The formation of polyadenylated 3' termini is essential to the biogenesis of eukaryotic mRNA. Defects in this processing decrease the amount of mRNA available for translation into protein, and thus interfere with normal cell function. Regulation at the level of polyadenylation can affect the amount and type of mRNA synthesized from a transcriptional unit. In this way, it becomes part of a cell's response to external stimuli governing growth, differentiation, and tissue-specific gene expression. For these reasons, it is important to understand the mechanism and regulation of polyadenylation. In mammals, maturation of the mRNA 3' end involves cleavage of precursor and addition of adenylate residues to the new end. This processing has been examined in vivo and in cell-free systems. This research has defined how precursor RNA is cleaved and polyadenylated and what signal sequences on precursor direct the processing. Further progress in characterizing the processing activities is severely limited by the lack of suitable molecular genetics in mammalian systems. However, little is known about 3' end formation in eukaryotes such as the yeast S. cerevisiae, which is more amenable to genetic analysis. The primary goal of this research is a thorough molecular analysis of polyadenylation in yeast using a combination of genetics and biochemistry. The results of these studies will allow us to compare the processing mechanism in yeast to that used in metazoans. Understanding the basic mechanism of polyadenylation will make it feasible to ask how the process is regulated as the physiological state of the cell changes, and how this regulation affects mRNA level globally or specifically.
The aims of this research are: I. Development of a genetic screen to identify the cis-acting RNA sequences and the trans-acting factors necessary for polyadenylation in yeast. Using primary a colorimetric assay, it should be possible to determine the minimal sequences needed for polyadenylation and to screen for mutants which do not recognize a functional polyadenylation signal. The mutant genes can then be identified and cloned by complementary transformation with wild type genes. II. Clarification of the role transcription termination in the formation of the mature 3' end of yeast mRNA. This experiment will use a nuclear run-on assay to detect any transcription beyond the poly(A) addition site. III. Development of an in vitro system in yeast which correctly polyadenylates precursor RNA. The goal of these experiments is to determine the molecular pathway of polyadenylation in yeast, to complement the genetic analysis of signal sequences and trans- acting factors, and to provide an assay for the purification of processing activities from crude extracts.
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