3′-Untranslated Region of Phosphoenolpyruvate Carboxykinase mRNA Contains Multiple Instability Elements That Bind AUF1
2005; Elsevier BV; Volume: 280; Issue: 31 Linguagem: Inglês
10.1074/jbc.m501204200
ISSN1083-351X
AutoresSachin Hajarnis, Jill M. Schroeder, Norman P. Curthoys,
Tópico(s)RNA modifications and cancer
ResumoPhosphoenolpyruvate carboxykinase (PEPCK) is regulated solely by alterations in gene expression that involve changes in rates of PEPCK mRNA transcription and degradation. A tetracycline-responsive promoter system was used to quantify the half-life of various chimeric β-globin-PEPCK (βG-PCK) mRNAs in LLC-PK -F+ cells. The control βG mRNA was extremely stable (t½ = 5 days). However, βG-PCK-1 mRNA, which contains the entire 3′-UTR of the PEPCK mRNA, was degraded with a half-life of 1.2 h. RNase H treatment indicated that rapid deadenylation occurred concomitant with degradation of the βG-PCK-1 mRNA. Previous studies indicate that PCK-7, a 50-nucleotide segment at the 3′-end of the 3′-UTR, binds an unidentified protein that may contribute to the rapid decay of the PEPCK mRNA. However, the chimeric βG-PCK-7 mRNA has a half-life of 17 h. Inclusion of the adjacent PCK-6 segment, a 23-bp AU-rich region, produced the βG-PCK-6/7 mRNA, which has a half-life of 3.6 h. The βG-PCK-3 mRNA that contains the 3′-half of 3′-UTR was degraded with the same half-life. Surprisingly, the βG-PCK-2 mRNA, containing the 5′-end of the 3′-UTR, was also degraded rapidly (t½ = 5.4 h). RNA gel shift analyses established that AUF1 (hnRNP D) binds to the PCK-7, PCK-6, and PCK-2 segments with high affinity and specificity. Mutational analysis indicated that AUF1 binds to a UUAUUUUAU sequence within PCK-6 and the stem-loop structure and adjacent CU-region of PCK-7. Thus, AUF1 binds to multiple destabilizing elements within the 3′-UTR that participate in the rapid turnover of the PEPCK mRNA. Phosphoenolpyruvate carboxykinase (PEPCK) is regulated solely by alterations in gene expression that involve changes in rates of PEPCK mRNA transcription and degradation. A tetracycline-responsive promoter system was used to quantify the half-life of various chimeric β-globin-PEPCK (βG-PCK) mRNAs in LLC-PK -F+ cells. The control βG mRNA was extremely stable (t½ = 5 days). However, βG-PCK-1 mRNA, which contains the entire 3′-UTR of the PEPCK mRNA, was degraded with a half-life of 1.2 h. RNase H treatment indicated that rapid deadenylation occurred concomitant with degradation of the βG-PCK-1 mRNA. Previous studies indicate that PCK-7, a 50-nucleotide segment at the 3′-end of the 3′-UTR, binds an unidentified protein that may contribute to the rapid decay of the PEPCK mRNA. However, the chimeric βG-PCK-7 mRNA has a half-life of 17 h. Inclusion of the adjacent PCK-6 segment, a 23-bp AU-rich region, produced the βG-PCK-6/7 mRNA, which has a half-life of 3.6 h. The βG-PCK-3 mRNA that contains the 3′-half of 3′-UTR was degraded with the same half-life. Surprisingly, the βG-PCK-2 mRNA, containing the 5′-end of the 3′-UTR, was also degraded rapidly (t½ = 5.4 h). RNA gel shift analyses established that AUF1 (hnRNP D) binds to the PCK-7, PCK-6, and PCK-2 segments with high affinity and specificity. Mutational analysis indicated that AUF1 binds to a UUAUUUUAU sequence within PCK-6 and the stem-loop structure and adjacent CU-region of PCK-7. Thus, AUF1 binds to multiple destabilizing elements within the 3′-UTR that participate in the rapid turnover of the PEPCK mRNA. Phosphoenolpyruvate carboxykinase (PEPCK) 1The abbreviations used are: PEPCK, phosphoenolpyruvate carboxykinase; βG-PCK, β-globin-PEPCK; bGH, bovine growth hormone; DRB, 5,6-dichloro-1-β-d-ribofuranosylbenzamidazole; AUF1, AU factor 1; Dox, doxycycline; nt, nucleotide; MOPS, 4-morpholinepropanesulfonic