An in vitro assay to study regulated mRNA stability

The examination of posttranscriptional regulation of mRNA in mammalian cells is critical to discovering the role that mRNA plays in the initiation and maintenance of cellular processes. The complexity of the system defies a holistic approach and, therefore, we have devised an in vitro mRNA turnover assay that enables us to elucidate the factors involved in mRNA deadenylation and degradation. Our system, using an S100 HeLa extract and in vitro transcribed RNAs, accurately mimics the end products of mRNA turnover, which have been previously described using in vivo studies and, in addition, allows for the detailed study of factors that may play a role in regulated deadenylation and degradation. Another important aspect of our system is the ease with which it can be manipulated. We can provide any synthetic RNA molecule to the assay to test for specific sequence activity. Furthermore, the results are clear and accurately interpretable. We have demonstrated that our in vitro system accurately deadenylates and decays a capped and polyadenylated RNA molecule in a processive manner without nonspecific nuclease activity. Finally, we have demonstrated regulated instability in vitro using the AU-rich elements (AREs) from tumor necrosis factor-alpha (TNF-alpha) and granulocyte macrophage colony stimulating factor (GM-CSF) embedded within the RNA molecule. The presence of the AREs increased the deadenylation and the decay rates seen in vivo. We feel that this system can be expanded and adapted to examine a variety of mRNA regulatory events in mammalian cells.