title: Site-directed deletion of a 10-nucleotide domain of the 3'-untranslated region of the GLUT1 glucose transporter mRNA eliminates cytosolic protein binding in human brain tumors and induction of reporter gene expression.
abstract: The posttranscriptional regulation of GLUT1 glucose transporter gene expression may be mediated by specific interactions between cytosolic trans-acting factors and regulatory cis-elements within the 3'-untranslated regions (UTRs) of the GLUT1 mRNA. Recent studies demonstrate that experimental and human brain tumors express an 80-kDa protein that reacts with a specific sequence around nucleotide 2,200 within the GLUT1 mRNA 3'-UTR. The 80-kDa protein is selectively expressed in hemangioblastoma, a tumor characterized by overexpression of GLUT1. The enhancer role of this GLUT1 3'-UTR cis-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 nucleotides 2,100-2,300 of the bovine GLUT1 3'-UTR inserted at the Pfl MI site within the luciferase 3'-UTR) results in a fivefold increase in luciferase gene expression. Deletion of nucleotides 2,181-2,190 of the bovine GLUT1 3'-UTR, i.e., the putative binding site of the 80-kDa protein, completely eliminated the enhancement of luciferase activity in the transfected cells. Luciferase mRNA containing the putative cis-element inserted in the 3'-UTR was transcribed, and after UV crosslinking, this mRNA complexed with the 80-kDa protein in cytosol of either C6 cells or hemangioblastoma. In contrast, this complex was undetected with either luciferase control mRNA or 10 nucleotide-deleted RNA. The present 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 posttranscriptional level.
authors.affiliation: Department of Medicine and Brain Research Institute, UCLA School of Medicine, Los Angeles, California 90095-1682, U.S.A.
authors.forename: H
authors.initials: H
authors.lastname: Tsukamoto
authors.forename: R J
authors.initials: RJ
authors.lastname: Boado
authors.forename: W M
authors.initials: WM
authors.lastname: Pardridge
mesh_terms: D000818:Animals; D001932:Brain Neoplasms / Q000378:metabolism*; D002417:Cattle; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D003600:Cytosol / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D017930:Genes, Reporter; D005910:Glioma; D051272:Glucose Transporter Type 1; D006801:Humans; D008156:Luciferases / Q000235:genetics; D009004:Monosaccharide Transport Proteins / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D010957:Plasmids; D011485:Protein Binding / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D003432:Cross-Linking Reagents; D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; D012333:RNA, Messenger; C495677:SLC2A1 protein, human; C495679:Slc2a1 protein, rat; D008156:Luciferases