Protein Kinase A-regulated Instability Site in the 3′-Untranslated Region of Lactate Dehydrogenase-A Subunit mRNA
1998; Elsevier BV; Volume: 273; Issue: 38 Linguagem: Inglês
10.1074/jbc.273.38.24861
ISSN1083-351X
AutoresDi Tian, Delai Huang, Sabine Short, Marc L. Short, Richard A. Jungmann,
Tópico(s)Protein Structure and Dynamics
ResumoExpression of the lactate dehydrogenase A subunit (LDH-A) gene can be controlled by transcriptional as well as posttranscriptional mechanisms. In rat C6 glioma cells, LDH-A mRNA is stabilized by activation and synergistic interaction of protein kinases A and C. In the present study, we aimed to identify the sequence domain which determines and regulates mRNA stability/instability by protein kinase A and focused our attention on the 3′-untranslated region (3′-UTR) of LDH-A mRNA. We have constructed various chimeric globin/lactate dehydrogenase (ldh) genes linked to the c-fos promoter and stably transfected them into rat C6 glioma cells. After their transfection, we determined the half-life of transcribed chimeric globin/ldh mRNAs. The results showed that at least three sequence domains within the LDH-A 3′-UTR consisting of nucleotides 1286–1351, 1453–1471, and 1471–1502 are responsible for the relatively rapid rate of LDH-A mRNA turnover in the cytoplasm. Whereas chimeric globin/ldh mRNAs containing the base sequences 1286–1351 and 1453–1471 were not stabilized by (S p)-cAMPS, an activator of protein kinase A, instability caused by the 1471–1502 domain was significantly reversed. Additional deletion and mutational analyses demonstrated that the 3′-UTR fragment consisting of the 22 bases 1478–1499 is a critical determinant for the (S p)-cAMPS-mediated LDH-A mRNA stabilizing activity. Because of its functional characteristics, we named the 22-base region "cAMP-stabilizing region." Expression of the lactate dehydrogenase A subunit (LDH-A) gene can be controlled by transcriptional as well as posttranscriptional mechanisms. In rat C6 glioma cells, LDH-A mRNA is stabilized by activation and synergistic interaction of protein kinases A and C. In the present study, we aimed to identify the sequence domain which determines and regulates mRNA stability/instability by protein kinase A and focused our attention on the 3′-untranslated region (3′-UTR) of LDH-A mRNA. We have constructed various chimeric globin/lactate dehydrogenase (ldh) genes linked to the c-fos promoter and stably transfected them into rat C6 glioma cells. After their transfection, we determined the half-life of transcribed chimeric globin/ldh mRNAs. The results showed that at least three sequence domains within the LDH-A 3′-UTR consisting of nucleotides 1286–1351, 1453–1471, and 1471–1502 are responsible for the relatively rapid rate of LDH-A mRNA turnover in the cytoplasm. Whereas chimeric globin/ldh mRNAs containing the base sequences 1286–1351 and 1453–1471 were not stabilized by (S p)-cAMPS, an activator of protein kinase A, instability caused by the 1471–1502 domain was significantly reversed. Additional deletion and mutational analyses demonstrated that the 3′-UTR fragment consisting of the 22 bases 1478–1499 is a critical determinant for the (S p)-cAMPS-mediated LDH-A mRNA stabilizing activity. Because of its functional characteristics, we named the 22-base region "cAMP-stabilizing region." lactate dehydrogenase untranslated region cAMP-stabilizing region base pair(s) adenosine 3′,5′ cyclic monophosphorothioate polymerase chain reaction. Analysis of the LDH1isoenzyme patterns in various cell types under a variety of physiologic conditions suggests complex regulatory mechanisms that determine specific isoenzyme expression (1Markert C.L. Ursprung H. Dev. Biol. 1962; 5: 363-381Crossref Scopus (156) Google Scholar, 2Richards A.H. Hilf R. Endocrinology. 