Characterization of GA mRNA turnover using an in vitro degradation assay

Abstract

During metabolic acidosis, the level of renal glutaminase (GA) is significantly increased. This adaptation contributes to the increased synthesis of NH4+ and HCO3− ions from glutamine to partially restore acid-base balance. The increased expression results from an increased stability of the GA mRNA that is mediated by two 8-base AU-sequences that function as a pH-response element (pHRE). This sequence binds ζ-crystallin/NADPH:quinone reductase (ζ-cryst) and AUF1 with high affinity and specificity. An in vitro mRNA decay assay was developed using a cytosolic extract of WKPT cells, an established line of rat renal proximal tubule cells. A 29-nt segment containing the pHRE of the GA mRNA was cloned into the pGemA0 and pGemA60 vectors. The two plasmids were transcribed in the presence of [α-32P]UTP and 500 μM 7metGpppG to produce mRNAs that either lack or contain a 60-nt poly(A) tail. As a control, the in vitro decay of pGemA60 and pGemTNFαARE-A60 were also analyzed. The turnover of the pGemGA-A60 mRNA was initiated by deadenylation or shortening of the poly-A tail. After deadenylation, the body of the transcript was rapidly degraded. In contrast, the pGem-A60 and the pGemTNFαARE-A60 mRNAs were relatively stable when incubated with the WKPT extract. Deadenylation and subsequent decay of the pGemGA-A60 mRNA was stimulated by addition of purified recombinant tristetraprolin (TTP) and was inhibited by addition of purified rat renal ζ-cryst. The addition of TTP also greatly enhanced deadenylation and degradation of the pGemTNFαARE-A60 mRNA, but not the pGem-A60 mRNA. The addition of ζ-cryst had little effect on the turnover of either of the control mRNAs. These data indicated that specific binding of ζ-cryst to the pHRE contributes to the enhanced stability of the GA mRNA during acidosis. Support: National Institute of Health Minority Supplement to Grant DK 37124