Cis-element/cytoplasmic protein interaction within the 3'-untranslated region of the GLUT1 glucose transporter mRNA.
The posttranscriptional regulation of glucose transporter GLUT1 gene expression may be mediated by specific interactions of cytosolic proteins and regulatory cis-elements within the untranslated regions (UTRs) of the GLUT1 mRNA. These putative cis/trans interactions were examined in the present studies with RNase T1 protection assays using 32P-labeled GLUT1 3'-UTR prepared from transcription plasmids and cytosolic proteins from C6 rat glioma cells. RNase T1 mapping studies localized a cis-element to nucleotides 2,170-2,207 on the bovine GLUT1 mRNA 3'-UTR. Ultraviolet cross-linking of RNA/protein complexes identified two complexes having molecular masses of 88 and 44 kDa. Competition studies with synthetic RNA and oligodeoxynucleotides showed the 88-kDa complex reacted with nucleotides 2,180-2,197 and that the 44-kDa complex reacted with sequences within nucleotides 1,717-2,132 of the bovine GLUT1 mRNA. The GLUT1 3'-UTR between nucleotides 2,100 and 2,300 was generated by polymerase chain reaction and subcloned at a unique Pfl/MI site within the 3'-UTR of a luciferase gene within the mammalian expression vector pGL2. Transfection of C6 rat glioma cells with the luciferase expression vector containing this portion of the GLUT1 3'-UTR resulted in a sixfold increase in luciferase gene expression in C6 cells. The identification of these cis/trans mechanisms provides support for the hypothesis that the posttranscriptional regulation of GLUT1 gene expression may be mediated by the interaction of specific cytosolic proteins with the GLUT1 mRNA 3'-UTR.
Dwyer
K J
KJ
Department of Medicine, UCLA School of Medicine 90095, USA.
Boado
R J
RJ
Pardridge
W M
WM
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003593:Cytoplasm / Q000378:metabolism; D005786:Gene Expression Regulation; D051272:Glucose Transporter Type 1; D008156:Luciferases / Q000235:genetics; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D015534:Trans-Activators / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D051272:Glucose Transporter Type 1; D015335:Molecular Probes; D009004:Monosaccharide Transport Proteins; D012333:RNA, Messenger; C495679:Slc2a1 protein, rat; D015534:Trans-Activators; D008156:Luciferases