1972; 91: 287-295Crossref PubMed Scopus (31) Google Scholar, 3Lee C. Oliver L. Coe E.L. Oyasu R. J. Natl. Cancer Inst. 1979; 62: 193-199PubMed Google Scholar, 4Jungmann R.A. Kelley D.C. Miles M.F. Milkowski D.M. J. Biol. Chem. 1983; 258: 5312-5318Abstract Full Text PDF PubMed Google Scholar, 5Matrisian L.M. Rautmann G. Magun B.E. Breathnach R. Nucleic Acids Res. 1985; 13: 711-726Crossref PubMed Scopus (102) Google Scholar, 6Short M.L. Huang D. Milkowski D.M. Short S. Kunstman K. Soong C.-J. Chung K.C. Jungmann R.A. Biochem. J. 1994; 304: 391-398Crossref PubMed Scopus (42) Google Scholar, 7Huang D. Jungmann R.A. Mol. Cell. Endocrinol. 1995; 108: 87-94Crossref PubMed Scopus (33) Google Scholar). The LDH-A subunit, for instance, is subject to regulation by a number of different effector agents such as estrogen (3Lee C. Oliver L. Coe E.L. Oyasu R. J. Natl. Cancer Inst. 1979; 62: 193-199PubMed Google Scholar, 8Li S.S. Hou E.W. Cell Biol. Int. Rep. 1989; 13: 619-624Crossref PubMed Scopus (11) Google Scholar), epidermal growth factor (5Matrisian L.M. Rautmann G. Magun B.E. Breathnach R. Nucleic Acids Res. 1985; 13: 711-726Crossref PubMed Scopus (102) Google Scholar), catecholamines (4Jungmann R.A. Kelley D.C. Miles M.F. Milkowski D.M. J. Biol. Chem. 1983; 258: 5312-5318Abstract Full Text PDF PubMed Google Scholar, 9Derda D.F. Miles M.F. Schweppe J.S. Jungmann R.A. J. Biol. Chem. 1980; 255: 11112-11121Abstract Full Text PDF PubMed Google Scholar), phorbol ester (7Huang D. Jungmann R.A. Mol. Cell. Endocrinol. 1995; 108: 87-94Crossref PubMed Scopus (33) Google Scholar), and c-Myc (10Shim H. Dolde C. Lewis B.C. Wu C.-S. Dang G. Jungmann R.A. Dalla-Favera R. Dang C.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6658-6663Crossref PubMed Scopus (866) Google Scholar), which change the isoenzyme pattern almost exclusively in favor of the LDH-5 (A4) isoenzyme. The functional importance of these LDH isoenzyme shifts is generally attributed to a need for increased A subunit-containing isoforms (such as LDH-4 or -5), which can derive more energy from the anaerobic pathway by reducing pyruvate to lactate. Investigations into the mechanism of LDH-A gene expression has identified two basic controls consisting of a transcription-regulatory cascade (4Jungmann R.A. Kelley D.C. Miles M.F. Milkowski D.M. J. Biol. Chem. 1983; 258: 5312-5318Abstract Full Text PDF PubMed Google Scholar, 6Short M.L. Huang D. Milkowski D.M. Short S. Kunstman K. Soong C.-J. Chung K.C. Jungmann R.A. Biochem. J. 1994; 304: 391-398Crossref PubMed Scopus (42) Google Scholar, 11Short S. Short M.L. Milkowski D.M. Jungmann R.A. J. Biol. Chem. 1991; 266: 22164-22172Abstract Full Text PDF PubMed Google Scholar) and a mechanism that regulates the half-life of LDH-A mRNA (4Jungmann R.A. Kelley D.C. Miles M.F. Milkowski D.M. J. Biol. Chem. 1983; 258: 5312-5318Abstract Full Text PDF PubMed Google Scholar, 12Huang D. Hubbard C.J. Jungmann R.A. Mol. Endocrinol. 1995; 9: 994-1004PubMed Google Scholar), both of which are major determinants of intracellular LDH-A mRNA levels. Messenger RNA turnover rates fluctuate over a wide range, and it is important to identify and characterize putative stability-regulating mRNA domains and their interacting factors that may be responsible for these functional effects. A great number of reports have demonstrated the existence of such domains and theirtrans-acting regulatory factors that are critical in determining the half-life of mRNA (13Ross J. Mol. Biol. Med. 1988; 5: 1-14PubMed Google Scholar). Several of these studies indicate that the stability of some, but not all, mRNA is determined by specific cis-acting AU-rich domains located in the 3′-UTR. For example, a number of mRNAs such as cytokine, lymphokine