Role of deadenylation and AUF1 binding in the pH-responsive stabilization of glutaminase mRNA.
During chronic metabolic acidosis, increased expression of renal glutaminase (GA) results from selective stabilization of the GA mRNA. This response is mediated by a direct repeat of an 8-base adenylate-uridylate (AU) sequence that binds zeta-crystallin and functions as a pH response element (pH-RE). A tetracycline-responsive promoter system was developed in LLC-PK(1)-F(+) cells to perform pulse-chase analysis of the turnover of a chimeric beta-globin (betaG) mRNA that contains 960 bp of the 3'-UTR of GA mRNA including the pH-RE. The betaG-GA mRNA exhibits a 14-fold increase in half-life when the LLC-PK(1)-F(+) cells are transferred to acidic medium. RNase H cleavage and Northern blot analysis of the 3'-ends established that rapid deadenylation occurred concomitantly with the rapid decay of the betaG-GA mRNA in cells grown in normal medium. Stabilization of the betaG-GA mRNA in acidic medium is associated with a pronounced decrease in the rate of deadenylation. Mutation of the pH-RE within the betaG-GA mRNA blocked the pH-responsive stabilization, but not the rapid decay, whereas insertion of only a 29-bp segment containing the pH-RE was sufficient to produce both a rapid decay and a pH-responsive stabilization. Various kidney cells express multiple isoforms of AUF1, an AU-binding protein that enhances mRNA turnover. RNA gel-shift assays demonstrated that the recombinant p40 isoform of AUF1 binds to the pH-RE with high affinity and specificity. Thus AUF1 may mediate the rapid turnover of the GA mRNA, whereas increased binding of zeta-crystallin during acidosis may inhibit degradation and result in selective stabilization.
Schroeder
Jill M
JM
Department of Biochemistry and Molecular Biology, Colorado State University, Campus Delivery 1870, Fort Collins, CO 80523-1870, USA.
Ibrahim
Hend
H
Taylor
Lynn
L
Curthoys
Norman P
NP
D000249:Adenosine Monophosphate / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D003600:Cytosol / Q000502:physiology; D005839:Gentamicins / Q000494:pharmacology; D005972:Glutaminase / Q000235:genetics*; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006921:Hygromycin B / Q000494:pharmacology; D007672:Kidney Cortex / Q000502:physiology*; D008297:Male; D016296:Mutagenesis; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016914:Ribonuclease H / Q000378:metabolism; D038204:beta-Crystallins / Q000378:metabolism
D005839:Gentamicins; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; C000630988:Hnrnpd protein, rat; D012333:RNA, Messenger; D038204:beta-Crystallins; D006921:Hygromycin B; D000249:Adenosine Monophosphate; C010680:antibiotic G 418; D016914:Ribonuclease H; D005972:Glutaminase