Abstract 9405434 A thorough understanding of eukaryotic gene expression requires knowledge not only of transcription rates but also of messenger RNA (mRNA) stability. In eukaryotes, regulation of RNA stability is mediated by a novel class of proteins which bind to mRNAs at specific sites. Yet, how these proteins work is poorly understood because few have been isolated or genetically studied. From mitochondria of the yeast, Saccharomyces cerevisiae, we have purified a site-specific RNA binding protein which specifically interacts with a dodecamer sequence found only at the 3' ends of all mitochondrial mRNAs. In vitro, this protein protects mRNAs from digestion by a unique, nucleoside triphosphate (NTP) dependent 3' exoribonuclease that was purified from yeast mitochondria. Since the pathway of mitochondrial RNA decay in yeast apparently initiates with an attack on an mRNA by that exoribonuclease, it appears that the binding protein is instrumental for the regulation of mRNA stability in the organelle. The objective of the proposed studies is to test the hypothesis that the mRNA binding protein regulates the activity of the 3' exoribonuclease towards mRNAs and thereby regulates the stability of mRNAs. The gene encoding the binding protein will be cloned and mutants of the binding protein will be isolated. RNA stability and processing in these mutants will be analyzed to investigate the function of the binding protein in vivo. The interaction of the purified protein with mitochondrial mRNAs and synthetic RNA oligonucleotides will be investigated in vitro in order to correlate its biochemical features with its functions in vivo. RNA processing and stability will be characterized in mutants of the NTP-dependent 3' exoribonuclease in order to further delineate the pathway of RNA decay in mitochondria and the role of the mRNA binding protein in that process. *** Most genes function by first being transcribed into messenger RNAs (mRNA) which exit the nucleus t o be translated into proteins in the cytoplasm. The activity of a gene is therefore determined mainly by three regulated processes: (i) the transcription rate of the gene, (ii) the translation rate of its mRNA, and (iii) the stability of that mRNA. Although much has been learned about the first two processes, little is known about the last. We will investigate the molecular mechanisms that regulate the life-time of mRNAs in the cell. Our model system is the mitochondria of yeast. The advantages of this model are (i) that mitochondrial RNA metabolism is considerable less complex than that for cytoplasmic mRNAs, and (ii) that yeast offer very powerful genetic methods for identifying and studying the genes and their products which are important for regulating mRNA stability. As for cytoplasmic mRNA stability, we have evidence that the life-time of mitochondrial mRNAs is regulated by their interaction with a member of a special class of proteins which bind to RNAs at specific locations. These site-specific RNA binding proteins are proving to be novel gene regulators whose mode of action is still unclear. We will identify the gene for the mitochondrial site-specific RNA binding protein. We will study its function genetically in the cell by examining gene mutants. We will study its function biochemically by characterizing its binding properties to chemically synthesized RNAs. We think that investigating the properties of this regulator of mitochondrial mRNA stability will provide important new information about this newly discovered class of gene regulators. %%%