and protooncogene mRNAs share a common sequence motif with a high content of A and U nucleotides in the 3′-UTR (14Ross J. Microbiol. Rev. 1995; 59: 423-450Crossref PubMed Google Scholar) and exhibit half-lives in the range of only a fraction of 1 h (15Shaw G. Kamen R. Cell. 1986; 46: 659-667Abstract Full Text PDF PubMed Scopus (3124) Google Scholar, 16Caput D. Beutler B. Hartog K. Thayer R. Brown-Shimer S. Cerami A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1670-1674Crossref PubMed Scopus (1218) Google Scholar, 17Cleveland D.W. Yen T.J. New Biol. 1989; 1: 121-126PubMed Google Scholar, 18Jones T.R. Cole M.D. Mol. Cell. Biol. 1987; 7: 4513-4521Crossref PubMed Scopus (213) Google Scholar). In addition, attention has focused on modulation of mRNA stability in response to a variety of physiological signals. For instance, histone mRNA stability is regulated by the cell cycle (19Schümperli D. Cell. 1986; 45: 471-472Abstract Full Text PDF PubMed Scopus (114) Google Scholar) and intracellular iron levels control the stability of transferrin receptor mRNA (20Casey J.L. Hentze M.W. Koeller D.M. Caughman S.W. Rouault T.A. Klausner R.D. Harford J.B. Science. 1988; 240: 924-928Crossref PubMed Scopus (510) Google Scholar, 21Malter J.S. Science. 1989; 246: 664-666Crossref PubMed Scopus (371) Google Scholar). Moreover, manipulation of cells with several different effector agents can alter the steady-state level of mRNA during cell growth or differentiation and may exhibit tissue-specific variations (14Ross J. Microbiol. Rev. 1995; 59: 423-450Crossref PubMed Google Scholar). This suggests that in addition to stability-regulating RNA domains effector agent-responsive protein factors may be involved in determining mRNA stability and that they could be subject to regulation by agents that activate second messenger signal pathways. It has recently been demonstrated that activators of protein kinases A and C are important effectors of mRNA stability regulation in a number of gene systems (14Ross J. Microbiol. Rev. 1995; 59: 423-450Crossref PubMed Google Scholar). Although it is known that increased levels of either cAMP or phorbol ester are sufficient signals for increased LDH-A mRNA stability, the molecular mechanisms mediating the effects of cAMP or phorbol ester on mRNA stability have not been defined in detail. Based on our previous data (4Jungmann R.A. Kelley D.C. Miles M.F. Milkowski D.M. J. Biol. Chem. 1983; 258: 5312-5318Abstract Full Text PDF PubMed Google Scholar, 12Huang D. Hubbard C.J. Jungmann R.A. Mol. Endocrinol. 1995; 9: 994-1004PubMed Google Scholar), we suggested that sequences within the non-coding regions of LDH-A mRNA together with protein kinase-regulated RNA-binding phosphoprotein(s) may play a pivotal role in determining the basal and regulated stability of mRNA (12Huang D. Hubbard C.J. Jungmann R.A. Mol. Endocrinol. 1995; 9: 994-1004PubMed Google Scholar, 22Tian, D., Huang, D., Brown, R. C., and Jungmann, R. A. (1998)J. Biol. Chem. 273, in pressGoogle Scholar). In the present study, we have chosen to identify putativecis-regulatory domains within the 3′-UTR of LDH-A mRNA that are involved in protein kinases A-mediated mRNA stability regulation. Our initial approach was to express transfected chimeric β-globin/ldh 3′-UTR constructs and to evaluate the functional effects of protein kinase A on chimeric mRNA stability. Furthermore, applying ribonuclease protection assay to determine the half-life of truncated and mutated fragments of LDH-A 3′-UTR, we systematically analyzed the 3′-UTR for the presence of (a) sequence domain(s) that cause mRNA instability, and (b) stability-regulatory domain(s) whose activity is modulated by protein kinase A. Our experiments demonstrate that several 3′-UTR fragments