EVIDENCE FOR TRANSLATIONAL CONTROL ELEMENTS WITHIN THE 5'-UNTRANSLATED REGION OF GLUT1 GLUCOSE-TRANSPORTER MESSENGER-RNA

Recent studies have indicated that the blood-brain barrier GLUT1 gluco se transporter is under posttranscriptional regulation. To begin funct ional mapping of the GLUT1 transcript, in the present investigation we studied the translational efficiency of capped full-length synthetic GLUT1 mRNA, and both 5'- and S'-untranslated regions (UTRs) deleted GL UT1 mRNAs. Deletion of 5'- and 5'-/3'-UTRs markedly reduced the transl ation efficiency of the human (h) GLUT1 transcript in the rabbit retic ulocyte lysate (RRL), and this effect was not modified by addition of microsomes to the translation system. The putative role of these hGLUT 1 5'-UTR cis-acting elements was studied using the luciferase expressi on vector pGL2. DNA corresponding to the hGLUT1 5'-UTR generated by PC R was subcloned at the HindIII site of the pGL2 located upstream of th e luciferase 5'-UTR. Transfection of brain endothelial cultured cells with pGL2 containing most of the hGLUT1 5'-UTR (nucleotides 1-171) mar kedly increased the expression of luciferase, and disruption of lucife rase-leading sequence with an unrelated 171-nucleotide fragment decrea sed its expression. Insertion of nucleotides 1-96 of the hGLUT1 5'-UTR retained most of the stimulatory effect, and nucleotides 123-171 prod uced 64% of maximal induction. On the contrary, clones containing nucl eotides 79-171 and 154-171 of bGLUT1 5'-UTR had marginal effects on lu ciferase expression. The present data provide evidence suggesting that the 5'-UTR of the GLUT1 mRNA contains cis-acting elements involved in the translational control of the GLUT1 gene in mammalian cells.