Control of Na+-K+-ATPase beta 1-subunit expression: role of 3'-untranslated region.
Using in vitro translation and cell transfection assays, we previously demonstrated that the Na+ -K+ -ATPase beta1 mRNA species containing its longest 3'-untranslated region (UTR) exhibited the lowest translational efficiency. Here, employing deletions and in vivo expression assays, using direct injection of plasmids into rat ventricular myocardium, we identified a 143-nt segment located in the distal 3'-UTR of beta1 mRNA that was associated with decreased luciferase expression; interestingly, this segment contains three AUUUA motifs. Using RNA-protein binding assays and UV cross-linking of cRNA with cytosolic proteins of rat heart, we identified an approximately 38-kDa protein that specifically bound to the cRNA encoding the 143-nt segment of beta1 mRNA 3'-UTR. Mutation of three nucleotides located in the middle region of the 143-nt segment, which was predicted to greatly disrupt a putative stem-loop structure of the cRNA in this region, was associated with reduced binding of the mutated cRNA to the protein migrating at approximately 38 kDa. The cRNA encoding a segment of cyclooxygenase-2 mRNA 3'-UTR containing six AUUUA sequences did not bind the protein migrating at approximately 38 kDa and did not compete with the binding of the wild-type 143-nt beta(1) cRNA to the protein. The above results suggest that the 143-nt segment in the distal segment of the 3'-UTR of beta1 mRNA may play an important role in the control of beta1-subunit expression.
Shao
Yvonne
Y
Departments of Medicine and Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4951, USA.
Ismail-Beigi
Faramarz
F
D020413:3' Untranslated Regions / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000502:physiology; D032383:Myocytes, Cardiac / Q000502:physiology; D009690:Nucleic Acid Conformation; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics*
D020413:3' Untranslated Regions; D018076:DNA, Complementary; D012333:RNA, Messenger; D008156:Luciferases; D000254:Sodium-Potassium-Exchanging ATPase