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 β-globin-PEPCK (βG-PCK) mRNAs in LLC-PK1-F+ cells. The control βG mRNA was extremely stable (t1/2 = 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 (t1/2 = 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. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.