evoked marked instability of the otherwise relatively stable β-globin mRNA. Most importantly, we were able to identify a uridine-rich cAMP-stabilizing region (CSR) responsible for regulating the rate of LDH-A mRNA turnover in response to activators of protein kinase A and the phosphatase inhibitor okadaic acid. Nucleic acid-modifying enzymes, acrylamide, nucleoside triphosphates were from Boehringer Mannheim. Radioisotopes were purchased from NEN Life Science Products. Other reagents were of molecular biology grade and purchased from Sigma. Cell culture products were purchased from Life Technologies, Inc. (S p)-Adenosine 3′,5′-cyclic monophosphorothioate ((S p)-cAMPS) and (R p)-adenosine 3′,5′-cyclic monophosphorothioate ((R p)-cAMPS) were from BIOLOG Life Science Institute. Synthesis and processing of synthetic DNA oligonucleotides and their ligation into the respective plasmid vectors were performed as described previously (23Kwast-Welfeld J. Soong C-J. Short M.L. Jungmann R.A. J. Biol. Chem. 1989; 264: 6941-6947Abstract Full Text PDF PubMed Google Scholar). Rat C6 glioma cells (ATCC CCL 107) were maintained as monolayers in Ham's F-10 nutrient medium supplemented with 10% dialyzed fetal calf serum, 50 units/ml penicillin, and 50 mg of streptomycin as described by us (9Derda D.F. Miles M.F. Schweppe J.S. Jungmann R.A. J. Biol. Chem. 1980; 255: 11112-11121Abstract Full Text PDF PubMed Google Scholar). All experiments were carried out at about 90% confluence, and serum was withdrawn 16–18 h prior to addition of various agents. A rat fibroblast LDH-A cDNA clone (pLDH-2), provided by Dr. Richard Breathnach, contains the full-length 1609-bp cDNA insert. The mRNA has a 103-nucleotide 5′-nontranslated region and a 510-nucleotide 3′-untranslated region, corresponding to nucleotides 1103 through 1613 (5Matrisian L.M. Rautmann G. Magun B.E. Breathnach R. Nucleic Acids Res. 1985; 13: 711-726Crossref PubMed Scopus (102) Google Scholar). The 3′-UTR contains the classic polyadenylation signal AAUAAA 18 nucleotides before the poly(A) sequence. A HinfI/BamHI fragment containing the entire LDH-A 3′-UTR (with 28-bp 5′ coding sequence and 100-bp pLDH-2 vector sequence) was inserted into pGem3Zf(−) at the BamHI site resulting in plasmid pLDH-5. Plasmid pLDH-6 was constructed to retain the complete 3′-UTR but eliminate the 28-bp LDH-A coding and 100-bp pLDH-2 vector sequences contained in pLDH-5. Using oligonucleotide primers, the complete 510-base pair 3′-UTR of LDH-A with 5′ BamHI and 3′ HindIII sites was amplified by polymerase chain reaction (PCR) from pLDH-5. The fragment was cloned into the BamHI/HindIII sites of pBluescript II KS+ (Stratagene). The rabbit β-globin expression vector pRc/FBB was constructed by Dr. D. Chagnovich (Northwestern University) in two steps from plasmids pRc/CMV (Invitrogen) and pBBB (kindly provided by Dr. M. E. Greenberg). To that purpose, pRc/CMV was linearized withBglII and blunt-ended with T4 DNA polymerase. After insertion of a decamer containing a SacII restriction site, the resulting vector was cut with SacII and HindIII to excise the cytomegalovirus promoter and to serve as acceptor for the modified pBBB. For this modification aSacII-Nru I fragment was excised from pBBB and replaced with HindIII linkers. After restriction withSacII, pRc/FBB was created by linking modified pBBB with the modified SacII-HindIII fragment of pRc/CMV. Plasmid pRc/FBB encodes a transcription unit consisting of β-globin coding region flanked by the β-globin 5′- and 3′- untranslated regions fused to the c-fos promoter. The sequence and correct orientation of all inserts were verified by restriction and DNA sequence analyses. Sequencing was carried out in both directions by the dideoxynucleotide chain terminator method