubated with cytosolic extracts from rat kidney
cortex, digested with RNase T1, and separated by electrophoresis on a
native polyacrylamide gel, two well-resolved bands that had a slower
mobility than the digested probe were observed (Fig. 2, lane 2).
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Competition studies.
The previously identified binding interactions of various proteins with
the 3'-nontranslated region of the PEPCK mRNA occurred with ambiguous
specificity (4, 5, 12). Thus competition studies were
performed to determine the specificity of the interaction of the
binding proteins detected in the rat renal cortical cytosolic extracts.
Samples containing 40 fmol of 32P-labeled PEPCK-6 RNA were
incubated with 3 µg of extract in the presence of increasing amounts
(10, 30, and 100-fold excess) of unlabeled PEPCK-5, PEPCK-6, PEPCK-7,
GA R-2I, and (AUUU)5A RNAs (Fig.
3). The GA R-2I RNA is a 29-base segment
of the 3'-nontranslated region of glutaminase mRNA that functions as a
pH-responsive element (18). The (AUUU)5A RNA
contains five reiterations of an AUUUA sequence. This is a common
recognition sequence for proteins that bind to AU-rich elements
(3). The band observed with the labeled PEPCK-6 RNA was
effectively competed with increasing amounts of unlabeled PEPCK-7, GA
R-2I, and (AUUU)5A RNAs, and it was only slightly competed
with increasing amounts of unlabeled PEPCK-5 and PEPCK-6 RNAs (Fig. 3).
The observation that unrelated RNAs [PEPCK-7, R-2I, and
(AUUU)5A] compete more effectively than the PEPCK-6 RNA
suggests that the binding interaction produced with this segment of the
PEPCK mRNA is nonspecific. Interestingly, the RNAs that compete most
effectively contain a high percentage of A and U residues. This is
particularly true for the GA R-2I and (AUUU)5A RNAs. Thus
the PEPCK-6 RNA may bind various proteins that interact with AU-rich
elements.
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Mapping of the instability element. The pLuc/Zeo plasmid was used to determine if the specific binding of a protein to the PEPCK-7 segment mediates the inherent instability of the PEPCK mRNA. This plasmid contains a chimeric gene that includes the coding sequence of the firefly luciferase and the 3'-nontranslated region and polyadenylation signal of the very stable bovine growth hormone mRNA. The plasmid also contains a large multicloning site within the 3'-nontranslated region to facilitate the cloning of cDNA fragments that encode potential mRNA instability elements. The insertion of short sequences within the 3'-nontranslated region is unlikely to affect transcription from the CMV promoter or translation of the chimeric mRNAs but may decrease the stability of the resulting transcript. Thus differences in the luciferase activities produced from the pLuc/Zeo plasmid and a derived construct will reflect the relative abundance of the two mRNAs that in turn should primarily reflect their relative stability.
To illustrate the reliability of this approach, a 507-bp segment of the rabbit
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DISCUSSION |
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Cytosolic proteins that bind to the 3'-nontranslated region of the
PEPCK mRNA are likely to play an important role in mediating the
degradation of this mRNA. Various protein/PEPCK mRNA interactions were
previously characterized using cytosolic extracts of hepatoma cells or
rat hepatocyes (4, 5, 12, 24). However, the function of
the characterized interactions was never tested directly. Furthermore,
none of the previously characterized interactions was found to be
specific. The detected binding interactions occurred with multiple
segments of the PEPCK mRNA and with unrelated RNAs. The first direct
evidence for the presence of a destabilizing cis-acting
element within the 3'-nontranslated region of the PEPCK mRNA was
obtained from experiments that measured the half-life of various
chimeric -globin mRNAs expressed in
LLC-PK1-F+ cells. The insertion of the complete
3'-nontranslated region of the PEPCK mRNA produced a chimeric mRNA that
decayed with a half-life of ~5 h compared with a half-life >30 h for
the parent construct (8).
