3′-Untranslated Region of Phosphoenolpyruvate Carboxykinase mRNA Contains Multiple Instability Elements That Bind AUF1

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) 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 constructs expressed from a binding assays demonstrated that AUF1 binds to of the segments that Mutational analyses identified specific within the PCK-6 and PCK-7 segments that The demonstrated that multiple AUF1 binding contribute to the rapid turnover of PEPCK mRNA. rats were from and were from The was from RNase RNA and were from or were from and the was from and for were from was from RNA was from was from RNase H was from and were from was from was a from was from and were from was from expression that the of AUF1 was from were from of and of various segments of the 3′-UTR of the PEPCK mRNA that were used in this are in The and and were 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 was by two and which was previously with and The of the and were and The the and The was by the with and The PCK-7 RNA was and the and were that was previously with and The and were by the with and and were also by and The for the was by the with and whereas the and were by of the with and of were with modifications of the of Y. Mol. Cell. Biol. PubMed Google Scholar). The were for the RNA gel shift following RNase were to a and to for 5 in a The were to and an of containing and was The were in for 5 and a The gel was and to a In transcription of and was 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 were using The were quantified by and was to the to the The of the was at and the of the was using from the RNA of rat renal 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 of protein J. Biol. Chem. Full Text PDF PubMed Google Scholar) or of AUF1 was for at in binding containing 1 of of and of A or of RNA was with the The was at for and 1 of RNase was and the were for at The were and the were and to a In the the various were with the rat renal cytosolic extract for at and to the binding of promoter system N. PubMed Scopus Google Scholar) was to the segments within the 3′-UTR of the PEPCK mRNA that A chimeric hormone gene was the to contains a the of β-globin (βG) and the and of bovine growth hormone The 3′-UTR of the rat PEPCK to was using and that and at the and The sequence of the was whereas that of the was the are the and The sequence was the of The was with and to a that was subsequently at the to was with and to a The was and to The was and that was previously with to containing the PCK-6, PCK-7, and (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 with and and in that was previously with to and Thus, of the chimeric βG-PCK mRNAs the 3′-UTR that is in the control βG-PCK mRNA. of cells were from and previously (8.Gstraunthaler G. Handler J.S. Am. J. Physiol. 1987; 252: C232-C238Crossref PubMed Google Scholar). that the and constructs were made by Mol. Cell. Biol. 1987; 7: PubMed Scopus Google Scholar) of the and a line of LLC-PK1-F+ cells that the protein. M. and N. h the was the cells were with and containing was h and containing and was individual were and the cells were the was to The were for by the cells for h in in the presence or absence of RNA was from the cells and a cells the βG-PCK-1 mRNA were in and to containing for h. level of was to to transcription from the The cells were two with and for h in to a of βG-PCK-1 mRNA. 1 was to RNA were at various following of and to analysis to the decay of the βG-PCK-1 mRNA. the various LLC-PK1-F+ cells were for in time 1 was to and h RNA was and to RNase H H treatment Curthoys N.P. Biochem. PubMed Scopus Google Scholar) was to the of the chimeric βG-PCK-1 mRNAs that were from the The that the of the PEPCK from was by A of was used to the of the mRNA. The RNA were from by the of to and of 5 at the RNA was by at for The were in of The was in a of containing of of RNase and of the with or of of The was at for and of RNase H and to was and the was at for of RNase H to 1 with RNase H was and the was at for of of of MOPS, and of was to the The was at for 5 and The RNase were to analysis previously (11.Hansen W.R. Barsic-Tress N. Taylor L. Curthoys N.P. Am. J. Physiol. 