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• W2159764578 abstract "Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor that is subject to regulation at the post-transcriptional level. At least two mRNA instability elements reside within the PAI-2 transcript; one in the coding region and another within the 3′-untranslated region (UTR). For the latter, a functional AU-rich motif (ARE) has been identified that provides a binding site for a number of cellular proteins, including the mRNA stability protein, HuR. In this study, we used the yeast three-hybrid system to screen a human leukocyte cDNA library to identify other proteins that associate with the PAI-2 ARE. This screen identified tristetraprolin (TTP) as a PAI-2 mRNA ARE-binding protein. UV cross-linking and immunoprecipitation experiments showed that TTP expressed in HEK293 cells could associate with the PAI-2 ARE in vitro. Co-transfection of plasmids expressing TTP and PAI-2 in HEK293 cells resulted in an increase in the decay rate of PAI-2 mRNA and loss of PAI-2 protein in a process that was dependent upon the PAI-2 3′-UTR. The 29-nt PAI-2 AU-rich element alone was also capable of conferring TTP-dependent mRNA instability to a reporter transcript. The extent of PAI-2 mRNA stability was remarkably sensitive to TTP since TTP-dependent PAI-2 mRNA decay occurred at TTP levels that were below Western blot detection limits. This study identifies TTP as a functional PAI-2 ARE-binding protein that modulates the post-transcriptional regulation of the PAI-2gene. Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor that is subject to regulation at the post-transcriptional level. At least two mRNA instability elements reside within the PAI-2 transcript; one in the coding region and another within the 3′-untranslated region (UTR). For the latter, a functional AU-rich motif (ARE) has been identified that provides a binding site for a number of cellular proteins, including the mRNA stability protein, HuR. In this study, we used the yeast three-hybrid system to screen a human leukocyte cDNA library to identify other proteins that associate with the PAI-2 ARE. This screen identified tristetraprolin (TTP) as a PAI-2 mRNA ARE-binding protein. UV cross-linking and immunoprecipitation experiments showed that TTP expressed in HEK293 cells could associate with the PAI-2 ARE in vitro. Co-transfection of plasmids expressing TTP and PAI-2 in HEK293 cells resulted in an increase in the decay rate of PAI-2 mRNA and loss of PAI-2 protein in a process that was dependent upon the PAI-2 3′-UTR. The 29-nt PAI-2 AU-rich element alone was also capable of conferring TTP-dependent mRNA instability to a reporter transcript. The extent of PAI-2 mRNA stability was remarkably sensitive to TTP since TTP-dependent PAI-2 mRNA decay occurred at TTP levels that were below Western blot detection limits. This study identifies TTP as a functional PAI-2 ARE-binding protein that modulates the post-transcriptional regulation of the PAI-2gene. Plasminogen activator inhibitor 2 (PAI-2) 1The abbreviations used are: PAI-2plasminogen activator inhibitor type 2TNFtumor necrosis factorILinterleukinAREAU-rich motifUTRuntranslated regionHAhemagglutininTTPtristetraprolinHEKhuman embryonic kidney cellsGM-CSFgranulocyte-macrophage colony-stimulating factor1The abbreviations used are: PAI-2plasminogen activator inhibitor type 2TNFtumor necrosis factorILinterleukinAREAU-rich motifUTRuntranslated regionHAhemagglutininTTPtristetraprolinHEKhuman embryonic kidney cellsGM-CSFgranulocyte-macrophage colony-stimulating factor is a member of the plasminogen activator family of proteins and controls the activity of urokinase plasminogen activator (u-PA) in the extracellular compartment (1Kruithof E.K. Baker M.S. Bunn C.L. Blood. 