Eukaryotic mRNAs generally are not replicas of the genes that encode them. Instead, mRNA is produced by a series of covalent modifications of a primary transcript including capping, splicing and polyadenylation. In principle, each maturation step provides a means of regulating mRNA formation. The ultimate objective of our work is to understand the molecular mechanism of mRNA synthesis and the means by which it is regulated. In this proposal, we focus on a form of regulated RNA processing that is critical in early development -- the addition and removal of poly (A) from maternal mRNAs. We propose to study these regulated RNA processing reactions in frog oocytes and embryos. We focus on the periods of oocyte maturation and fertilization, and use cyclin and c-mos mRNAs as models. In the last grant period, we laid the groundwork for the work proposed here. We identified sequences that regulate poly (A) addition and removal, and characterized the enzymes involved. We developed assays for poly (A) addition and removal, both in vivo, by microinjection, and in vitro, using extracts of frog eggs. In addition, we elucidated key features of the mechanism by which poly (A) is added in the nucleus of all cells; this knowledge provides an invaluable intellectual framework and critical reagents for the work we now propose In the next grant period, sequences that are critical for poly (A) addition and removal during development will be identified precisely. We will characterize and purify the enzymes involved, both by classical means and molecular cloning, and determine how those activities are regulated during development. We will test our hypothesis that changes in the length of the poly (A) tail of c-mos mRNA help control the embryonic cell cycle. By in vitro complementation tests, we will determine whether factors that direct cytoplasmic polyadenylation during development are functionally interchangeable with those that add poly (A) in the nucleus. The work proposed will elucidate fundamental mechanisms of gene expression, and therefore has important practical applications. Changes in poly (A) length occur throughout development, and in the embryos of all animal species so far examined. Our detailed investigations of the regulation of c-mos, a proto-oncogene normally expressed only in the germ line, bear on how cell division is controlled both in normal and abnormal growth.
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