acid; UTR, untranslated region. catalyzes the conversion of oxaloacetate to phosphoenolpyruvate, a rate-limiting step in gluconeogenesis. However, this activity is not regulated by allosteric mechanisms or by covalent modifications (1.Hanson R.W. Patel Y.M. Adv. Enzymol. Relat. Areas Mol. Biol. 1994; 69: 203-281PubMed Google Scholar). Instead, it is regulated by mechanisms that affect the level of the PEPCK mRNA and subsequently determine the level of the PEPCK protein. In liver, transcription of the PEPCK gene is inhibited by insulin and is activated by glucocorticoids, thyroid hormone, and glucagon, which acts through cAMP. Within the renal proximal tubule, parathyroid hormone and angiotensin II increase cAMP levels and cause an increased transcription of the PEPCK gene (2.Liu X. Curthoys N.P. Am. J. Physiol. 1996; 271: F347-F355Crossref PubMed Google Scholar, 3.Watford M. Mapes R.E. Arch. Biochem. Biophys. 1990; 282: 399-403Crossref PubMed Scopus (13) Google Scholar). The level of the PEPCK mRNA in rat kidney is also increased rapidly following the onset of metabolic acidosis (4.Hwang J.J. Curthoys N.P. J. Biol. Chem. 1991; 266: 9392-9396Abstract Full Text PDF PubMed Google Scholar). The latter adaptation is initiated within 1 h and reaches a 6-fold induction within 7 h. The 6-fold induced level of PEPCK mRNA is sustained in the kidneys of rats that are made chronically acidotic (4.Hwang J.J. Curthoys N.P. J. Biol. Chem. 1991; 266: 9392-9396Abstract Full Text PDF PubMed Google Scholar). The time required for an mRNA to change from one steady state to another is usually proportional to its half-life (5.Hargrove J.L. FASEB J. 1993; 7: 1163-1170Crossref PubMed Scopus (37) Google Scholar). Thus, rapid induction of an mRNA is feasible only if the mRNA has a rapid turnover. Previous studies have shown that the PEPCK mRNA is degraded rapidly in liver and in hepatoma cells (6.Hod Y. Hanson R.W. J. Biol. Chem. 1988; 263: 7747-7752Abstract Full Text PDF PubMed Google Scholar), in rat kidney cortex (1.Hanson R.W. Patel Y.M. Adv. Enzymol. Relat. Areas Mol. Biol. 1994; 69: 203-281PubMed Google Scholar), and in LLC-PK1-F+ cells (7.Holcomb T. Liu W. Snyder R. Shapiro R. Curthoys N.P. Am. J. Physiol. 1996; 271: F340-F346PubMed Google Scholar), a line of porcine proximal tubule-like cells selected for their ability to grow in the absence of glucose (8.Gstraunthaler G. Handler J.S. Am. J. Physiol. 1987; 252: C232-C238Crossref PubMed Google Scholar). Previous studies have also demonstrated that the half-life of the PEPCK mRNA is increased in liver in response to cAMP (9.Liu J. Hanson R.W. Mol. Cell. Biochem. 1991; 104: 89-100PubMed Google Scholar) and glucocorticoids (10.Petersen D.D. Magnuson M.A. Granner D.K. Mol. Cell. Biol. 1988; 8: 96-104Crossref PubMed Scopus (86) Google Scholar). Increased stability may also contribute to the sustained induction of renal PEPCK mRNA during chronic acidosis (4.Hwang J.J. Curthoys N.P. J. Biol. Chem. 1991; 266: 9392-9396Abstract Full Text PDF PubMed Google Scholar). Therefore, selective mRNA stabilization may play an important role in the physiological regulation of PEPCK gene expression. However, the mechanisms that cause the rapid turnover and selective stabilization of the PEPCK mRNA are unknown. The presence of a destabilizing cis-element within the 3′-UTR of the PEPCK mRNA was previously demonstrated using DRB, a polymerase II inhibitor, to determine the half-life of a chimeric β-globin-PEPCK mRNA expressed in LLC-PK1-F+-cells (11.Hansen W.R. Barsic-Tress N. Taylor L. Curthoys N.P. Am. J. Physiol. 