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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Control of Na⁺-K⁺-ATPase ₁-subunit expression: role of 3'-untranslated region

Departments of Medicine and of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4951

Submitted 31 March 2003 ; accepted in final form 21 October 2003

Using in vitro translation and cell transfection assays, we previously demonstrated that the Na⁺-K⁺-ATPase ₁ 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 ₁ 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 38-kDa protein that specifically bound to the cRNA encoding the 143-nt segment of ₁ 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 38 kDa. The cRNA encoding a segment of cyclooxygenase-2 mRNA 3'-UTR containing six AUUUA sequences did not bind the protein migrating at 38 kDa and did not compete with the binding of the wild-type 143-nt ₁ cRNA to the protein. The above results suggest that the 143-nt segment in the distal segment of the 3'-UTR of ₁ mRNA may play an important role in the control of ₁-subunit expression.

RNA-protein binding; AUUUA sequence; plasmid expression in heart; direct myocardial injection; cardiac expression

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