CIS-ELEMENT CYTOPLASMIC PROTEIN-INTERACTION WITHIN THE '-UNTRANSLATEDREGION OF THE GLUT1 GLUCOSE-TRANSPORTER MESSENGER-RNA/

The posttranscriptional regulation of glucose transporter GLUT1 gene e xpression may be mediated by specific interactions of cytosolic protei ns 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 P-32-lab eled GLUT1 3'-UTR prepared from transcription plasmids and cytosolic p roteins 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'-UT R. Ultraviolet cross-linking of RNA/ protein complexes identified two complexes having molecular masses of 88 and 44 kDa. Competition studie s with synthetic RNA and oligodeoxynucleotides showed the 88-kDa compl ex reacted with nucleotides 2,180-2,197 and that the 44-kDa complex re acted with sequences within nucleotides 1,717-2,132 of the bovine GLUT 1 mRNA, The GLUT1 3'-UTR between nucleotides 2,100 and 2,300 was gener ated by polymerase chain reaction and subcloned at a unique Pf/MI site within the 3'-UTR of a luciferase gene within the mammalian expressio n vector pGL2. Transfection of C6 rat glioma cells with the luciferase expression vector containing this portion of the GLUT1 3'-UTR resulte d 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 expr ession may be mediated by the interaction of specific cytosolic protei ns with the GLUT1 mRNA 3'-UTR.