1995; 86: 4007-4024Crossref PubMed Google Scholar). PAI-2 is also a member of the OV-serpin family of serine protease inhibitors and has been considered as the most enigmatic member of this group of proteins (2Bachmann F. Thromb. Haemost. 1995; 74: 172-179Crossref PubMed Scopus (47) Google Scholar). One of the most striking features of PAI-2 is that it exists in two molecular forms: a predominant non-glycosylated protein and a less abundant secreted glycosylated protein. The predominant intracellular location of PAI-2 has suggested additional functions for this u-PA inhibitor, and growing evidence has indicated an involvement for PAI-2 in the intracellular events associated with differentiation (3Jensen P.J. Wu Q. Janowitz P. Ando Y. Schechter N.M. Exp. Cell Res. 1995; 217: 65-71Crossref PubMed Scopus (77) Google Scholar), proliferation (4Hibino T. Matsuda Y. Takahashi T. Goetinck P.F. J. Invest. Dermatol. 1999; 112: 85-90Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 5Yu H. Maurer F. Medcalf R.L. Blood. 2002; 99: 2810-2818Crossref PubMed Scopus (55) Google Scholar), signal transduction (6Shafren D.R. Gardner J. Mann V.H. Antalis T.M. Suhrbier A. J. Virol. 1999; 73: 7193-7198Crossref PubMed Google Scholar) and possibly apoptosis (7Dickinson J.L. Bates E.J. Ferrante A. Antalis T.M. J. Biol. Chem. 1995; 270: 27894-27904Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 8Dickinson J.L. Norris B.J. Jensen P.H. Antalis T.M. Cell Death Differ. 1998; 5: 163-171Crossref PubMed Scopus (64) Google Scholar). PAI-2 has also generated interest because of its impressive regulatory profile. PAI-2 gene transcription rates are markedly increased in response to the tumor promoter phorbol 12-myristate 13-acetate (PMA) (9Schleuning W-D. Medcalf R.L. Hession C. Rothenbühler R. Kruithof E.K.O. Mol. Cell. Biol. 1987; 7: 4564-4567Crossref PubMed Scopus (98) Google Scholar), the phosphatase inhibitor, okadaic acid (10Medcalf R.L. J. Biol. Chem. 1992; 267: 12220-12226Abstract Full Text PDF PubMed Google Scholar), tumor necrosis factor-α (TNF) (11Medcalf R.L. Kruithof E.K.O. Schleuning W-D. J. Exp. Med. 1988; 168: 751-759Crossref PubMed Scopus (82) Google Scholar, 12Pytel B.A. Peppel K. Baglioni C. J. Cell. Physiol. 1990; 144: 416-422Crossref PubMed Scopus (34) Google Scholar) and lipopolysaccharide (LPS) (13Schwartz B.S. Monroe M.C. Bradshaw J.D. Blood. 1989; 73: 2188-2195Crossref PubMed Google Scholar). plasminogen activator inhibitor type 2 tumor necrosis factor interleukin AU-rich motif untranslated region hemagglutinin tristetraprolin human embryonic kidney cells granulocyte-macrophage colony-stimulating factor plasminogen activator inhibitor type 2 tumor necrosis factor interleukin AU-rich motif untranslated region hemagglutinin tristetraprolin human embryonic kidney cells granulocyte-macrophage colony-stimulating factor Notwithstanding the important contribution of transcriptional control (14Dear A.E. Costa M. Medcalf R.L. FEBS Lett. 1997; 402: 265-272Crossref PubMed Scopus (23) Google Scholar, 15Mahony D. Stringer B.W. Dickinson J.L. Antalis T.M. Eur. J. Biochem. 1998; 256: 550-559Crossref PubMed Scopus (5) Google Scholar) to the regulation of PAI-2 expression, significant regulation also occurs at the post-transcriptional level, most notably at the level of PAI-2 mRNA stability (16Maurer F. Medcalf R.L. J. Biol. Chem. 1996; 271: 26074-26080Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 17Maurer F. Tierney M. Medcalf R.L. Nucleic Acids Res. 1999; 27: 1664-1673Crossref PubMed Scopus (62) Google Scholar). Post-transcriptional regulation of gene expression has proven to be a particularly important component in the global control of gene regulation. It is now well established that the 3′-untranslated region (3′-UTR) of mRNA is critical in the decision-making process of transcript longevity (18Ross J. Trends Genet. 