In the present study, potential binding to the entire 3'-nontranslated
region was tested using cytosolic extracts of rat kidney cortex, and
the two observed binding activities were then mapped to two unique RNA
segments. Competition studies demonstrated that binding to the PEPCK-7
RNA exhibits a high degree of specificity. Thus the experiments
reported in the present study are the first to demonstrate a specific
interaction of a cytosolic protein with a discrete segment of the
3'-nontranslated region of the PEPCK mRNA. The PEPCK-7 RNA (Fig.
6A) has a stretch of 21 nucleotides in which 19 are either A or U residues. However, the
remainder of the PEPCK-7 RNA is not enriched for AU nucleotides.
Sequence analysis revealed a direct repeat of 8 bases that are
separated by a single nucleotide and a potential secondary structure
that contains an 8-base stem with 2 mismatches and an 11-base loop (22). The free energy for the formation of this structure
was predicted to be 12.7 kJ at 37°C. It is unknown whether one or more of these potential elements constitute the site that binds a
specific protein from rat kidney cortex.
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The interaction of another rat kidney protein was also detected with the PEPCK-6 segment that is also rich in AU nucleotides (Fig. 6B). However, competition with different RNAs demonstrated that this binding was nonspecific. This protein may have a weak affinity for AU-rich sequences, since it was preferentially competed by RNAs that contain a high percentage of A and U nucleotides. Thus the binding observed may represent a nonspecific or low-affinity interaction with one or more of the broad range of AU-rich RNA-binding proteins (3).
The results of the mapping experiments performed with the pLuc/Zeo plasmid indicate that neither the PEPCK-6 nor the PEPCK-7 segment is sufficient to mediate the instability inherent in the full-length 3'-nontranslated region of the PEPCK mRNA. However, incorporation of both segments in the pLuc/Zeo plasmid produced a decrease in luciferase activity that was nearly equal to that observed with the pLuc-PEPCK-1/Zeo plasmid. Thus the high affinity and specific binding of a protein to the PEPCK-7 sequence are not sufficient to mediate the turnover of the PEPCK mRNA. Instead, the initial binding of a protein to the PEPCK-7 segment may recruit or enhance the binding of one or more proteins that interact in part with the AU-rich PEPCK-6 sequence to form a complex. It is likely that this complex is what mediates the turnover of the PEPCK mRNA. This hypothesis is also supported by the RNA gel shift data shown in Fig. 2. When incubated with a rat renal cortical cytosolic extract, both the PEPCK-1 and the PEPCK-3 RNAs produce two discrete bands in an RNA gel shift. In both cases, the more intense band has the slower mobility. It is the protein binding represented by this band that maps to the PEPCK-6 segment. However, with the shorter PEPCK-6 segment, protein binding is substantially reduced. This observation suggests that the binding observed with the longer segments is facilitated by interaction with the protein that binds specifically to the PEPCK-7 segment. The high affinity and specificity of the binding observed with the PEPCK-7 RNA suggest that it should be feasible to use this sequence as an affinity ligand to purify the associated binding protein. Once identified, the PEPCK-7 RNA binding protein may be used to identify the additional proteins that are necessary to mediate the turnover of the PEPCK mRNA.
RNA-protein interactions are also critical in mediating physiological changes in mRNA turnover that contribute to the regulation of gene expression (26). Various hormones and effectors have been reported to affect the stability of the PEPCK mRNA. For example, treatment with glucocorticoids (25) or cAMP (20) caused an increase in the half-life of the PEPCK mRNA in liver. It was initially observed that the increase in PEPCK mRNA levels after treatment with cAMP persisted after the activation of transcription had returned to basal levels (17, 27). Subsequently, the effect of cAMP was characterized further by direct measurement of the half-life of the PEPCK mRNA in hepatoma cells (13). The addition of cAMP caused a four- to sixfold increase in the half-life of the PEPCK mRNA (13, 20). Similar observations suggest that acidosis may increase the stability of the PEPCK mRNA in kidney (15, 16), whereas treatment of LLC-PK1-F+ cells with phorbol esters caused a decrease in the half-life of the PEPCK mRNA (19).