1996; 271: F126-F131PubMed Google Scholar). The parent β-globin (βG) mRNA decayed with a half-life of >30 h, whereas the chimeric βG-PCK-1 mRNA, containing the complete 3′-UTR of PEPCK mRNA, decayed with a half-life of 5 h. RNA gel shift assays using a cytosolic extract of rat renal cortex identified two protein binding interactions, but only one exhibited properties characteristic of specific binding (12.Laterza O.F. Taylor L. Unnithan S. Nguyen L. Curthoys N.P. Am. J. Physiol. Renal Physiol. 2000; 279: F866-F873Crossref PubMed Google Scholar). The high affinity and specific interaction mapped to the PCK-7 segment of the 3′-UTR of the PEPCK mRNA, whereas the low affinity and nonspecific interaction mapped to the PCK-6 segment. However, functional studies using luciferase constructs suggested that both segments contribute to the rapid decay of the PEPCK mRNA (12.Laterza O.F. Taylor L. Unnithan S. Nguyen L. Curthoys N.P. Am. J. Physiol. Renal Physiol. 2000; 279: F866-F873Crossref PubMed Google Scholar). In the present study, the contribution of individual segments of the 3′-UTR to the stability of the PEPCK mRNA were quantified by analyses of the half-lives of multiple βG-PCK reporter constructs expressed from a tetracycline (Tet)-regulated promoter. Direct binding assays demonstrated that AUF1 binds to each of the segments that function as instability elements. Mutational analyses identified specific sequences within the PCK-6 and PCK-7 segments that bind AUF1. The results demonstrated that multiple AUF1 binding sites contribute to the rapid turnover of PEPCK mRNA. Materials—Male Sprague-Dawley rats were purchased from Charles River Breeding Laboratories. [α-32P]UTP and [α-32P]dCTP (3,000 Ci/mmol) were purchased from MP Biochemicals. The oligo labeling kit was from Ambion. Restriction enzymes, RNase T1, T7 RNA polymerase, and yeast tRNA were acquired from Roche Applied Science, New England Biolabs, or MBI Fermentas. GENECLEAN kits were obtained from Bio101, Inc., and the PCRScript cloning kit was obtained from Stratagene. Micro Bio-spin columns and chemicals for acrylamide gels were purchased from Bio-Rad. RNAsin was obtained from Fischer. An RNA standard was purchased from Invitrogen. Dulbecco's modified Eagle's medium/F-12 base medium was purchased from Sigma. Hybridase™ Thermostable RNase H was purchased from Epicenter. Geneticin (G418) and hygromycin B were obtained from Mediatech. TRIzol® reagent was purchased from Invitrogen. Anti-AUF1 monoclonal antibody (5B9) was a gift from Dr. Gideon Dreyfuss. Polyclonal anti-AUF1 antibody was purchased from Upstate Biotechnology. Anti-CP1 and anti-CP2 antibodies were obtained from Dr. Stephen Liebhaber. Anti-HuR antibody was purchased from Santa Cruz Biotechnology. An expression plasmid that encodes the p40 isoform of AUF1 was obtained from Dr. Jeffrey Wilusz. All other biochemicals were purchased from Sigma. Construction of pBSSK-PCK Transcription Vectors and Generation of DNA Templates—The various segments of the 3′-UTR of the PEPCK mRNA that were used in this study are illustrated in Fig. 1. The pBSSK-PCK-1, pBSSK-PCK-2, pBSSK-PCK-6, and pBSSK-PCK-7 plasmids and templates were prepared as described previously (12.Laterza O.F. Taylor L. Unnithan S. Nguyen L. Curthoys N.P. Am. J. Physiol. Renal Physiol. 2000; 279: F866-F873Crossref PubMed Google Scholar). The pBSSK-PCK-6/7 plasmid was constructed by annealing two oligonucleotides and ligating into pBlueScriptII-SK(–), which was previously digested with Asp718 and XbaI. The sequences of the forward and reverse oligonucleotides were 5′-GTACCGTATGTTTAAATTATTTTTATACACTGCCCTTTCTTACCTTTCTTTACATAATTGAAATAGGTATCCTGACCA-3′ and 5′-CTAGTGGTCAGGATACCTATTTCAATTATGTAAAGAAAGGTAAGAAAGGGCAGTGTATAAAAATAATTTAAACATACG-3′, respectively. The underlined bases represent the partial Asp718 and XbaI sites. The pBSSK-PCK-6/7 template was obtained by digesting the plasmid with BssHII and SpeI. The PCK-7 RNA was divided