1996; 12: 171-175Abstract Full Text PDF PubMed Scopus (298) Google Scholar). Most studies have focused on conserved AU-rich elements within the 3′-UTR of unstable cytokine or proto-oncogene transcripts. Typical elements are mostly of the AUUUA or UUAUUUAUU type or variants thereof (19Shaw G. Kamen R. Cell. 1986; 46: 659-667Abstract Full Text PDF PubMed Scopus (3103) Google Scholar, 20Bakheet T. Frevel M. Williams B.R. Greer W. Khabar K.S. Nucleic Acids Res. 2001; 29: 246-254Crossref PubMed Scopus (340) Google Scholar), and are mRNA destabilizing motifs that act, in part, by their ability to attract specialized RNA-binding proteins to elicit the decay process. These same elements have also been linked to other aspects of mRNA metabolism and processing including RNA trafficking (21Snee M. Kidd G.J. Munro T.P. Smith R. J. Cell Sci. 2002; 115: 4661-4669Crossref PubMed Scopus (44) Google Scholar) and translation (22Zhang T. Kruys V. Huez G. Gueydan C. Biochem. Soc. Trans. 2001; 30: 952-958Crossref Google Scholar), indicating that a given AU-rich element has pleiotropic effects. A number of AU-rich element-binding proteins have been identified and shown to interact with these elements in many of these unstable transcripts including AUF-1 (23Zhang W. Wagner B.J. Ehrenman K. Schaefer A.W. DeMaria C.T. Crater D. DeHaven K. Long L. Brewer G. Mol. Cell. Biol. 1993; 13: 7652-7665Crossref PubMed Scopus (491) Google Scholar), AUH (24Nakagawa J. Waldner H. Meyer-Monard S. Hofsteenge J. Jeno P. Moroni C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2051-2055Crossref PubMed Scopus (124) Google Scholar), the ELAV protein family members (25Good P.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4557-4561Crossref PubMed Scopus (271) Google Scholar), and tristetraprolin (TTP) (26Carballo E. Lai W.S. Blackshear P.J. Science. 1998; 281: 1001-1005Crossref PubMed Google Scholar). AUF-1 for example, engages AU-rich elements in a variety of these genes, and the affinity of interaction correlates with the ability of these sequences to serve as mRNA instability elements (27DeMaria C.T. Brewer G. J. Biol. Chem. 1996; 271: 12179-12184Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Members of the ELAV family including HuD, HuC, and HuR (HuA) can also interact with these same elements, but at least for HuR, this interaction is associated more with stabilization rather than destabilization of mRNA (28Levy N.S. Chung S. Furneaux H. Levy A.P. J. Biol. Chem. 1998; 273: 6417-6428Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar, 29Fan X.C. Steitz J.A. EMBO J. 1998; 17: 3448-3460Crossref PubMed Scopus (740) Google Scholar, 30Brennan C.M. Steitz J.A. Cell. Mol. Life Sci. 2001; 58: 266-277Crossref PubMed Scopus (870) Google Scholar). Arguably the most powerful mRNA destabilizing protein identified is TTP. This protein is a critical regulator of TNF-α and GM-CSF mRNA instability (26Carballo E. Lai W.S. Blackshear P.J. Science. 1998; 281: 1001-1005Crossref PubMed Google Scholar, 31Lai W.I Carballo E. Strum J.R. Kennington E.A. Phillips R.S. Blackshear P.J. Mol. Cell. Biol. 1999; 19: 4311-4323Crossref PubMed Scopus (619) Google Scholar, 32Carballo E. Lai W.S. Blackshear P.J. Blood. 2000; 95: 1891-1899Crossref PubMed Google Scholar). This has been decisively born out in TTP −/− mice, which develop a severe inflammatory phenotype because of the increase in cytokine expression resulting from the loss of negative regulation at the post-transcriptional level (26Carballo E. Lai W.S. Blackshear P.J. Science. 