1996; 271: F126-F131PubMed Google Scholar), that of a RNase H gel 1 and of was to the and a gel containing MOPS, 1 and 1 was The was with the RNA was using the and the RNA was by the at A of β-globin was by R. PubMed Scopus Google Scholar) with and A of the RNA from was by PubMed Scopus Google Scholar) with and A was from with The were a and using the The of and analysis were previously (11.Hansen W.R. Barsic-Tress N. Taylor L. Curthoys N.P. Am. J. Physiol. 1996; 271: F126-F131PubMed Google Scholar). The were to a and the of the of was quantified using The level of the chimeric β-globin mRNA was by the level of to for in half-life the of were the time the of The are the of from The line the of the was by a that its of βG-PCK-1 promoter was used to determine the half-life of the βG-PCK-1 mRNA. cells were in the presence of to of the βG-PCK-1 mRNA. A was by from the for h and was subsequently by 1 to transcription of the mRNA. RNA were from the cells at various following of the and the levels of βG-PCK-1 mRNA and were quantified by analysis analysis demonstrated that degradation of βG-PCK-1 mRNA is initiated a and with a rapid half-life (t½ = 1.2 h). of βG-PCK-1 degradation of mRNAs is usually initiated by the binding of specific to within the 3′-UTR R. Mol. Biol. PubMed Scopus Google Scholar, Biochem. Full Text PDF PubMed Scopus Google Scholar). The subsequently a M. J. Mol. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) and the R. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar) to the and a rapid RNase H treatment of the from the βG-PCK-1 mRNA half-life analysis was to determine deadenylation the decay of the PEPCK mRNA. of the RNA the with a and by treatment with RNase produced a to the of the 3′-end of the βG-PCK-1 mRNA. of the same RNA in the presence of only the produced containing Thus, the βG-PCK-1 mRNA a treatment of from the time demonstrated that the βG-PCK-1 mRNA a rapid deadenylation that concomitant with the decay of the mRNA. of the within the 3′-UTR of half-life analyses were using cells in the absence of to expression of the chimeric PEPCK decay of the mRNA was subsequently initiated by the of 1 analysis was using from cells that the parent analysis that the control βG mRNA is extremely stable and with a half-life of analysis also indicated that the βG-PCK-1 mRNA was degraded with a half-life of h, with the Previous RNA gel shift analysis that a protein in cytosolic of rat kidney cortex binds to PCK-7, a 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). the PCK-7 segment was to cause the rapid of the PEPCK mRNA, the turnover of the βG-PCK-7 mRNA was analyses that the βG-PCK-7 mRNA was degraded with a half-life of 17 h Therefore, of the PCK-7 segment only the chimeric mRNA and was not to the rapid turnover with βG-PCK-1 mRNA. Thus, within the 3′-UTR may to the complete of the PEPCK mRNA. analysis of the of and PEPCK mRNAs the presence of a AU sequence that is of the PCK-7 segment. sequence is of a segment 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 the parent to LLC-PK1-F+ cells were with this and half-life analyses were The mRNA decayed with a half-life of only h determine the PCK-6 and PCK-7 segments cells that were to half-life analysis demonstrated that the βG-PCK-6/7 mRNA decayed with a half-life of 3.6 h half-life was that with βG-PCK-7 or the mRNA but was that with the segment. Therefore, within the 3′-UTR may to the complete of the PEPCK mRNA. this cells constructs containing segments of the 3′-UTR of the PEPCK mRNA were The segment contains from the 3′-end of the PEPCK mRNA that the segment analysis using from cells the βG-PCK-3 mRNA established that this mRNA decayed with a half-life of 3.6 h, the same with the βG-PCK-6/7 mRNA Thus, the segment contains of the elements within the 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 Previous RNA gel shift assays with PCK-2 RNA and rat renal cytosolic to the of a (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, expressed in the porcine kidney decayed with a half-life of 5.4 h Thus, the half-life analyses of the PCK-6, PCK-7, and PCK-2 constructs that the 3′-UTR of the PEPCK mRNA contains multiple elements that contribute to the rapid turnover that is with the chimeric mRNA. AUF1 within the 3′-UTR of the PEPCK analysis was to the within rat renal cytosolic that within the segment. with that from to were to with the RNA