SITE-DIRECTED DELETION OF A 10-NUCLEOTIDE DOMAIN OF THE 3'-UNTRANSLATED REGION OF THE GLUT1 GLUCOSE-TRANSPORTER MESSENGER-RNA ELIMINATES CYTOSOLIC PROTEIN-BINDING IN HUMAN BRAIN-TUMORS AND INDUCTION OF REPORTER GENE-EXPRESSION

The posttranscriptional regulation of GLUT1 glucose transporter gene e xpression may be mediated by specific interactions between cytosolic t rans-acting factors and regulatory cis-elements within the 3'-untransl ated regions (UTRs) of the GLUT1 mRNA. Recent stud ies demonstrate tha t experimental and human brain tumors express an 80-kDa protein that r eacts with a specific sequence around nucleotide 2,200 within the GLUT 1 mRNA 3'-UTR. The 80-kDa protein is selectively expressed in hemangio blastoma, a tumor characterized by overexpression of GLUT1. The enhanc er role of this GLUT1 3'-UTR cia-element was confirmed in the present studies using the luciferase expression vector pGL2 and site-directed deletion. Transfection of C6 glioma cells with pGL2 (containing nucleo tides 2,100-2,300 of the bovine GLUT1 3'-UTR inserted at the PflMI sit e within the luciferase 3'-UTR) results in a fivefold increase in luci ferase gene expression. Deletion of nucleotides 2,181-2,190 of the bov ine GLUT1 3'-UTR, i.e., the putative binding site of the 80-kDa protei n, completely eliminated the enhancement of luciferase activity in the transfected cells. Luciferase mRNA containing the putative cia-elemen t inserted in the 3'-UTR was transcribed, and after UV crosslinking, t his mRNA complexed with the 80-kDa protein in cytosol of either C6 cel ls or hemangioblastoma. In contrast, this complex was undetected with either luciferase control mRNA or 10 nucleotide-deleted RNA. The prese nt study provides evidence that nucleotides 2,181-2,190 of the bovine GLUT1 mRNA 3'-UTR forms a complex with brain tumor cytosolic proteins that serves to increase GLUT1 gene expression at the posttranscription al level.