Specificity and functional analysis of the pH-responsive element within renal glutaminase mRNA.
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-PK(1)-F(+) cells, the half-life of a beta-globin reporter construct, betaG-phosphoenolpyruvate carboxykinase, was only slightly affected (1.3-fold) by growth in acidic (pH 6.9, 10 mM HCO(-)(3)) vs. normal (pH 7.4, 25 mM HCO(-)(3)) 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 betaG-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 betaG-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.
Laterza
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Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins 80523-1870, USA.
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D000138:Acidosis / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D017930:Genes, Reporter; D005914:Globins / Q000235:genetics; D005972:Glutaminase / Q000235:genetics*; D007668:Kidney / Q000201:enzymology / Q000378:metabolism*; D018374:LLC-PK1 Cells; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D013552:Swine
D017931:DNA Primers; D012333:RNA, Messenger; D005914:Globins; D004247:DNA; D005972:Glutaminase