into three subfragments, PCK-8 (bp 2546–2572), PCK-9 (bp 2558–2587), and PCK-10 (bp 2567–2595). Double-stranded oligonucleotides encoding the PCK-8, PCK-9, and PCK-10 RNAs were cloned into pBlueScriptII-SK(–) that was previously digested with Asp718 and XbaI. The pBSSK-PCK-8, pBSSK-PCK-9, and pBSSK-PCK-10 templates were obtained by digesting the plasmids with BssHII, SacI, and XbaI. pBSSK-PCK-Mut-6, pBSSK-PCK-Mut-1, pBSSK-PCK-Mut-2, pBSSK-PCK-Mut-3, and pBSSK-PCK-Mut-4 were also constructed by annealing double-stranded oligonucleotides and ligating them into pBlueScriptII-SK(–). The template for the pBSSK-PCK-Mut-6 construct was obtained by digesting the plamids with BssHII, SacI, and XbaI, whereas the PCK-Mut-1, Mut-2, Mut-3, and Mut-4 templates were obtained by digestion of the plasmids with BssHII and XbaI. UV Cross-linking of RNA-Protein Complexes—The cross-linking experiments were performed with minor modifications of the procedure of You et al. (13.You Y. Chen C.Y. Shyu A.B. Mol. Cell. Biol. 1992; 12: 2931-2940Crossref PubMed Google Scholar). The samples were prepared as described for the RNA gel shift experiments, except that, following RNase T1 digestion, they were transferred to a 96-well microtiter plate and exposed to shortwave (254 nm) radiation for 5 min in a UV Stratalinker 2400 (Stratagene). The samples were transferred to 1.5-ml microfuge tubes, and an equal volume of 2× SDS sample buffer containing 10% (v/v) glycerol, 5% (v/v) β-mercaptoethanol, 3% (w/v) SDS, 184 mm Tris-Cl (pH 6.8), and 0.2% (w/v) bromphenol blue was added. The samples were heated in boiling water for 5 min and then resolved on a 10% SDS-polyacrylamide gel. The gel was dried and exposed to a PhosphorImager screen. In Vitro Transcription—In vitro transcription of 32P-labeled and unlabeled RNAs was performed as described previously (12.Laterza O.F. Taylor L. Unnithan S. Nguyen L. Curthoys N.P. Am. J. Physiol. Renal Physiol. 2000; 279: F866-F873Crossref PubMed Google Scholar), and the products were purified using Bio-Rad Micro Bio-Spin P30 columns. The 32P-labeled RNAs were quantified by scintillation counting, and diethylpyrocarbonate-treated water was added to adjust the sample to the desired concentration. The absorbance of the unlabeled RNAs was measured at 260 nm, and the concentration of the transcripts was calculated using extinction coefficients determined from the nucleotide composition. RNA Electrophoretic Mobility Shift Assay—An aliquot of rat renal cortical extract (12.Laterza O.F. Taylor L. Unnithan S. Nguyen L. Curthoys N.P. Am. J. Physiol. Renal Physiol. 2000; 279: F866-F873Crossref PubMed Google Scholar) containing ∼1–8 μg of protein (14.Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) or 40–400 ng of recombinant AUF1 was preincubated for 10 min at room temperature in binding buffer containing 0.5% Nonidet P-40, 1 mm dithiothreitol, 2 μg of yeast tRNA, 40 units of RNAsin, 10% glycerol, and 10–40 fmol of 32P-labeled RNA. A 30-, 200-, 300-, or 500-fold excess of competitor RNA was added with the labeled RNA. The reaction mixture was incubated at room temperature for 20 min, and then 1 μl of RNase T1 was added and the samples were incubated for 10 min at room temperature. The samples were separated on 5% native polyacrylamide gels, and the gels were dried and exposed overnight to a PhosphorImager screen. In the supershift experiments, the various antibodies were preincubated with the rat renal cytosolic extract for 20 min at room temperature and then added to the binding buffer. Construction of TetβG-PCK Vectors—A tetracycline-regulated promoter system (15.Xu N. Loflin P. Chen C.Y. Shyu A.B. Nucleic Acids Res. 1998; 26: 558-565Crossref PubMed Scopus (136) Google Scholar) was utilized to map the segments within