Eukaryotic mRNAs can be controlled at many different steps. In the nucleus, transcription and mRNA processing are required to produce an mRNA that can be translated. In the cytoplasm, mature mRNAs can be regulated at the level of stability, translational activity and cellular location. The objective of the proposed research is to understand the mechanism of mRNA processing and function in animal cells. Although nuclear polyadenylation is well understood, the mechanisms and control of cytoplasmic polyadenylation are not, despite their great importance. Regulated changes in poly(A) lengh occur in the cytoplasm throughout development, affect many mRNAs, and occur in many if not all animal species. Our primary goal is to understand, in molecular terms, how cytoplasmic polyadenylation is controlled, and how that control contributes to critical events in early development. The approach taken is to identify those regions of the mRNA that are critical for cytoplasmic polyadenylation, and then to determine how they exert their effects. Using a combination of in vivo and in vitro approaches, we propose to elucidate how the cytoplasmic polyadenylation apparatus is assembled and controlled. We will indentify sequences and trans-acting factors that activate polyadenylation, and others that repress it. Throughout, we will examine the biological significance of the reactions by manipulating endogenous mRNAs through novel methods. We focus on the periods of meiotic maturation and fertilization, and use frog oocytes as a model. We use molecular genetics to determine how cytoplasmic polyadenylation is triggered, and how it is repressed. We exploit a range of assays and methods that we have helped develop, in which each process of interest can be studied both in vivo, in an intact oocyte, and in a cell-free system using either crude extracts or purified components. The work proposed will elucidate fundamental mechanisms of gene expression, and therefore has important applications. Our detailed investigations of c-mos mRNA, a proto-oncogene normally expressed only in the germ line, will bear on the regulation of cell division in both normal and abnormal growth. Further, we have developed a facile molecular genetic method, termed the three-hybrid system, with which RNA-protein interactions can be readily dissected in vivo. This method may have practical application, including the analysis and dissection of viral RNA-protein interactions, and screens for inhibitors that might mitigate their infectivity. Here, the method is used to facilitate our studies of how cytoplasmic polyadenylation is specifically repressed.
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