Preliminary experiments indicate that LLC-PK1-F+ cells also express a protein that exhibits a high affinity and specific binding interaction with the PEPCK-7 segment of the PEPCK mRNA. This interaction was increased in extracts of LLC-PK1-F+ cells that were treated with either acidic media or with cAMP. However, neither treatment produced a change in luciferase activity in cells transiently transfected with the pLuc-PEPCK-1/Zeo construct that was significantly different from the change observed with the parent pLuc/Zeo construct. Thus the observed changes in luciferase activity were due primarily to the effects of cAMP or of acidic media on the activity of the CMV promoter that drives expression of the luciferase gene. Therefore, it will be necessary to develop alternative approaches to investigate whether the identified instability element also contributes to the mechanism by which the half-life of the PEPCK mRNA is altered in response to chronic treatment with different physiological stimuli.
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ACKNOWLEDGEMENTS |
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This research was supported in part by National Institutes of Diabetes and Digestive and Kidney Diseases Grant DK-43704 awarded to N. P. Curthoys.
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FOOTNOTES |
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Address for reprint requests and other correspondence: N. Curthoys, Dept. of Biochemistry and Molecular Biology, Colorado State Univ., Ft. Collins, CO 80523-1870 (E-mail: NCurth{at}lamar.ColoState.edu).
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.
Received 20 September 1999; accepted in final form 19 July 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alberta, JA,
Rundell K,
and
Stiles CD.
Identification of an activity that interacts with the 3'-untranslated region of c-myc mRNA and the role of its target sequence in mediating rapid mRNA degradation.
J Biol Chem
269:
4532-4538,
1994
2.
Chen, C,
and
Okayama H.
High-efficiency transformation of mammalian cells by plasmid DNA.
Mol Cell Biol
7:
2745-2752,
1987[ISI][Medline].
3.
Chen, C-YA,
and
Shyu A-B.
AU-rich elements: characterization and importance in mRNA degradation.
Trends Biochem Sci
20:
465-470,
1995[ISI][Medline].
4.
Christ, B,
Heise T,
and
Jungermann K.
Binding of cytosolic protein from cultured rat hepatocytes to the 3'-end of phosphoenolpyruvate carboxykinase mRNAsignificance for protein-mediated mRNA stabilization.
Biochem Biophys Res Commun
177:
1273-1282,
1991[ISI][Medline].
5.
Christ, B,
and
Nath A.
The glucagon-insulin antagonism in the regulation of cytosolic protein binding to the 3' end of phosphoenolpyruvate carboxykinase mRNA in cultured rat hepatocytes. Possible involvement in the stabilization of the mRNA.
Eur J Biochem
215:
541-547,
1993[Abstract].
6.
Gorman, C,
Padmanabhan R,
and
Howard BH.
High efficiency DNA mediated transformation of primate cells.
Science
221:
551-553,
1983[ISI][Medline].
7.
Gstraunthaler, G,
and
Handler JS.
Isolation, growth and characterization of a gluconeogenic strain of renal cells.
Am J Physiol Cell Physiol
252:
C232-C238,
1987
8.
Hansen, WR,
Barsic-Tress N,
Taylor L,
and
Curthoys NP.
The 3'-nontranslated region of the rat renal glutaminase mRNA contains a pH-responsive stability element.
Am J Physiol Renal Fluid Electrolyte Physiol
271:
F126-F131,
1996
9.
Hanson, RW,
and
Patel YM.
Phosphoenolpyruvate carboxykinase (GTP): the gene and the enzyme.
Adv Enzymol Relat Areas Mol Biol
69:
203-281,
1995[ISI].
10.
Hanson, RW,
and
Reshef L.
Regulation of phosphoenolpyruvate carboxykinase (GTP) gene expression.
Annu Rev Biochem
66:
581-611,
1997[ISI][Medline].
11.
Hargrove, JL.
Microcomputer-assisted kinetic modeling of mammalian gene expression.
FASEB J
7:
1163-1170,
1993
12.
Heise, T,
Nath A,
Jungermann K,
and
Christ B.
