title: 3'-Untranslated region of phosphoenolpyruvate carboxykinase mRNA contains multiple instability elements that bind AUF1.
abstract: 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 beta-globin-PEPCK (betaG-PCK) mRNAs in LLC-PK -F(+) cells. The control betaG mRNA was extremely stable (t(1/2) = 5 days). However, betaG-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 betaG-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 betaG-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 betaG-PCK-6/7 mRNA, which has a half-life of 3.6 h. The betaG-PCK-3 mRNA that contains the 3'-half of 3'-UTR was degraded with the same half-life. Surprisingly, the betaG-PCK-2 mRNA, containing the 5'-end of the 3'-UTR, was also degraded rapidly (t((1/2)) = 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870, USA.
authors.forename: Sachin
authors.initials: S
authors.lastname: Hajarnis
authors.forename: Jill M
authors.initials: JM
authors.lastname: Schroeder
authors.forename: Norman P
authors.initials: NP
authors.lastname: Curthoys
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D004795:Enzyme Stability; D006207:Half-Life; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015183:Restriction Mapping; D016415:Sequence Alignment; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid
chemical_list: D020413:3' Untranslated Regions; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; C000630988:Hnrnpd protein, rat; D012333:RNA, Messenger; D019764:Phosphoenolpyruvate Carboxykinase (ATP)