the 3′-UTR of the PEPCK mRNA that function as instability elements. A chimeric β-globin/growth hormone gene was cloned into the pTRE2 plasmid to yield pTetβG. This construct contains a Tet-responsive promoter, the coding region of rabbit β-globin (βG) gene, and the 3′-nontranslated region and polyadenylation site of bovine growth hormone (bGH) cDNA. The 3′-UTR of the rat PEPCK cDNA (corresponding to nucleotides 2008–2595) was PCR-amplified using forward and reverse oligonucleotide primers that introduced SpeI and XbaI restriction sites at the 5′- and 3′-ends, respectively. The sequence of the forward primer was 5′-GCACTAGTGGGCGAATTGGGTACTAG-3′, whereas that of the reverse primer was 5′-CGTGGTCTAGATACCTATTTCAATTATGTAAAGAAAGG-3′, where the underlined sequences are the SpeI and XbaI sites, respectively. The amplified sequence was then cloned into the SrfI site of pPCRScript-SK(+). The resulting plasmid pPCR-PCK-1 was then digested with SpeI and XbaI to yield a 604-bp fragment that was subsequently cloned into pTetβG at the SpeI site to yield pTβG-PCK-1. pTβG-PCK-1 was digested with BssHII and EcoRV restriction enzymes to release a 224-bp 3′-fragment. The remaining plasmid was blunted and re-ligated to produce pTβG-PCK-2. The 224-bp fragment was blunted and inserted into pTetβG that was previously linearized with EcoRV to produce pTβG-PCK-3. Double-stranded oligonucleotides containing the PCK-6, PCK-7, and PCK-6/7 sequences (12.Laterza O.F. Taylor L. Unnithan S. Nguyen L. Curthoys N.P. Am. J. Physiol. Renal Physiol. 2000; 279: F866-F873Crossref PubMed Google Scholar) were synthesized with SpeI and XbaI overhangs and inserted in pTet-βG that was previously digested with SpeI to yield pTβG-PCK-6, pTβG-PCK-7, and pTβG-PCK-6/7, respectively (Fig. 1). Thus, all of the chimeric βG-PCK mRNAs retained the bGH 3′-UTR that is contained in the control βG-PCK mRNA. Creation of TβG-PCK Stable Cell Lines—LLC-PK1-F+ cells were obtained from Dr. Gerhard Gstraunthaler and cultured as previously described (8.Gstraunthaler G. Handler J.S. Am. J. Physiol. 1987; 252: C232-C238Crossref PubMed Google Scholar). Cell lines that stably express the TβG-PCK-1, TβG-PCK-2, TβG-PCK-3, TβG-PCK-6, TβG-PCK-7, and TβG-PCK-6/7 constructs were made by calcium phosphate cotransfection (16.Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4821) Google Scholar) of the pTβG-PCK plasmid and pcDNA 3.1/Hygro (Invitrogen) into 8C cells, a line of LLC-PK1-F+ cells that overexpress the tTA protein. 2J. M. Schroeder and N. P. Curthoys, unpublished data. At 24 h post-transfection, the medium was removed, the cells were washed twice with phosphate-buffered saline, and then fresh medium containing 0.2 mg/ml G418 was added. At 48 h post-transfection and every 2 days thereafter, fresh medium containing 0.2 mg/ml G418 and 0.6 mg/ml hygromycin B was added. After 14–21 days, individual colonies were isolated and expanded. After the cells were split onto 10-cm plates, the hygromycin B concentration was reduced to 0.2 mg/ml. The cell lines were tested for Dox responsiveness by maintaining the cells for 48 h in medium in the presence or absence of 0.5 μg/ml Dox. Total RNA was harvested from the cells and analyzed on a Northern blot. Half-life Analysis—For pulse-chase analysis, cells expressing the βG-PCK-1 mRNA were initially grown in medium minus Dox and then transferred to medium containing 50 ng/ml Dox for 48 h. This level of Dox was determined to be sufficient to completely shut off transcription from the Tet promoter. The cells were then washed two times with phosphate-buffered saline and grown for 3 h in fresh medium minus Dox to create a pulse of βG-PCK-1 mRNA. Subsequently, 1 μg/ml Dox was added to inhibit transcription. RNA samples were isolated at various times following re-addition of Dox and subjected to Northern analysis to follow the decay of the newly synthesized βG-PCK-1 mRNA. Alternatively, the various TβG-PCK-expressing LLC-PK1-F+ cells were grown for 5–7 days in medium minus Dox. At time zero, 1 μg/ml Dox was added to each plate. At 0, 3, 6, 9, and 12 h post-Dox treatment, RNA was isolated and subjected to Northern analysis. RNase H Treatment—RNase H treatment (17.Porter D. Curthoys N.P. Anal. Biochem. 