Different Na, K-ATPase mRNA(beta1) 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 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 beta1-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 beta1-cRNA and under an equivalent (standard) condition. The longest beta1-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 beta1-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 beta1-mRNAs may be due to their 3'-UTRs. To further define the role of beta1-3'-UTR, chimeric luciferase constructs containing different segments of the beta1-3'-UTR were transiently transfected into Clone 9 cells. Compared to the chimeric construct containing the shortest beta1-3'-UTR segment (ending at the first poly(A) site), the construct containing the full-length beta1-3'-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 beta1-3'-UTR region was 0.17 that in cells expressing chimeric luciferase mRNA containing beta1-3'-UTR up to the first poly(A) signal (P < 0.05). We conclude that the translational efficiency of the different beta1-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 beta1-mRNA.
Shao
Y
Y
Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4951, USA.
Ismail-Beigi
F
F
D020413:3' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D002999:Clone Cells; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D066298:In Vitro Techniques; D008156:Luciferases / Q000235:genetics; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012156:Reticulocytes / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
D020413:3' Untranslated Regions; D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D000254:Sodium-Potassium-Exchanging ATPase