with specific synthetic oligonucleotides as primers. One day prior to transfection, cell cultures were prepared by seeding 1 × 105 cells/60-mm plate in medium containing 10% fetal calf serum. Each plate was treated with 100 μg of Lipofectin in 2 ml of Opti-MEM I for 20 min after which 10 μg of supercoiled plasmid DNA were added. After 16 h, the Lipofectin solution was replaced with 3 ml of DMEM supplemented with 2% fetal calf serum. Two days following transfection, cells were trypsinized and replated at several dilutions between 1:5 and 1:10 in selective medium containing 0.5 mg/ml G418 (Geneticin). Cells were fed with selective medium every third day until resistant colonies were clearly visible (after about 2 weeks). Individual drug-resistant colonies were subcloned and expanded under selection conditions. Total cytoplasmic RNA was prepared as described (12Huang D. Hubbard C.J. Jungmann R.A. Mol. Endocrinol. 1995; 9: 994-1004PubMed Google Scholar). Transcripts derived from the glyceraldehyde-3-phosphate dehydrogenase gene and the various transfected chimeric β-globin/ldh 3′-UTR vectors were detected by RNase protection analysis of 5 μg of RNA isolated at various times after serum stimulation and addition of effector agents using32P-labeled complementary rabbit β-globin antisense probes (24Rodger J.R. Johnson M.L. Rosen J.M. Methods Enzymol. 1985; 109: 572-592Crossref PubMed Scopus (47) Google Scholar). The complete antisense β-globin probe was synthesized with the MAXIscript in vitro transcription kit from the rabbit β-globin gene cloned into pBluescript. Radioactivity corresponding to the protected fragments was visualized by autoradiography and quantified using a BAS III FUJI radioanalytic imaging scanner. The half-life of LDH-A mRNA and various chimeric globin/ldh mRNAs was calculated by a nonlinear regression analysis of the results using the InPlot program (GraphPAD Software, San Diego, CA). The decay of chimeric globin/ldh mRNAs from the linker-scanning studies was quantitated using Real-Time PCR detection with a 7700 ABI PRISM Sequence Detection system using the TaqMan PCR reagent kit (Perkin-Elmer) with two fluorogenic probes: 6-carboxyfluorescein as the reporter probe and 6-carboxy-tetramethylrhodamine as the quencher probe (25Heid C.A. Stevens J. Livak K.J. Williams P.M. Genome Res. 1996; 6: 986-994Crossref PubMed Scopus (5038) Google Scholar). Isolation of genomic DNA and Southern blot hybridization analysis were carried out as described (26Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1987)Current Protocols in Molecular Biology, Vols. 1–2, John Wiley & Sons, New YorkGoogle Scholar). It is known that the presence of AU-rich regions in the 3′-UTR may function as destabilizing elements in several mRNAs. Sequence analysis of the LDH-A 3′-UTR identifies a 99-nucleotide stretch (nucleotides 1450–1549), which is relatively AU-rich when compared with the overall nucleotide composition of the 3′-UTR. Whether or not this AU-rich region contains a site(s) that determines LDH-A mRNA stabilizing/destabilizing activity and can additionally be modulated through protein kinase signal pathways has so far not been determined. As a first step to experimentally identify stability/instability elements in the 3′-UTR of LDH-A mRNA, we replaced the 3′-UTR of β-globin mRNA in plasmid pRc/FBB with the entire LDH-A 3′-UTR and then determined the rate of decay and half-life of the chimeric globin/ldh mRNA under various experimental conditions. By choosing an expression vector (pRc/FBB) with a serum-inducible c-fospromoter (15Shaw G. Kamen R. Cell. 1986; 46: 659-667Abstract Full Text PDF PubMed Scopus (3124) Google Scholar, 27Greenberg M.E. Ziff E.B. Nature. 1984; 311: 433-438Crossref PubMed Scopus (2009) Google Scholar, 28Shyu A.