In mammalian cells, the c-fos proto-oncogene is a representative of a class of approximately 50-100 growth factor inducible genes, termed immediate early genes, whose transcripts decay rapidly in the cytoplasm with a short half-life of less than 30 min. The instability of this class of mRNA distinguishes it from most mammalian messages, which typically are much more stable with half-lives ranging from hours to days. This proposal focuses on elucidating immediate-early-gene mRNA degradation in mammalian cells, using the c-fos proto-oncogene as a model system. Four important questions are addressed. First, what are the functionally important features of the two major c-fos determinants of mRNA instability? Second, what is the nature of the c-fos coding region determinant of instability? How its destabilizing function is coupled to protein translation? Third, what are the cytoplasmic factors that interact with c- fos instability determinants? Fourth, are the mechanisms by which the c- fos mRNA is targeted for rapid and selective degradation also responsible for the decay of other immediate-early-gene mRNAs? Examination of the structural features of cis-acting determinants of c-fos mRNA and the nature of the c-fos coding region determinant of instability will involve creating defined mutations and fusions of the human c-fos and the rabbit beta-globin genes in vitro, introducing these altered genetic constructs into mouse fibroblasts, and analyzing the decay of the resulting mRNAs in vivo. The identification and characterization of cytoplasmic mRNA degradation factors will involve both reconstituting mRNA decay in an in vitro system and performing in vitro mRNA binding assays. Finally, mRNA decay of three immediate-early-gene mRNAs will be investigated to test for generalization of the decay mechanisms of c-fos mRNA. The results of these studies should enhance our knowledge of a fundamental aspect of gene expression that presently is poorly understood. Because the marked instability of the c-fos proto-oncogene mRNA helps to prevent oncogenic transformation by this gene, identification and characterization of the cellular factors that are involved in the decay of c-fos mRNA has the potential to define a new class of cancer of suppressor genes. Therefore, the knowledge learned form the proposed studies should ultimately be of value to understanding molecular mechanisms of oncogenesis.
Showing the most recent 10 out of 31 publications
