Characterization of the pH-responsive stabilization of glutamate dehydrogenase and glutaminase messenger RNAs

Abstract

During chronic metabolic acidosis, the adaptive increase in rat renal ammoniagenesis and gluconeogenesis from glutamine is sustained, in part, by increased expression of the mitochondrial glutaminase (GA) and glutamate dehydrogenase (GDH) enzymes. The increased enzyme levels result from the pH-responsive stabilization of their respective mRNAs. GA mRNA contains a pH-response element (pHRE) within its 3’-untranslated region (3'-UTR) that contains a direct repeat of an 8-base AU-rich sequence (ARE) and binds ζ-crystallin/NADPH:quinone reductase (ζ-crys/Nqr) with high affinity and specificity. ζ-crys/Nqr also binds to the full-length 3'-UTR of the GDH mRNA, but with less affinity. The 3'-UTR of GDH mRNA contains four 8-base AREs that are 88% identical to one of the GA AREs. The individual 8-base ARE from the GA pHRE bind the ζ-crys/Nqr with high affinity. The four individual GDH AREs bind ζcrys/Nqr with less affinity than the GA AREs. Competition studies confirmed the relative order of affinities observed in the direct binding studies. Experiments were performed to determine whether the GDH AREs are functional pHREs. Insertion of the GDH 3’-UTR into the stable β-globin plasmid (pβG) produced a chimeric mRNA that was stabilized in acidic medium. In functional studies. GDH4 was seen to function as a pHRE.

A tetracycline responsive expression system was created that can activate or inhibit transcription of a single gene within the cell so that the in vivo decay of the GA mRNA could be studied. An 8C line of LLC-PK1-F+ cells stably expressing the tTA protein, a recombinant transcriptional activator protein, was produced. When pTRE2-βG-GA-bGH was stably transfected into the 8C cell line, a homogeneous population of PGGA mRNA was synthesized using a transcriptional pulse. After creating a transcriptional pulse a pH-responsive stabilization of the βG-GA mRNA was observed. The deadenylation and decay of the βG-GA mRNA over time was monitored to determine the type of decay that occurs under conditions mimicking metabolic acidosis. RNase H treatment was used to obtain a shorter RNA so deadenylation could be easily monitored. Biphasic decay of the βG-GA mRNA was observed under normal and acidic conditions using a synchronous, distributive deadenylation mechanism in the initial phase.