Characterization of the instability elements present within the 3' UTR of the glutaminase mRNA in WKPT kidney cells

Abstract

During metabolic acidosis, the level of glutaminase (GA) is increased 8-fold within the renal proximal convoluted tubule. This adaptation contributes to the ability of the kidney to generate NH4+ and HCO3- ions from glutamine to facilitate the excretion of acids and partially restore acid-base balance. The increased GA expression results from increased stability of the GA mRNA that is mediated by two 8-base AU-sequences that function as pH-response elements (pHRE). This sequence binds ζ-cryst and p40-AUF1 with high affinity and specificity. An in vitro mRNA decay assay was developed using cytosolic extracts of WKPT cells, a line of rat renal proximal tubule cells. A 28-nt segment containing the pHRE of GA mRNA was cloned into pGem-A0 and pGem-A60 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. The GemGA-A60 mRNA was used as a substrate for an in vitro mRNA turnover assay. As controls, in vitro decay of Gem-A60 and GemARE-A60 were also analyzed. The latter mRNA contains the AU-rich element that mediates the rapid turnover of TNFa mRNA. The turnover of GemGA-A60 mRNA was enhanced by addition of poly-A RNA and inhibited by p40-AUF1 and HuR. By contrast, the Gem-A60 mRNA was relatively stable when incubated with the WKPT extracts and its decay was not affected by addition of various RNA binding proteins. The addition of tristetraprolin (TTP) greatly enhanced deadenylation and degradation of GemARE-A6o mRNA, but only slightly stimulated the decay of GemGA-A60 mRNA. The turnover rate of GemGA-A60 was greatly enhanced in a cytosolic extracts of a clonal line of WKPT cells that over express ζ-cryst by 36-fold. Omission of ATP and phosphocreatine also increased the rate of deadenylation and resulted in the accumulation of the fully deadenylated product. The data indicate that in vitro decay of GemGA-A60 mRNA is initiated by deadenylation and that this reaction is enhanced by endogenously expressed ζ-cryst and TTP and inhibited by p40-AUF1 and HuR. Subsequent degradation of the deadenylated RNA required ATP and thus, may be mediated by the exosome. An in vitro decapping assay demonstrated that both the 5' → 3' and 3' → 5' degradation pathways are involved in the turnover of the GA mRNA. Determination of whether 5' → 3' or 3' → 5' decay pathway predominates in vivo will require further analysis. Antibodies against ζ-cryst, total eIF2α and phospho-eIF2α were used in immunostaining experiments to reveal that phospho-eIF2α and ζ-cryst may co-localize in stress granules. The treatment of WKPT-ζ+ cells with pH 6.9 medium activated the ER stress signaling pathway and resulted in the initial, but transient, association of ζ-cryst with stress granules. The completed experiments demonstrated the differential role of ARE binding proteins on the regulation of the GA mRNA, and the importance of localization of RNA:protein complexes as a possible determining factor of mRNA turnover.