-B. Greenberg M.E. Belasco J.G. Genes Dev. 1989; 3: 60-72Crossref PubMed Scopus (452) Google Scholar), we also avoided artifacts that potentially occur when commonly used transcriptional inhibitors such as actinomycin D (29Harrold S. Genovese C. Kobrin B. Morrison S.L. Milcarek C. Anal. Biochem. 1991; 198: 19-29Crossref PubMed Scopus (104) Google Scholar, 30Scott W.A. Tomkins G.M. Methods Enzymol. 1975; 40: 273-293Crossref PubMed Scopus (22) Google Scholar) are used to stop ongoing transcription. To perform the decay studies, the pRc/FBB/ldh construct was introduced into rat C6 glioma cells by stable transfection. G418-resistant colonies were pooled to ensure a heterogeneity of integration sites. Quantitative ribonuclease protection assays for each chimeric vector were performed to measure mRNA half-lives. After serum-starving the transfected cells for 25–30 h, pulse induction of the chimeric globin/ldh gene under the control of the c-fos promoter was done by addition of fetal calf serum. Total cytoplasmic RNA was isolated at subsequent time points and ribonuclease protection assays were done. As shown in Fig. 1 (panel A, lane wt β-globin), wild-type β-globin mRNA was remarkably stable and persisted in the cytoplasm with a half-life of about 21 h (extrapolated from Fig. 1, panel B), similar to data obtained by others (28Shyu A.-B. Greenberg M.E. Belasco J.G. Genes Dev. 1989; 3: 60-72Crossref PubMed Scopus (452) Google Scholar, 31Aviv H. Valloch V. Bastos R. Levy S. Cell. 1976; 8: 495-503Abstract Full Text PDF PubMed Scopus (109) Google Scholar, 32Lowenhaupt K. Lingrel J.B. Cell. 1978; 14: 337-344Abstract Full Text PDF PubMed Scopus (46) Google Scholar, 33Volloch V. Housman D. Stamatoyannopoulos G. Nienhuis A.N. Organization and Expression of Globin Genes. Alan R. Liss, Inc., New York1981: 251-257Google Scholar). In stark contrast, the chimeric β-globin/ldh mRNA (panel A, lane Control) decayed at a much faster rate (t 1/2 ≈ 70 min) (Fig. 1, panel B), similar to the rapid rate of decay of wild-type LDH-A mRNA (t 1/2 ≈ 55 min) in glioma (4Jungmann R.A. Kelley D.C. Miles M.F. Milkowski D.M. J. Biol. Chem. 1983; 258: 5312-5318Abstract Full Text PDF PubMed Google Scholar, 12Huang D. Hubbard C.J. Jungmann R.A. Mol. Endocrinol. 1995; 9: 994-1004PubMed Google Scholar). For each chimeric vector the decay data consistently obeyed first-order kinetics. Since the basal mRNA levels may reflect the copy number of integrated globin genes after transfection, we analyzed the copy number of integrated globin genes by Southern blot analysis (not shown). We found that the average number of integrated globin copies is nearly the same for all transfected cells. By treating the transfected cells with the protein kinase A agonist, (S p)-cAMPS, we found that the activated protein kinase had the ability to alter the stability of chimeric globin/ldh mRNA. (S p)-cAMPS significantly prolonged the half-life of hybrid globin/ldh mRNA (panel A, lane Sp-cAMPS) but not that of wild-type globin mRNA (not shown). For instance, the half-life of globin/ldh mRNA increased from about 70 min in untreated cells to about 8 h in (S p)-cAMPS-treated cells (seepanel B). Inasmuch as the above data suggested the involvement of protein kinase A and, hence, protein phosphorylation, we sought additional insight into the significance of potential phosphorylation events by preventing protein phosphorylation through the use of inhibitors of protein kinase A. We used the selective protein kinase A antagonist (R p)-cAMPS to prevent protein kinase activation. The results summarized in Table I show that (R p)-cAMPS blocks protein kinase A-mediated stabilization of globin/ldh mRNA. Furthermore, we used okadaic acid, an inhibitor of protein phosphatases-1 and -2A (34Haystead T.A.J. Sim A.T.R. Carling D. Honnor R.C. Tsukitani Y. Cohen P. Hardie D.G. Nature. 