1998; 281: 1001-1005Crossref PubMed Google Scholar). TTP is therefore clearly a mRNA destabilizing protein that acts at least in part by promoting deadenylation and by attracting the exosome to the transcript for rapid decay (33Chen C-Y. Gherzi R. Ong S.E. Chan E.L. Raijmakers R. Prujin Ger J.M. Stoecklin G. Moroni C. Mann M. Karin M. Cell. 2001; 107: 451-464Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar). Previous studies have addressed the mechanisms underlying the post-transcriptional regulation of the PAI-2 gene. Two functional mRNA instability elements have been identified within the PAI-2 transcript, one within the 3′-UTR of PAI-2 mRNA (16Maurer F. Medcalf R.L. J. Biol. Chem. 1996; 271: 26074-26080Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 17Maurer F. Tierney M. Medcalf R.L. Nucleic Acids Res. 1999; 27: 1664-1673Crossref PubMed Scopus (62) Google Scholar), and the other within exon 4 of the coding region (34Tierney M. Medcalf R.L. J. Biol. Chem. 2001; 276: 13675-13684Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Closer analysis of the 3′-UTR region of PAI-2 mRNA led to the identification of a nonameric AU-rich element (UUAUUUAUU) (ARE), 304-nt upstream from the poly(A) tail, as a mRNA destabilizing determinant (16Maurer F. Medcalf R.L. J. Biol. Chem. 1996; 271: 26074-26080Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). This element was subsequently shown to provide a binding site for a number of cytoplasmic and nuclear proteins (17Maurer F. Tierney M. Medcalf R.L. Nucleic Acids Res. 1999; 27: 1664-1673Crossref PubMed Scopus (62) Google Scholar), one of which was HuR, although the role of HuR in the post-transcriptional control of PAI-2 remains to be determined. To identify and characterize additional proteins that assemble on the PAI-2 ARE, a human leukocyte cDNA library was screened for PAI-2 AU-rich element-binding proteins using the yeast three-hybrid approach. This screen identified tristetraprolin (TTP) as a specific PAI-2 ARE-binding protein that associates with the PAI-2 AU-rich element and participates in the PAI-2 mRNA decay process. Since TTP is widely considered as a post-transcriptional regulator of cytokine gene expression in response to inflammatory signals our study suggests that the biological targets of TTP are not restricted to cytokines and implicates PAI-2 in a broader context of the inflammatory response associated with TTP. The reporter yeast strain L40-coat and the RNA bait plasmid vectors pIIIA/MS2-1 and pIIIA/MS2-2 (containing selection marker URA3, ADE2) (35SenGupta D.J. Zhang B. Kraemer B. Pochart P. Fields S. Wickens M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8496-8501Crossref PubMed Scopus (435) Google Scholar,36Zhang B. Gallegos M. Puoti A. Durkin E. Fields S. Kimble J. Wickens M.P. Nature. 1997; 390: 477-484Crossref PubMed Scopus (428) Google Scholar) were generous gifts from Dr. Marv Wickens of the University of Wisconsin. The TTP mammalian expression plasmid CMV.hTTPtag (31Lai W.I Carballo E. Strum J.R. Kennington E.A. Phillips R.S. Blackshear P.J. Mol. Cell. Biol. 1999; 19: 4311-4323Crossref PubMed Scopus (619) Google Scholar) (hereafter referred to as pTTP-HA) was provided by Dr. Paul Bohjanen (University of Minnesota). The wild-type PAI-2 expression plasmid pCI-PAI-2 contains the 1872-bp PAI-2 cDNA (9Schleuning W-D. Medcalf R.L. Hession C. Rothenbühler R. Kruithof E.K.O. Mol. Cell. Biol. 1987; 7: 4564-4567Crossref PubMed Scopus (98) Google Scholar) inserted into theEcoR1 site of the expression plasmid pCl-neo (Promega, Madison, WI) (34Tierney M. Medcalf R.L. J. Biol. Chem. 2001; 276: 13675-13684Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The RNA bait plasmids were constructed as follows. The 5′-phosphorylated oligonucleotides of the ARE region (29 bp) of the human PAI-2 gene, were synthesized by Sigma Genosys: 5′-TTTTACTTTGTTATTTATTATTTTATATA-3′ and 5′-TATATAAAATAATAAATAACAAAGTAAAA-3′. After annealing, the double-stranded ARE oligonucleotide was inserted into theSmaI site of the pIIIA/MS2-2 and pIIIA/MS2-1, creating ARE/MS2-2 and ARE/MS2-1, respectively (36Zhang B. Gallegos M. Puoti A. Durkin E. Fields S. Kimble J. Wickens M.P. Nature. 