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

The specificity and the functional significance of the binding of a specifi c 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 s equence is not an AUUUA binding protein. When expressed in LLC-PK1-F+ cells , the half-life of a beta-globin reporter construct, beta G-phosphoenolpyru vate carboxykinase, was only slightly affected (1.3-fold) by growth in acid ic (pH 6.9, 10 mM HCO3-) vs. normal (pH 7.4, 25 mM HCO3-) medium. However, insertion of short segments of GA mRNA containing the direct repeat or a si ngle eight-base AU sequence was sufficient to impart a fivefold pH-responsi ve stabilization to the chimeric mRNA. Furthermore, site-directed mutation of the direct repeat of the 8-base AU sequence in a beta G-GA mRNA, which c ontains 956 bases of the 3'-nontranslated region of the GA mRNA, completely abolished the pH-responsive stabilization of the wild-type beta G-GA mRNA. Thus either the direct repeat or a single eight-base AU sequence is both s ufficient and necessary to create a functional pH-response element.