1997; 247: 279-286Crossref PubMed Scopus (10) Google Scholar) was performed to selectively cleave the 3′-ends of the chimeric βG-PCK-1 mRNAs that were isolated from the pulse-chase experiments. The 21-nt PCK oligo 5′-GCCCAAGATTTTTTTCTCCCC-3′ that encodes the template strand of the PEPCK cDNA from bp 2199–2219 was synthesized by Macromolecular Resources (Fort Collins, CO). A 30-mer of oligo(dT) was used to remove the poly(A) tail of the mRNA. The RNA samples were precipitated from Formazol by adjusting the final concentration of NaCl to 0.2 m and adding 4 volumes of ethanol. After 5 min at room temperature, the RNA was pelleted by centrifugation at 10,000 × g for 10 min. The pellets were resuspended in 25 μl of DEPC-treated water. The cleavage reaction was performed in a final volume of 12.5 μl containing 10 μg of RNA, 1.0 μl of RNase H, and 50 pmol of the PCK oligo with or without addition of 50 pmol of oligo(dT). The mixture was incubated at 55 °C for 60 s, and then 1.5 μl of 10× RNase H buffer (0.5 m Hepes, pH 7.4, 1.0 m NaCl, 20 mm MgCl2, and 10 mm dithiothreitol), prewarmed to 62 °C, was added, and the reaction was incubated at 62 °C for 15 min. An additional unit of RNase H (freshly diluted to 1 unit/μl with 1× RNase H buffer) was added, and the sample was incubated at 62 °C for 15 min. Thereafter, 29 μl of denaturing buffer (20 μl of Formazol, 3 μl of 10× MOPS, and 6 μl of 37% formaldehyde solution) was added to stop the reaction. The sample was incubated at 55 °C for 5 min and then stored on ice. The RNase H-treated RNAs were subjected to Northern blot analysis as described previously (11.Hansen W.R. Barsic-Tress N. Taylor L. Curthoys N.P. Am. J. Physiol. 1996; 271: F126-F131PubMed Google Scholar), except that 10 μl of a RNase H gel loading buffer (50% glycerol, 1 mm EDTA, 0.25% bromphenol blue, 0.25% xylene cyanol, and 25 μl/ml of 10 mg/ml ethidium bromide) was added to the samples, and a 1.2% agarose gel containing 20 mm MOPS, pH 7.0, 1 mm EDTA, and 1 mm sodium acetate was used. The blot was hybridized with the bGH probe. Northern Analyses—Total cellular RNA was isolated using the TRIzol® reagent, and the RNA concentration was determined by measuring the absorbance at 260 nm. A 507-bp fragment of rabbit β-globin cDNA was excised by restricting pRSV-βG (18.Gorman C. Padmanabhan R. Howard B.H. Science. 1983; 221: 551-553Crossref PubMed Scopus (450) Google Scholar) with HindIII and BglII. A 2.0-kb fragment of the 18 S ribosomal RNA cDNA from Acanthamoeba castellanii was excised by restricting pAr2 (19.D'Alessio J.M. Harris G.H. Perna P.J. Paule M.R. Biochemistry. 1981; 20: 3822-3827Crossref PubMed Scopus (23) Google Scholar) with HindIII and EcoRI. A 228-bp bGH fragment was excised from pPCRScript-bGH with SphI. The fragments were separated on a 1% agarose gel, excised, and purified using the GENECLEAN kit. The synthesis of oligo-labeled cDNA probes and Northern analysis were performed as described previously (11.Hansen W.R. Barsic-Tress N. Taylor L. Curthoys N.P. Am. J. Physiol. 