Mapping and functional analysis of an instability element in phosphoenolpyruvate carboxykinase mRNA

Phosphoenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme of ren al gluconeogenesis. The 3'-nontranslated region of the PEPCK mRNA contains an instability element that facilitates its rapid turnover and contributes to the regulation of PEPCK gene expression. Such processes are mediated by specific protein-binding elements. Thus RNA gel shift analysis was used to identify proteins in rat renal cortical cytosolic extracts that bind to the 3'-nontranslated region of the PEPCK mRNA. Deletion constructs were then u sed to map the binding interactions to two adjacent RNA segments (PEPCK-6 a nd PEPCK-7). However, competition experiments established that only the bin ding to PEPCK-7 was specific. Functional studies were performed by cloning similar segments in a luciferase reporter construct, pLuc/Zeo. This analysi s indicated that both PEPCK-6 and PEPCK-7 segments were necessary to produc e a decrease in luciferase activity equivalent to that observed with the fu ll-length 3'-nontranslated region. Thus the PEPCK-7 segment binds a specifi c protein that may recruit one or more proteins to form a complex that medi ates the rapid decay of the PEPCK mRNA.