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

Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado

Submitted 14 June 2005 ; accepted in final form 11 October 2005

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 -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 -globin (G) mRNA that contains 960 bp of the 3'-UTR of GA mRNA including the pH-RE. The G-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 G-GA mRNA in cells grown in normal medium. Stabilization of the G-GA mRNA in acidic medium is associated with a pronounced decrease in the rate of deadenylation. Mutation of the pH-RE within the G-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 -crystallin during acidosis may inhibit degradation and result in selective stabilization.

renal ammoniagenesis; metabolic acidosis; posttranscriptional regulation

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