1996; 271: F126-F131PubMed Google Scholar). The blots were exposed to a PhosphorImager screen, and the intensity of the resulting digital image of each band was quantified using Molecular Dynamics software. The level of the chimeric β-globin mRNA was divided by the corresponding level of 18 S rRNA to correct for errors in sample loading. For half-life studies, the log of normalized data were then plotted versus the time after the addition of doxycycline. The reported values are the mean ± S.E. of data obtained from triplicate samples. The line representing the best fit of the data points was determined by a KaleidaGraph program that weights each data point based upon its standard deviation. Half-life Analysis of βG-PCK-1 mRNA—A tetracycline-regulated promoter (Tet-off system) was used to determine the half-life of the βG-PCK-1 mRNA. Stably transformed cells were maintained in the presence of 50 ng/ml doxycycline (Dox) to suppress synthesis of the βG-PCK-1 mRNA. A transcriptional pulse was created by removing Dox from the medium for 3 h and was subsequently chased by adding 1 μg/ml Dox to selectively inhibit transcription of the TβG-PCK-1 mRNA. RNA samples were isolated from the cells at various intervals following initiation of the chase, and the levels of βG-PCK-1 mRNA and 18 S rRNA were quantified by Northern analysis (Fig. 2A). This analysis demonstrated that degradation of βG-PCK-1 mRNA is initiated after a brief lag (30 min) and then proceeds with a very rapid half-life (t½ = 1.2 h). Deadenylation of βG-PCK-1 mRNA—Rapid degradation of mammalian mRNAs is usually initiated by the binding of specific protein(s) to unique element(s) within the 3′-UTR (20.Parker R. Song H. Nat. Struct. Mol. Biol. 2004; 11: 121-127Crossref PubMed Scopus (647) Google Scholar, 21.Chen C.Y. Shyu A.B. Trends Biochem. Sci. 1995; 20: 465-470Abstract Full Text PDF PubMed Scopus (1680) Google Scholar). The RNA-binding protein(s) subsequently recruit a poly(A)-specific ribonuclease (22.Gao M. Fritz D.T. Ford L.P. Wilusz J. Mol. Cell. 2000; 5: 479-488Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar) and the exosome (23.van Hoof A. Parker R. Cell. 1999; 99: 347-350Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) to remove the poly(A) tail and accomplish a rapid 3′ → 5′ exonucleolytic degradation, respectively. RNase H treatment of the RNAs isolated from the βG-PCK-1 mRNA half-life analysis was performed to determine whether deadenylation precedes the decay of the PEPCK mRNA. Incubation of the RNA isolated immediately after the 3-h pulse (0-h sample) with a complementary oligonucleotide and oligo(dT), followed by treatment with RNase H, produced a 315-nt fragment (Fig. 2B). This fragment corresponded to the expected length of the fully deadenylated 3′-end of the βG-PCK-1 mRNA. Digestion of the same RNA sample in the presence of only the complementary oligonucleotide produced larger fragments containing ∼500 nt. Thus, the newly synthesized βG-PCK-1 mRNA contained a significant poly(A) tail. Identical treatment of RNAs isolated from the later time points demonstrated that the βG-PCK-1 mRNA undergoes a rapid deadenylation that occurs concomitant with the decay of the mRNA. Mapping of the Instability Elements within the 3′-UTR of PEPCK—Further half-life analyses were performed using cells grown in the absence of Dox to maximally induce expression of the chimeric PEPCK mRNAs. Selective decay of the reporter mRNA was subsequently initiated by the addition of 1 μg/ml Dox. Northern blot analysis was performed using RNAs isolated from 8C cells that stably express the parent TetβG construct. This analysis determined that the control βG mRNA is extremely stable and decays with a half-life of ∼5 days (Fig. 3). Northern analysis also indicated that the βG-PCK-1 mRNA was degraded with a half-life of 1.8 h, consistent with the previous pulse-chase analysis. Previous RNA gel shift analysis indicates that a protein in cytosolic extracts of rat kidney cortex binds specifically to PCK-7, a 50-nt segment of the 3′-UTR of the PEPCK mRNA (12.Laterza O.F. Taylor L. Unnithan S. Nguyen L. Curthoys N.P. Am. J. Physiol. Renal Physiol. 