Specificity and functional analysis of the pH-responsive element within renal glutaminase mRNA

Abstract

The specificity and the functional significance of the binding of a specific cytosolic protein to a direct repeat of an eight-base AU sequence within the 3′-nontranslated region of the glutaminase (GA) mRNA were characterized. Competition experiments established that the protein that binds to this sequence is not an AUUUA binding protein. When expressed in LLC-PK1-F+cells, the half-life of a β-globin reporter construct, βG-phospho enolpyruvate carboxykinase, was only slightly affected (1.3-fold) by growth in acidic (pH 6.9, 10 mM [Formula: see text]) vs. normal (pH 7.4, 25 mM [Formula: see text]) medium. However, insertion of short segments of GA mRNA containing the direct repeat or a single eight-base AU sequence was sufficient to impart a fivefold pH-responsive stabilization to the chimeric mRNA. Furthermore, site-directed mutation of the direct repeat of the 8-base AU sequence in a βG-GA mRNA, which contains 956 bases of the 3′-nontranslated region of the GA mRNA, completely abolished the pH-responsive stabilization of the wild-type βG-GA mRNA. Thus either the direct repeat or a single eight-base AU sequence is both sufficient and necessary to create a functional pH-response element.