The formation of mRNA 3' ends is an essential step in mRNA synthesis in eukaryotes. It involves recognition of processing signals on the primary transcript, cleavage of this RNA, and addition of adenylate residues to the new end. The primary goal of this research is a thorough molecular analysis of the first two steps of this processing event, using a combination of biochemistry, molecular biology and genetics in the yeast S. cerevisiae. In this organism, three factors (CFIA, CFIB, and CF II) are sufficient for these two events. An understanding of the mechanism of polyadenylation will provide the basis from which to ask questions about how the process is regulated as the physiological state of the cell changes and how polyadenylation interacts with other processes involved in mRNA synthesis, utilization and metabolism. To accomplish this goal, we propose as our first specific aim to further characterize the cis- and trans-acting factors necessary for recognition and cleavage of yeast polyadenylation precursor, and second, to understand how the cleavage complex is assembled and how these interactions promote cleavage. These experiment will determine what constitutes the core, or minimal cleavage complex and what is the essential role(s) of each subunit. These roles include binding to polyadenylation signal sequences at and upstream of the cleavage site, interaction with other proteins to assemble and/or activate the cleavage complex, and enzymatic function as the endonuclease. We will also investigate the role of ATP in assembly and cleavage. These experiments should provide insight into the mechanism of this reaction a nd increase our understanding of what features are conserved with the mammalian reaction, and which are different. Our additional aims will use this information to explore how polyadenylation in yeast might promote transport of mRNA and to examine possible examples of regulated polyadenylation in yeast. We are in a strong position to embark upon the proposed research because we now have purified factors and genes encoding the components of these factors, as well as specific antibodies, to use in studying the molecular biology of this process. We will also take advantage of the genetics available in yeast to confirm and expand upon important features identified by the in vitro experiments.
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