1989; 337: 78-81Crossref PubMed Scopus (706) Google Scholar), to shift the overall balance of phospho-/dephosphoproteins in favor of the phosphorylated proteins. Exposure of transfected cells to okadaic acid (20 nm) resulted in a much slower decay and a markedly enhanced stability of globin/ldh mRNA (see Fig. 2).Table IEffect of an activator and inhibitor of protein kinase A on stability regulation of chimeric globin/ldh mRNATreatmentHalf-life (t½)None69 ± 4 min(S p)-cAMPS (500 μm)8.7 ± 1.25 h(R p)-cAMPS (500 μm)78 ± 4 minRat C6 glioma cells were stably transfected with pRc/FBB in which the globin 3′-UTR was replaced with the entire LDH 3′-UTR fragment. Cells were treated for 6 h with the indicated concentrations of agents. After addition of serum, RNA was isolated at various time points up to 12 h. Globin/ldh mRNA decay was assessed by ribonuclease protection assay, and half-lives were determined as described under "Experimental Procedures." Results are expressed as mean and S.E. of four separate experiments. Open table in a new tab Rat C6 glioma cells were stably transfected with pRc/FBB in which the globin 3′-UTR was replaced with the entire LDH 3′-UTR fragment. Cells were treated for 6 h with the indicated concentrations of agents. After addition of serum, RNA was isolated at various time points up to 12 h. Globin/ldh mRNA decay was assessed by ribonuclease protection assay, and half-lives were determined as described under "Experimental Procedures." Results are expressed as mean and S.E. of four separate experiments. Thus, the studies show that the half-life of chimeric globin/ldh mRNA and its regulation are similar, if not identical, to that of wild-type LDH-A mRNA and that the 3′-UTR contains all elements needed to (a) destabilize LDH-A mRNA and (b) to convey protein kinase-mediated stability to globin mRNA. We conclude that regulation of LDH-A mRNA stability is an inherent function of its 3′-UTR and that it is not, or is only to some minor degree, affected by other regions of the mRNA (such as coding regions and 5′-UTR). Based on above data, we proceeded to identify putative regions within the LDH-A 3′-UTR that are (a) instrumental in determining instability of LDH-A mRNA and (b) responsible for the protein kinase-mediated stabilization of the mRNA. To that purpose, we generated a series of systematically truncated 3′-UTR fragments that were inserted into the unique BglII site located at the junction of the β-globin translated and 3′-untranslated regions in pRc/FBB (35van Ooyen A. van den Berg J. Mantei N. Weissmann C. Science. 1979; 206: 337-344Crossref PubMed Scopus (162) Google Scholar) (see Fig. 3). This produced vectors with a globin reporter gene, which, upon transcription, yielded unique chimeric globin/ldh mRNAs whose decay characteristics and half-lives were determined. As in each vector the promoter (c-fos) and globin coding regions are identical, the transcripts differ from each other only in their 3′-UTR sequence. In our studies, the density of autoradiographic bands corresponding to various mRNAs was normalized to that of glyceraldehyde-3-phosphate dehydrogenase mRNA, since this mRNA was stable under the experimental conditions regardless of the presence or absence of effector agent. The normalized band of highest density was taken as the starting decay time point. The basal half-lives of the different truncated chimeric globin/ldh mRNAs are shown in Fig. 3. The half-life of several chimeric mRNAs was only slightly lower (between 18 and 20 h) than that of wild-type β-globin mRNA (about 21 h), and they decayed less than 10% over the 12-h time course of the experiment (time course not shown). In