2000; 279: F866-F873Crossref PubMed Google Scholar). To test whether the PCK-7 segment was sufficient to cause the rapid destabilization of the PEPCK mRNA, the turnover of the βG-PCK-7 mRNA was determined. Northern blot analyses revealed that the βG-PCK-7 mRNA was degraded with a half-life of 17 h (Fig. 3). Therefore, insertion of just the PCK-7 segment only partially destabilized the chimeric mRNA and was not sufficient to produce the rapid turnover observed with βG-PCK-1 mRNA. Thus, additional sequences within the 3′-UTR may be needed to constitute the complete instability element of the PEPCK mRNA. Sequence analysis of the 3′-UTRs of human, rat, and mouse PEPCK mRNAs revealed the presence of a conserved 16-nt AU sequence that is located immediately upstream of the PCK-7 segment. This sequence is part of a 23-nt segment termed PCK-6 that binds a protein in a rat renal cytosolic extract with low affinity (12.Laterza O.F. Taylor L. Unnithan S. Nguyen L. Curthoys N.P. Am. J. Physiol. Renal Physiol. 2000; 279: F866-F873Crossref PubMed Google Scholar). Therefore, the PCK-6 segment was inserted into the parent TetβG vector to generate pTβG-PCK-6. LLC-PK1-F+ cells were stably transfected with this construct, and half-life analyses were performed. The βG-PCK-6 mRNA decayed with a half-life of only 6 h (Fig. 3). To determine whether the PCK-6 and PCK-7 segments act synergistically, cells that stably express pTβG-PCK-6/7 were subjected to half-life analysis. Northern blot analysis demonstrated that the βG-PCK-6/7 mRNA decayed with a half-life of 3.6 h (Fig. 4). This half-life was significantly lower than that observed with either βG-PCK-7 or the βG-PCK-6 mRNA but was still greater than that observed with the full-length PCK-1 segment. Therefore, additional sequences within the 3′-UTR may be needed to constitute the complete instability element of the PEPCK mRNA. To test this hypothesis, cells expressing constructs containing longer segments of the 3′-UTR of the PEPCK mRNA were generated. The PCK-3 segment contains 224 nt from the 3′-end of the PEPCK mRNA that includes the PCK-6/7 segment (Fig. 1). Half-life analysis performed using RNAs isolated from cells expressing the βG-PCK-3 mRNA established that this mRNA decayed with a half-life of 3.6 h, the same as observed with the βG-PCK-6/7 mRNA (Fig. 4). Thus, the PCK-6/7 segment contains all of the instability elements within the PCK-3 segment. The half-life of the βG-PCK-2 mRNA that contains the 5′-end of the 3′-UTR of the PEPCK mRNA was also determined. Previous RNA gel shift assays with 32P-labeled PCK-2 RNA and rat renal cytosolic extracts failed to demonstrate the formation of a RNA-protein complex (12.Laterza O.F. Taylor L. Unnithan S. Nguyen L. Curthoys N.P. Am. J. Physiol. Renal Physiol. 2000; 279: F866-F873Crossref PubMed Google Scholar). However, the βG-PCK-2 mRNA, when expressed in the porcine kidney cells, decayed with a half-life of 5.4 h (Fig. 4). Thus, the half-life analyses of the PCK-1, PCK-6, PCK-7, and PCK-2 constructs establish that the 3′-UTR of the PEPCK mRNA contains multiple instability elements that contribute to the rapid turnover that is observed with the full-length chimeric mRNA. AUF1 Binds within the 3′-UTR of the PEPCK mRNA—UV cross-linking analysis was performed to identify the proteins within rat renal cytosolic extracts that bind within the PCK-6/7 segment. Proteins with apparent molecular mass values that range from 40 to 100 kDa were found to be associated with the PCK-6/7 RNA (Fig. 5A). Identical patterns of labeled proteins were observed when PCK-1 and PCK-7 were incubat
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