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.
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.
Tsukamoto
H
H
Department of Medicine and Brain Research Institute, UCLA School of Medicine, Los Angeles, California 90095-1682, U.S.A.
Boado
R J
RJ
Pardridge
W M
WM
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
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