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.