Different Na, K-ATPase mRNAβ1 Species Exhibit Unique Translational Efficiencies

We have previously identified five Na, K-ATPase β1-mRNA species that are expressed in the rat heart, kidney, and brain. These mRNAs which are unequal in their abundance have an identical coding region but differ in the length of their 5′- and 3′-untranslated regions (UTRs). In this study we examined the possibility that the β1-mRNA species exhibit differential translational efficiencies. We constructed expression plasmids encoding each of the five mRNAs and transcribed and translated them in vitro. Using rabbit reticulocyte system we determined the translation of the different mRNAs under conditions optimized for each β1-cRNA and under an equivalent (standard) condition. The longest β1-cRNA species (initiating at the first transcription start site and ending at the last [fifth] poly(A) site) exhibited the lowest relative translational efficiency averaging 0.2 ± 0.05 units/mol of cRNA compared to the shortest β1-cRNA species initiating at the first transcription start site and ending at the first poly(A) signal (with an assigned relative value of 1.0). These results suggested that the different translation rates of β1-mRNAs may be due to their 3′-UTRs. To further define the role of β1-3′-UTR, chimeric luciferase constructs containing different segments of the β1-3′-UTR were transiently transfected into Clone 9 cells. Compared to the chimeric construct containing the shortest β1-3′-UTR segment (ending at the first poly(A) site), the construct containing the full-length β1-3′-UTR exhibited a luciferase expression of 0.23 ± 0.04. To control for potential changes in the abundance of 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 above constructs. The ratio of luciferase activity/chimeric luciferase-mRNA content in cells expressing the construct containing the entire β1-3′-UTR region was 0.17 that in cells expressing chimeric luciferase mRNA containing β1-3′-UTR up to the first poly(A) signal (P < 0.05). We conclude that the translational efficiency of the different β1-mRNA species is negatively regulated by the 3′-UTR of the mRNA and that a regulating region appears to be localized between the second and fifth poly(A) signals of β1-mRNA.