of were and PCK-7 were with the renal cytosolic extract and to not to of this and were used in gel of the cytosolic extract with of the PCK-7 In and two J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), or an protein Mol. PubMed Scopus Google Scholar), A was the PCK-6, and were with not the AUF1 binding interactions, gel shift assays were using various RNA The of AUF1 high affinity and specific binding to the RNA The AUF1 protein also with the PCK-6 and PCK-7 the PCK-2 RNA a with However, AUF1 to a with a RNA from not A analysis was to the of AUF1 binding to the various The interaction with RNA was by the of of or PCK-7 but not by the of a RNA from A demonstrated the of AUF1 binding to the A of not an the binding AUF1 binds to the PCK-2 RNA the binding is also specific Therefore, AUF1 specific binding to segment of the PEPCK 3′-UTR that contains an Mutational analyses were to the specific within the RNA that PCK-6 contains a UUAUUUUAU sequence that is to the AU-rich in mRNA that binds AUF1 Y. G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). this sequence in PCK-6 RNA was by multiple binding of AUF1 was of AUF1 to PCK-6 and The PCK-6 and were in the absence and presence of The were with RNase to the a and a PCK-7 contains a of an sequence by a a structure containing an and an and a in which of the are A and the AUF1 binding the PCK-7 RNA was contains the RNA of the AU-rich and RNA the stem-loop with a rat renal cytosolic the and not the a not the AUF1 binds to only the and Therefore, the the stem-loop structure AUF1 binding of the PCK-7 PCK-7 PCK-7 and PCK-7 were to binding to the individual or A PCK-7 was to binding to the sequence that the of the stem-loop AUF1 binds to PCK-7 RNA but not to of the constructs Therefore, within the PCK-7 AUF1 binds to the to the or to both Previous using DRB, a specific of RNA polymerase to the half-life of the βG-PCK-1 mRNA in LLC-PK1-F+ cells demonstrated that the 3′-UTR of the PEPCK mRNA contains a destabilizing (11.Hansen W.R. Barsic-Tress N. Taylor L. Curthoys N.P. Am. J. Physiol. 1996; 271: F126-F131PubMed Google Scholar). In the study, a promoter system was used to quantify the in decay rates and to the of multiple elements within various chimeric βG-PCK this the half-life for the βG-PCK-1 mRNA was that in the transcription of polymerase II it may the of that in the of mRNA turnover. In with the the transcription of the gene of is inhibited by the of a level of Therefore, the half-life of the βG-PCK-1 mRNA of h is a of its turnover in LLC-PK1-F+ cells. The analysis with multiple βG-PCK constructs that the PEPCK mRNA contains multiple elements that are within the PCK-6, and PCK-7 segments of the are the important in the levels of PEPCK mRNA that in response to various and to changes in are made feasible by the rapid decay of the PEPCK mRNA. The decay of mRNAs that AU-rich elements is by the of the from the 3′-end of the mRNA N. Mol. Cell. Biol. PubMed Scopus Google Scholar). in a or an deadenylation in the of a containing the mRNA N. Mol. Cell. Biol. PubMed Scopus Google Scholar), this is the decay of the of the mRNA is In deadenylation of the factor mRNA N. Mol. Cell. Biol. PubMed Scopus Google Scholar) in the of a mRNA that is rapidly The PEPCK mRNA a or but contains a sequence within the PCK-6 segment of its RNase H assays indicated that the βG-PCK-1 mRNA a rapid and deadenylation that is complete within h However, of the βG-PCK-1 mRNA was degraded within h. Therefore, the degradation of the βG-PCK-1 mRNA may by a in which a of the chimeric mRNA a rapid deadenylation that is complete within whereas the a The the βG-PCK mRNAs produced only a low level of the chimeric mRNA during a a it was to quantify the decay of the mRNAs using the Therefore, the half-lives of the various βG-PCK mRNAs containing segments of the 3′-UTR of the PEPCK mRNA were by to cells that were in the absence of A with this high levels of expression from the promoter the decay and cause an decay of the mRNA N. PubMed Scopus Google Scholar). However, expression of the βG-PCK-1 mRNA that of the and the half-lives of the βG-PCK-1 mRNA by the two were the half-lives for the βG-PCK constructs the decay rates of the the it was not to the of deadenylation of the various constructs of the βG-PCK-1 mRNA. Previous studies have that cytosolic of rat hepatoma cells (6.Hod Y. Hanson R.W. J. Biol. Chem. 1988; 263: 7747-7752Abstract Full Text PDF PubMed Google Scholar) and T. Biochem. Biophys. 