Different Na, K-ATPase mRNA(beta 1) species exhibit unique translational efficiencies

We have previously identified five Na, K-ATPase beta1-mRNA species that are expressed in the rat heart, kidney, and brain. These mRNAs which are unequ al in their abundance have an identical coding region but differ in the len gth of their 5 '- and 3 ' -untranslated regions (UTRs). In this study we ex amined the possibility that the beta1-mRNA species exhibit differential tra nslational efficiencies. We constructed expression plasmids encoding each o f the five mRNAs and transcribed and translated them in vitro. Using rabbit reticulocyte system we determined the translation of the different mRNAs u nder conditions optimized for each beta1-cRNA and under an equivalent (stan dard) condition. The longest beta1-cRNA species (initiating at the first tr anscription start site and ending at the last [fifth] poly(A) site) exhibit ed the lowest relative translational efficiency averaging 0.2 +/- 0.05 unit s/mol of cRNA compared to the shortest beta1-cRNA species initiating at the first transcription start site and ending at the first poly(A) signal (wit h an assigned relative value of 1.0). These results suggested that the diff erent translation rates of beta1-mRNAs may be due to their 3 ' -UTRs. To fu rther define the role of beta1-3 ' -UTR, chimeric luciferase constructs con taining different segments of the beta1-3 ' -UTR were transiently transfect ed into Clone 9 cells. Compared to the chimeric construct containing the sh ortest beta1-3 ' -UTR segment (ending at the first poly(A) site), the const ruct containing the full-length beta1-3 ' -UTR exhibited a luciferase expre ssion of 0.23 +/- 0.04. To control for potential changes in the abundance o f the expressed chimeric mRNAs which may lead to differences in luciferase expression, luciferase activity was normalized against chimeric luciferase- mRNA content measured in mixtures of cells stably transfected with the abov e constructs. The ratio of luciferase activity/chimeric luciferase-mRNA con tent in cells expressing the construct containing the entire beta1-3 ' -UTR region was 0.17 that in cells expressing chimeric luciferase mRNA containi ng beta1-3 ' -UTR up to the first poly(A) signal (P < 0.05). We conclude th at the translational efficiency of the different beta1-mRNA species is nega tively regulated by the 3 ' -UTR of the mRNA and that a regulating region a ppears to be localized between the second and fifth poly(A) signals of beta 1-mRNA. (C) 2001 Academic Press.