Regulation of mRNA stability plays a key role in controlling the expression of many growth factors, cytokines and transcription factors. Regulated mRNA turnover, therefore, plays an important role in cell growth, differentiation and development, and can have a significant impact on several disease states, including cancer. The mechanisms and factors that mediate regulated mRNA turnover are, however, generally unclear. We have discovered a novel enzymatic activity in mammalian cells that specifically removes the 5' cap from mRNAs. Furthermore, this decapping activity is highly regulated in our in vitro assay system. The poly (A) tail of the transcript, in conjuction with poly (A) binding protein, specifically represses decapping through a novel, elF4E-independent complex. AU-rich instability elements, which regulate the stability of many short-lived mRNAs in vivo, dramatically stimulate decapping in our in vitro assays. In this application, we will build upon these observations and investigate the underlying mechanisms that regulate decapping of mRNAs in mammalian cells. First, we propose to identify and characterize the novel human decapping enzyme. Second, we will investigate the mechanism of poly(A)-mediated repression of decapping. Finally, we will address the factors and mechanisms involved in the stimulation of decapping by AU-rich instability elements. In total, these studies should provide pivotal insights into this important area of post-transcriptional regulation of gene expression in mammalian cells.
