Evidence for direct and specific physical interaction between the stress-inducible CHOP mRNA and intracellular proteins

Abstract

The metabolism of messenger RNA plays an important role in regulating the expression of specific genes under a diversity of extracellular conditions. The CHOP gene is specifically induced to express when cells encounter a variety of stressful conditions. Although it has been demonstrated that stress-induced expression of CHOP gene is regulated at both the transcriptional and the post-transcriptional level, little is known about the proteins that are involved in the metabolism of CHOP mRNA. To address this issue, a [3H]-Uridine labeled RNA probe of 335bp synthesized using a fragment of the CHOP gene as a template was incubated with intracellular proteins isolated from HeLa cells by affinity adsorption to heparin-agarose beads. The formation of RNA-protein complexes were revealed by the difference in sedimentation velocity of the free and the protein-bound RNA probe on sucrose density gradient ultracentrifugation. Our results demonstrated that certain proteins extracted from a lamin-positive cellular fraction were able to bind to the CHOP RNA probe. Binding of these proteins to the labeled RNA probe was not detected in the presence of an excess of the unlabelled RNA probe. The formation of labeled RNA-protein complexes could still be detected if a 15bp sequence element in the coding segment, but not an AU-rich element in the 3’UTR, was deleted from the unlabelled RNA used in the competition experiment. These results suggest that specific proteins will bind to specific sequence sites of the CHOP mRNA. The functions of these CHOP mRNA binding proteins are presently unknown.