Investigating ornithine decarboxylase posttranscriptional regulation via a pulldown assay using biotinylated transcripts

Ornithine decarboxylase (ODC) is the first rate-limiting enzyme in the polyamine biosynthetic pathway. It has been well documented that ODC is tightly regulated at the levels of transcription, posttranscriptional changes in RNA, and protein degradation during normal conditions and that these processes are dysregulated during tumorigenesis. Moreover, it has been recently shown that ODC is posttranscriptionally regulated by RNA binding proteins (RBPs) which can bind to the ODC mRNA transcript and alter its stability and translation. Using a mouse skin cancer model, we show that the RBP human antigen R (HuR) is able to bind to synthetic mRNA transcripts through a pulldown assay which utilizes a biotin-labeled ODC 30-untranslated region (UTR). The details of this method are described here. A better understanding of the mechanism(s) which regulates ODC is critical for targeting ODC in chemoprevention.