1997; 390: 477-484Crossref PubMed Scopus (428) Google Scholar). The insertion and orientation of the ARE in the plasmids was confirmed by PCR and sequence analysis (sequencing primer: 5′-CTGTCTCTATACTCCCCTATAG-3′). Plasmid pCMV-glo (hereafter referred to as pCI-glo), contains the rabbit β-globin gene driven by the CMV promoter (37Nanbu R. Menoud P-A. Nagamine Y. Mol. Cell. Biol. 1994; 14: 4920-4928Crossref PubMed Google Scholar). Plasmid pCI-glo-PAI-2 is the same as pCI-glo, but contains the entire 3′-UTR of the PAI-2 mRNA inserted into the 3′-UTR of the globin reporter gene (16Maurer F. Medcalf R.L. J. Biol. Chem. 1996; 271: 26074-26080Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Plasmid pCI-glo-ARE and pCI glo-2ARE are the same as pCI-glo, but contain either a single copy or two copies of the 29-nt PAI-2 ARE inserted into the 3′-UTR of the globin reporter gene, respectively. To prepare these plasmids, complementary oligonucleotides containing the single (29 nt) or tandem (58 nt) copy of the PAI-2 AU-rich element were synthesized. These oligonucleotides also included restriction sites for XhoI at each end to enable subcloning into the XhoI site of pCI-glo. Oligonucleotides were annealed, digested with XhoI, and inserted into theXhoI site of pCI-glo using standard techniques. The yeast three-hybrid screening procedures were performed essentially as described elsewhere (35SenGupta D.J. Zhang B. Kraemer B. Pochart P. Fields S. Wickens M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8496-8501Crossref PubMed Scopus (435) Google Scholar, 38Park Y.W. Tan S.L. Katze M.G. BioTechniques. 1999; 26: 1102-1106Crossref PubMed Scopus (15) Google Scholar, 39Bernstein D.S. Buter N. Stumpf C. Wickens M. Methods. 2002; 26: 123-141Crossref PubMed Scopus (113) Google Scholar). The plasmids pIIIA/ARE-MS2-2 and pIIIA/ARE-MS2-1 were introduced into yeast L40-coat by transformation. A human leukocyte cell cDNA library (250 μg; Clontech) was transformed into L40/pIIIA/ARE-MS2-2 and L40/pIIIA/ARE-MS2-1. Double transformants (Trp+, Leu+, Ura+) were then plated onto Trp−, Leu−, His−, Ura− plates containing 5 mm 3-aminotriazole (3-AT). As a negative selection procedure, candidate yeast clones were transferred onto plates containing 0.1% 5-fluoro-orotic acid (5-FOA) to eliminate the majority of RNA-independent false positive colonies (38Park Y.W. Tan S.L. Katze M.G. BioTechniques. 1999; 26: 1102-1106Crossref PubMed Scopus (15) Google Scholar). The same yeast clones were also transferred onto Trp−, Leu−, His− Ura− plates containing 5 mm 3-AT (“master plates”). The 5-FOA-sensitive colonies identified on the master plates were subsequently patched onto non-selective plates (Trp−, Leu−) to select for colonies in which the RNA plasmid had been removed (“RNA-dropout colonies”): After 3–4 days growth, single yeast colonies that had the RNA plasmid dropped out (pink color) were spotted back onto Leu− Trp− plates containing 0.1% 5-FOA to confirm that the RNA plasmid had been removed and were able to grow. In parallel, yeast were also transferred to Leu−, His− plates containing 5 mm 3-AT to retest for the RNA-dependent activation of HIS3. In order to test candidate colonies for RNA sequence-specific activation of HIS3, the RNA dropout colonies expressing the putative RNA-binding protein cDNA, were retransformed with four different RNA plasmids: ARE/MS2−2, ARE/MS2−1, PAI-2 exon 4/MS2−2 as an unrelated RNA bait, and RNA bait vector pIIIA/MS2-2. After