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Evidence for Translational Control Elements Within the 5′‐Untranslated Region of GLUT1 Glucose Transporter mRNA
Author(s) -
Boado Ruben J.,
Tsukamoto Haruhisa,
Pardridge William M.
Publication year - 1996
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1996.67041335.x
Subject(s) - glut1 , luciferase , untranslated region , messenger rna , microbiology and biotechnology , five prime untranslated region , biology , glucose transporter , three prime untranslated region , translational regulation , translation (biology) , nucleotide , ribosomal binding site , transfection , gene , biochemistry , insulin , endocrinology
Recent studies have indicated that the blood‐brain barrier GLUT1 glucose transporter is under post‐transcriptional regulation. To begin functional 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 3′‐untranslated regions (UTRs) deleted GLUT1 mRNAs. Deletion of 5′‐ and 5′‐/3′‐UTRs markedly reduced the translation efficiency of the human (h) GLUT1 transcript in the rabbit reticulocyte lysate (RRL), and this effect was not modified by addition of microsomes to the translation system. The putative role of these hGLUT1 5′‐UTR cis ‐acting elements was studied using the luciferase expression vector pGL2. DNA corresponding to the hGLUT1 5′‐UTR generated by PCR was subcloned at the Hin dIII site of the pGL2 located upstream of the luciferase 5′‐UTR. Transfection of brain endothelial cultured cells with pGL2 containing most of the hGLUT1 5′‐UTR (nucleotides 1–171) markedly increased the expression of luciferase, and disruption of luciferase‐leading sequence with an unrelated 171‐nucleotide fragment decreased its expression. Insertion of nucleotides 1–96 of the hGLUT1 5′‐UTR retained most of the stimulatory effect, and nucleotides 123–171 produced 64% of maximal induction. On the contrary, clones containing nucleotides 79–171 and 154–171 of bGLUT1 5′‐UTR had marginal effects on luciferase 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.