Purification of a RNA-binding protein from rat liver. Identification as ferritin L chain and determination of the RNA/protein binding characteristics.
J Biol Chem
32:
20222-20229,
1997.
13.
Hod, Y,
and
Hanson RW.
Cyclic AMP stabilizes the mRNA for phosphoenolpyruvate carboxykinase (GTP) against degradation.
J Biol Chem
236:
7747-7752,
1988.
14.
Holcomb, T,
Liu W,
Snyder R,
Shapiro R,
and
Curthoys NP.
Promoter elements which mediate the pH-response of phosphoenolpyruvate carboxykinase mRNA in LLC-PK1-F+ cells.
Am J Physiol Renal Fluid Electrolyte Physiol
271:
F340-F346,
1996
15.
Hwang, J-J,
and
Curthoys NP.
Effect of acute alterations in acid-base balance on rat renal glutaminase and phosphoenolpyruvate carboxykinase gene expression.
J Biol Chem
266:
9392-9396,
1991
16.
Kaiser, S,
and
Curthoys NP.
Effect of pH and bicarbonate on phosphoenolpyruvate carboxykinase and glutaminase mRNA levels in cultured renal epithelial cells.
J Biol Chem
266:
9397-9402,
1991
17.
Lamers, WH,
Hanson RW,
and
Meisner HM.
cAMP stimulates transcription of the gene for cytosolic phosphoenolpyruvate carboxykinase in rat liver nuclei.
Proc Natl Acad Sci USA
79:
5137-5141,
1982[Abstract].
18.
Laterza, OF,
Hansen WR,
Taylor L,
and
Curthoys NP.
Identification of an mRNA-binding protein and the specific elements that may mediate the pH-responsive induction of renal glutaminase mRNA.
J Biol Chem
272:
22481-22488,
1997
19.
Liu, W,
Feifel E,
Holcomb T,
Liu X,
Spitaler N,
Gstraunthaler G,
and
Curthoys NP.
PMA and staurosporine affect expression of the phosphoenolpyruvate carboxykinase gene in LLC-PK1-F+ cells.
Am J Physiol Renal Physiol
275:
F361-F369,
1998
20.
Liu, J,
and
Hanson RW.
Regulation of phosphoenolpyruvate carboxykinase (GTP) gene transcription.
Mol Cell Biochem
104:
89-100,
1991[ISI][Medline].
21.
Lowry, OH,
Rosebrough NJ,
Farr AL,
and
Randall RJ.
Protein measurement with Folin-phenol reagent.
J Biol Chem
193:
265-275,
1951
22.
Matzura, O,
and
Wennborg A.
RNAdraw: an integrated program for RNA secondary structure calculation and analysis under 32-bit Microsoft Windows.
Comput Appl Biosci
12:
247-249,
1996[Abstract].
23.
Melton, DA,
Kreig PA,
Rebogliati MR,
Manniatis T,
Zinn K,
and
Green MR.
Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter.
Nucleic Acids Res
12:
7035-7056,
1984[Abstract].
24.
Nachaliel, N,
Jain D,
and
Hod Y.
A cAMP-regulated RNA-binding protein that interacts with phosphoenolpyruvate carboxykinase (GTP) mRNA.
J Biol Chem
268:
24203-24209,
1993
25.
Petersen, DD,
Magnuson MA,
and
Granner DK.
3' Noncoding region of phosphoenolpyruvate carboxykinase mRNA contains a glucocorticoid-responsive mRNA-stabilizing element.
Mol Cell Biol
8:
96-104,
1988[ISI][Medline].
26.
Ross, J.
mRNA stability in mammalian cells.
Microbiol Rev
59:
423-450,
1995[Abstract].
27.
Sasaki, K,
Cripe TP,
Koch SR,
Andreone TL,
Peterson DD,
Beale EG,
and
Granner DK.
Multihormonal regulation of phosphoenolpyruvate carboxykinase gene transcription. The dominant role of insulin.
J Biol Chem
259:
15242-15251,
1984