transformation, the single yeast colonies were picked and spotted onto Ura−, Trp−, Leu− plates to confirm the success of transformation and also onto Trp−, Ura−, Leu−, His− plates containing 5 mm3-AT to test for RNA sequence-specific activation of HIS3. Positive colonies should grow only with RNA bait plasmids containing the PAI-2 ARE sequence, but not with baits containing irrelevant RNA sequence or empty RNA bait vector alone. cDNA plasmids from positive clones were isolated and subjected to DNA sequencing analysis (GAL4 AD sequencing primer: 5′-TACCACTACAATGGAT-3′). The cDNA sequences were then BLAST-searched for gene homology with the GenBankTM databases at NCBI. HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal calf serum, 2 mm glutamine, and 1× penicillin and streptomycin at 37 °C under 5% CO2. HEK293 cells were transiently co-transfected with the PAI-2 expression plasmid pCI-PAI-2 (17Maurer F. Tierney M. Medcalf R.L. Nucleic Acids Res. 1999; 27: 1664-1673Crossref PubMed Scopus (62) Google Scholar) and the TTP expression plasmid pTTP-HA, or globin/PAI-2 3′-UTR and TTP expression plasmids, by the calcium phosphate method (40Costa M. Shen Y. Maurer F. Medcalf R.L. Eur. J. Biochem. 1998; 258: 123-131Crossref PubMed Scopus (44) Google Scholar), except that the transfection mixture was allowed to incubate with the cells for 16–24 h, and the glycerol shock was omitted (31Lai W.I Carballo E. Strum J.R. Kennington E.A. Phillips R.S. Blackshear P.J. Mol. Cell. Biol. 1999; 19: 4311-4323Crossref PubMed Scopus (619) Google Scholar). Fresh DMEM medium containing 10% heat-inactivated fetal calf serum was then added and cells maintained for a further 24 h. Total RNA was extracted for Northern blotting as described below. Bluescript plasmids harboring the 29-nt PAI-2 AU-rich element (17Maurer F. Tierney M. Medcalf R.L. Nucleic Acids Res. 1999; 27: 1664-1673Crossref PubMed Scopus (62) Google Scholar) were used to transcribe the PAI-2 ARE RNA probes in vitro. Following linearization withXbaI, 1 μg of template was incubated for 2 h at 37 °C in the presence of 50 μCi of [α-32P]UTP (PerkinElmer Life Sciences), 10 μm UTP, 0.5 mm ATP, 0.5 mm GTP, 0.5 mm CTP, 20 units of RNase inhibitor (Promega) and 50 units of T7 RNA polymerase. RNA probes were purified on a 6% polyacrylamide-urea denaturing gel, eluted in a 500 mm NH4CH3COO, 1 mm EDTA solution for 6 h at room temperature, ethanol precipitated at −80 °C, and resuspended in water (500–1000 cps/μl) as previously described (16Maurer F. Medcalf R.L. J. Biol. Chem. 1996; 271: 26074-26080Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). To prepare protein extracts for the UV cross-linking and immunoprecipitation, confluent cells were collected by trypsinization, washed three times with phosphate-buffered saline, then lysed for 5 min on ice in 100 μl/106 cells of cytoplasmic extraction buffer (CEB; 10 mm HEPES, pH 7.1, 3 mmMgCl2, 14 mm KCl, 0.2% Nonidet P-40, 5% glycerol, 1 mm dithiothreitol, 2 μg/ml aprotinin, 0.5 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 1 mm sodium orthovanadate, and 1 mm NaF). The nuclei were pelleted for 1 min at 1000 × g at 4 °C, and the supernatant containing the cytosolic fraction was aliquoted, snap frozen in liquid nitrogen, and stored at −80 °C. Nuclear protein extracts were prepared from isolated nuclei as previously described (41Costa M. Medcalf R.L. Eur. J. Biochem. 