1991; PubMed Scopus Google Scholar) that to multiple segments of the 3′-UTR of PEPCK mRNA. the binding were by of the cells with cAMP and the of the the binding elements The of affinity the protein in rat liver that binds to the 3′-UTR of PEPCK mRNA T. Biochem. Biophys. 1991; PubMed Scopus Google Scholar). However, the physiological of this interaction to In the study, AUF1, a was shown to with high affinity and to PCK-6, and PCK-7, the same segments that contribute to the rapid turnover of the PEPCK mRNA. AUF1 binds to the of G. RNA in Scholar), J. Y. Y. G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), G. R. Cell. Full Text PDF PubMed Scopus Google Scholar), and M. G. J. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). AUF1 was also identified Y. G. PubMed Scopus Google Scholar), a protein that the and the G. S. Biochem. 1993; PubMed Scopus Google Scholar). of AUF1 and are produced by of the AUF1 Y. G. PubMed Scopus Google Scholar). The AUF1 is was in the AUF1 has also shown to RNA in cells G. M. R. Mol. Cell. Biol. PubMed Scopus Google Scholar) and it in cells N. PubMed Scopus Google Scholar). Thus, its mRNA stability the AU-rich and the expressed in a line N. Mol. Cell. Biol. PubMed Scopus Google Scholar). of by AUF1 the RNA is for the of AU-rich mRNA J. Biol. Chem. PubMed Scopus Google Scholar) and W. L. G. Mol. Cell. Biol. 1993; PubMed Scopus Google Scholar) have shown that AUF1 with a of and an activity in a role of in decay J. Biol. Chem. PubMed Scopus Google Scholar). the the factor the protein and the protein also with AUF1 G. R. G. PubMed Scopus Google Scholar) and may its Thus, it to determine of are also in decay of the PEPCK mRNA. The high affinity protein binding with rat renal to the PCK-7 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). Thus, it was to that the PCK-7 segment not a destabilizing with the Inclusion of the adjacent PCK-6 segment, which contains a UUAUUUUAU in a decay with that with the Thus, binding of AUF1 at both may to the that the rapid decay of the PEPCK mRNA. is by analysis of the 3′-UTR with rat renal analysis the presence of multiple that renal binding to this However, analyses using and not to in the of binding in PEPCK mRNA turnover is Previous studies using a rat renal extract to a with the PCK-2 RNA that the 5′-end of the 3′-UTR of 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). However, this segment also binds AUF1 and a destabilizing the β-globin mRNA. analysis the presence of a of and the 5′-end of also contains a sequence that is in the and PEPCK mRNAs AUF1 within this However, the affinity of the AUF1 binding with this segment is with that with the region. Thus, protein may to this segment and participate in the turnover of the PEPCK mRNA. analysis of the and PEPCK mRNAs identified two AU-rich within the segments of their The is a sequence that is in the PCK-6 segment. Mutational analysis that AUF1 binds to the AU-rich within this sequence The is a sequence that is within the PCK-7 segment. The latter sequence the entire of a stable stem-loop The that AUF1 not to the PCK-7 RNA that it also the sequence that the of the stable stem-loop The sequence of the PCK-7 RNA was using to that the in the sequence the ability to the stem-loop structure the of an The PCK-7 also to a with The PCK-7 and constructs in the and in both of the However, the two also the ability of the to a stable stem-loop Thus, the to to may indicate that the stem-loop structure is also for AUF1 binding to the sequence that the However, the PCK-7 is to the same stem-loop that the are also required for binding of the is the that AUF1 binding may by an adjacent The of this demonstrated that are at destabilizing elements within the 3′-UTR of the PEPCK mRNA. The destabilizing elements are within the of the 3′-UTR, whereas a within the of the the the following for PEPCK mRNA degradation AUF1 and various may and to the and within the segment. AUF1 and may also to the sequence or the of and within the PCK-2 segment. The latter binding may or may by binding of AUF1 to the the of the AUF1 may the and the which subsequently the PEPCK mRNA in the