The experiments proposed here ask what role mRNA processing plays in regulating the structure, stability and translation of messenger RNAs. Specifically, we intend to identify both the cellular factors involved in mRNA 3' end cleavage and polyadenylation and the genes that encode them. We will then be able to ask physiological and biochemical questions about the roles of these factors in the regulation of mRNA levels during cellular growth and at different stages of the cell cycle. We will employ a direct biochemical assay to screen a large collection of conditional lethal mutants for enzymatic defects in mRNA 3' end formation that affect cell viability or growth. We will clone and characterize the genes using classical genetic and molecular biological techniques. We will search for second site revertants in the expectation of identifying other genes whose products play a role in mRNA processing. These mutants will also be used in temperature shift studies to determine the effects of each defect on the stability, nucleocytoplasmic transport and translation of mRNAs. The isolated genes will be used to determine the effect of knockout mutations, site directed alterations and controlled expression on cell growth and the ultimate fate of the abnormally processed mRNAs. In a parallel biochemical approach, we will begin to purify the factors which catalyze specific cleavage and polyadenylation of yeast mRNAs. This is a proposal to investigate a very poorly understood part of the process by which genetic information is decoded and expressed, viz. the assembly of a finished messenger RNA molecule from its precursor, and the transport of the messenger RNA from the nucleus to the cytoplasm.