1996; 237: 532-538Crossref PubMed Scopus (30) Google Scholar). Protein concentration of cell extracts was determined by the Bio-Rad protein dye reagent. For the binding assays, 4 μg of protein extract were preincubated with 150 μg of heparin and 40 units of RNase inhibitor (RNasin;Promega) for 10 min at room temperature in CEB buffer before addition of the RNA probe (200 cps) in a total volume of 20 μl. After a further 30 min incubation at room temperature, samples were transferred to a 96-well microplate, which was placed on ice and irradiated for 15 min at a distance of 5 cm from a UV source (Ultra LUM model UVB-20). RNA not associated with protein was digested with 100 units of RNase T1 (Roche Molecular Biochemicals) for 20 min at room temperature. In some experiments the RNase T1-resistant RNA-protein complexes were further digested with 1 μg of RNase A (Roche Molecular Biochemicals) at 37 °C for 15 min. The samples were diluted to 0.4 ml in CEB buffer, and protein A-Sepharose (Amersham Biosciences) added (total volume of 0.45 ml). These were incubated overnight at 4 °C in the presence of either preimmune rabbit serum (1:150 dilution) or crude polyclonal rabbit anti-HA (HA.11) antiserum (1:150 dilution) (BabCO, Richmond, CA). Immune complexes were recovered by centrifugation, washed three times with CEB buffer, resuspended in 50 μl of 2× SDS sample buffer, and subjected to SDS-PAGE on 10% acrylamide gels and autoradiography. Total RNA was purified from transfected cells as described by Chomczynski and Sacchi (42Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62909) Google Scholar). 20-μg aliquots of RNA were electrophoresed through 1% agarose gels containing 20% formaldehyde and subsequently transferred to Hybond-N+ membranes (Amersham Biosciences). The filters were hybridized with32P-labeled DNA probes as described (17Maurer F. Tierney M. Medcalf R.L. Nucleic Acids Res. 1999; 27: 1664-1673Crossref PubMed Scopus (62) Google Scholar). Membranes were processed by standard techniques and exposed to Kodak BioMax film (Eastman Kodak) at −80 °C with two intensifying screens. The labeled cDNA probes used for hybridization were obtained as follows: 752 bp of the exon 1–6 PCR-amplified fragment of PAI-2 cDNA (9Schleuning W-D. Medcalf R.L. Hession C. Rothenbühler R. Kruithof E.K.O. Mol. Cell. Biol. 1987; 7: 4564-4567Crossref PubMed Scopus (98) Google Scholar); the 1.1-kb HindIII TTP cDNA fragment of pTTP-HA (CMV.hTTPtag (31Lai W.I Carballo E. Strum J.R. Kennington E.A. Phillips R.S. Blackshear P.J. Mol. Cell. Biol. 1999; 19: 4311-4323Crossref PubMed Scopus (619) Google Scholar)); and the 380-bpHindIII/BamHI fragment of pCMV-glo containing the globin cDNA fragment (37Nanbu R. Menoud P-A. Nagamine Y. Mol. Cell. Biol. 1994; 14: 4920-4928Crossref PubMed Google Scholar). Western blotting was performed as previously described (34Tierney M. Medcalf R.L. J. Biol. Chem. 2001; 276: 13675-13684Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Cytoplasmic extracts (up to 50 μg) prepared from cells were separated by SDS-PAGE under reducing conditions and transferred to nylon membranes. Membranes were hybridized with antibodies directed against TTP (1:1000 dilution; kind gift from Dr. Andrew Clark) or PAI-2 (1:10,000 dilution; American Diagnostica). Secondary antibodies coupled to horseradish peroxidase were added (1:2,000 dilution) and the immunocomplexes assessed by enhanced chemiluminescence. The yeast three-hybrid approach was used to screen a human leukocyte cDNA library for proteins that interacted with the functional 29-nt AU-rich element within the 3′-UTR of PAI-2 mRNA. This genetic approach yielded seven positive clones that interacted with the PAI-2 AU-rich RNA bait in both an RNA-dependent and RNA sequence-dependent manner. Sequence analysis revealed that three of seven clones were identical, and all clones contained partial cDNA sequences (1399–1436 bp) that were homologous to human TTP (Fig. 1). The 5′-end point of the clones ended between −310 and −354 of the TTP cDNA. All clones had the identical 3′ structure, with the exception of one clone (clone 5) that contained a 