contrast, insertion of fragments 1286–1351, 1453–1470, 1463–1502, 1463–1527, and 1471–1502 had a marked destabilizing effect and the corresponding chimeric mRNAs exhibited significantly reduced half-lives. From these data, we infer that LDH-A 3′-UTR contains at least three destabilizing elements located in fragments 1286–1351, 1453–1470, and 1471–1502. The regulatory effect of protein kinase A on the stability of short-lived chimeric globin/ldh mRNAs (see Fig. 3) was analyzed after stimulation of transfected cells with the second messenger (S p)-cAMPS. The results are shown in Table II. Chimeric mRNAs transcribed from vectors with inserted fragments 1286–1351 and 1453–1470 were destabilized, but their stability was not affected by treatment of cells with (S p)-cAMPS. In contrast, cells transfected with a vector containing fragment 1453–1527, 1463–1502, or 1463–1527 transcribed a chimeric mRNA that was not only destabilized but responded to (S p)-cAMPS treatment of cells with a significant stabilization of the corresponding globin/ldh mRNAs. When fragment 1463–1502 was further shortened to bases 1471–1502, the resulting chimeric globin/ldh mRNA was destabilized and cells responded to (S p)-cAMPS treatment with stabilization of the mRNA. Further truncation resulted in a loss of (S p)-cAMPS responsiveness (see below). Thus, the data show that fragment 1471–1502 contains the sequences that are necessary for the stabilizing effect of (S p)-cAMPS.Table IIEffect of (Sp)-cAMPS on the half-life of chimeric globin/ldh mRNAsFragment inserted (base no.)Half-life(S p)-cAMPS/controlControl(S p)-cAMPSh1103–16101.2 ± 0.68.3 ± 0.97.11286–13513.9 ± 0.53.1 ± 0.70.81453–14702.9 ± 0.93.6 ± 1.21.21453–15274.5 ± 0.315.6 ± 2.13.51463–15024.1 ± 0.617.5 ± 2.14.31463–15273.7 ± 0.716.9 ± 2.44.61471–15023.9 ± 0.817.8 ± 2.54.6Rat C6 glioma cells were stably transfected with pRc/FBB in which the listed fragments of LDH-A 3′-UTR (with 5′ and 3′ BglII ends) had been inserted into the BglII site of pRc/FBB (see Fig. 3). Cells were treated in serum-free medium for 6 h with 0.5 mm (S p)-cAMPS. After addition of serum, RNA was isolated at various time points up to 12 h. Globin/ldh mRNA decay was assessed by ribonuclease protection assay, and half-lives were determined as described under "Experimental Procedures." Results are expressed as mean and S.E. of four separate experiments. Open table in a new tab Rat C6 glioma cells were stably transfected with pRc/FBB in which the listed fragments of LDH-A 3′-UTR (with 5′ and 3′ BglII ends) had been inserted into the BglII site of pRc/FBB (see Fig. 3). Cells were treated in serum-free medium for 6 h with 0.5 mm (S p)-cAMPS. After addition of serum, RNA was isolated at various time points up to 12 h. Globin/ldh mRNA decay was assessed by ribonuclease protection assay, and half-lives were determined as described under "Experimental Procedures." Results are expressed as mean and S.E. of four separate experiments. To expand and confirm the above data, we carried out a stability analysis of LDH-A 3′-UTR in which partial base sequences had systematically been deleted from the 3′-UTR. Globin 3′-UTR in pRc/FBB was removed byBglII/HindIII digestion and replaced with truncated LDH-A 3′-UTR fragments. After stable transfection of the vectors into rat C6 glioma cells, the vectors were transcribed, and the decay characteristics of the chimeric globin/ldh mRNAs were analyzed. The half-lives determined by this analysis are summarized in Table III. While deletion of various fragments had little effect on destabilization and (S p)-cAMPS responsiveness, deletion of fragment 1453–1527 and the even shorter fragment 1478–1502 resulted in increased stability and loss of
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