Role of deadenylation and AUF1 binding in the pH-responsive stabilization of glutaminase mRNA

Abstract

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 ζ-crystallin and functions as a pH response element (pH-RE). A tetracycline-responsive promoter system was developed in LLC-PK1-F+cells to perform pulse-chase analysis of the turnover of a chimeric β-globin (βG) mRNA that contains 960 bp of the 3′-UTR of GA mRNA including the pH-RE. The βG-GA mRNA exhibits a 14-fold increase in half-life when the LLC-PK1-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 βG-GA mRNA in cells grown in normal medium. Stabilization of the βG-GA mRNA in acidic medium is associated with a pronounced decrease in the rate of deadenylation. Mutation of the pH-RE within the βG-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 ζ-crystallin during acidosis may inhibit degradation and result in selective stabilization.