43-bp deletion at the terminus of the 3′-UTR (Fig. 1). TTP contains 326 amino acids with three tetraprolin repeats and two zinc fingers of the 2 Cys-Cys-Cys-His class. All clones contained the two zinc finger domains and two of the three tetraprolin domains suggesting the functional importance of these domains for PAI-2 AU-rich RNA binding activity. To confirm that TTP could directly interact with the PAI-2 ARE, RNA probes containing a single copy of the PAI-2 AU-rich element were incubated with cytoplasmic extracts prepared from either mock-transfected HEK293 cells, or cells transiently transfected with 5 μg of plasmid pTTP-HA. Samples were UV cross-linked then immunoprecipitated with anti-HA antibodies or preimmune serum, and subjected to SDS-PAGE (Fig.2, lanes 1–4). Replicate samples were digested with RNase T1 either alone (lanes 5–8) or in combination with RNase A (lanes 9–12) prior to immunoprecipitation. As shown, extracts prepared from cells transfected with 5.0 μg of pTTP-HA did not produce an immunoprecipitable complex in the presence of preimmune serum (lane 2). However, protein-RNA complexes were detected when samples were immunoprecipitated with the anti-HA antibodies (lane 4). Following RNase T1 digestion, two complexes of ∼120 and 55 kDa still remained (lane 8). When samples were digested with both RNase T1 and RNase A only a single complex of molecular mass 52 kDa was present (lane 12), although the 120-kDa complex was still present after longer exposure of gel to x-ray film. The 120-kDa complex most likely represents TTP associating with another protein and is more sensitive to RNase A possibly due to conformational changes. The slight reduction in molecular mass of the HA-containing RNA complex from 55 to 52 kDa is consistent with the further trimming of the RNA probe from the HA-tagged TTP protein by RNase A. Extracts derived from mock-transfected cells did not contain any HA-containing immune complexes. Taken together, these data indicate that TTP expressed in cells recognizes the AU-rich element in the PAI-2 3′-UTR. It was important to determine whether TTP could influence the post-transcriptional regulation of the PAI-2 gene. TTP has previously been shown to influence cytokine gene expression by associating with AU-rich elements and accelerating the decay rate of the cytokine transcript. To determine whether TTP could act in a similar manner on PAI-2 mRNA, plasmids expressing PAI-2 (pCI-PAI-2) and TTP (pTTP-HA) were co-transfected into HEK293 cells. In this experiment, cells were transfected with a fixed amount of pCI-PAI-2 (5 μg) and increasing amounts (0, 0.005, 0.01, 0.05, 0.1, 1.0, 5.0 μg) of plasmid pTTP-HA. As shown in Fig. 3panel A, cells transfected with pCI-PAI-2 expressed a PAI-2 transcript (lane 2, middle blot). However, the intensity of this transcript decreased as the expression levels of TTP increased (lanes 3–9). It is also evident that levels of PAI-2 mRNA were significantly reduced at relatively low levels of TTP mRNA expression (e.g. lane 6). Furthermore, higher concentrations of TTP resulted in the formation of a stable but truncated PAI-2 transcript (lanes 7–9; indicated witharrow). The significance of the formation of this truncated PAI-2 transcript is unclear, but it is unlikely to be of physiological significance" @default.
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• W2159764578 date "2003-04-01" @default.
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• W2159764578 title "Inherent Instability of Plasminogen Activator Inhibitor Type 2 mRNA Is Regulated by Tristetraprolin" @default.
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• W2159764578 doi "https://doi.org/10.1074/jbc.m213027200" @default.
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