Demonstration of specific physical interaction between CHOP mRNA and intracellular proteins

Abstract

The ability of a cell to respond precisely to environmental stress depends on the expression of a large number of genes in a finely coordinated manner. One of such genes is CHOP that encodes the CCAAT/Enhancer-Binding Protein Homologous Protein. CHOP is usually expressed to mediate apoptosis under the condition of excessive stress. The expression of CHOP therefore has to be stringently regulated as its expression will determine the fate of a cell under stress. The expression of many genes is regulated at the posttranscriptional level through the metabolism of their mRNA, such as maturation, transport, storage, and degradation of mRNA. Many metabolic processes of mRNA are known to be mediated by RNA-binding proteins that specifically interact with the mRNA. RNA-binding proteins that interact with the CHOP mRNA have until present not been identified. The aim of this study is to investigate what proteins may bind specifically to CHOP mRNA. The study will enable further understanding regarding how the expression of CHOP is regulated in cellular stress response.

Proteins extracted from HeLa cells were incubated with a 335bp [3H]-labelled CHOP RNA probe that spans over a part of the coding region and the 3’UTR of CHOP mRNA. Sucrose density gradient ultracentrifugation revealed that after incubation with proteins extracted from HeLa cells, the sedimentation rate of the [3H]-CHOP RNA probe was significantly higher than that of the free [3H]-RNA probe. The formation of heavy molecular complexes involving the [3H]-CHOP RNA probe was therefore suggested. However, no increase in sedimentation rate of the [3H]-CHOP RNA probe was observed in the presence of an excess of unlabelled CHOP RNA probe. Similar observations were made when the experiments were performed using proteins isolated from cells treated with As2O3.

Two putative sequence elements, the Adenylate-Uridylate-Rich Element (ARE) and the Putative Regulatory Element (PRE) located respectively in the 3’UTR and coding region of the CHOP mRNA were then examined for their involvement in RNA-protein interaction. The deletion of ARE and/or PRE, from the [3H]-CHOP RNA probe had little effect on the binding of the RNA probe to the HeLa cell proteins. Consistently, unlabelled CHOP RNA probes with the same deletions were only slightly weaker in competing with the intact [3H]-CHOP RNA probe to bind to HeLa cell proteins. Human Antigen R (HuR) was identified by Western blot analysis to be present in the proteins that were obtained by pull-down assays using biotinylated CHOP RNA as a probe. The deletion of ARE and/or PRE resulted in a slight reduction of HuR obtained by pull down assays.

This study provides the first evidence that physical binding interaction occurs between intracellular RNA-binding proteins and CHOP mRNA. More importantly, one such protein is HuR. Data suggest that HuR binding to the CHOP mRNA is mediated by sequences in the CHOP mRNA other than ARE and PRE.