We propose to continue our studies on N-terminal acetylation and on the DRN (degradation of mRNA in the nucleus) pathway in Saccharomyces cerevisiae, two areas of research that are conceptually unrelated, but that originated from investigations with the iso-1 -cytochrome c system. N-terminal acetylation is the one of most common protein modification in eukaryotes, occurring on approximately 85% of the different varieties of cytosolic mammalian proteins, on approximately 50% of yeast proteins, but rarely on prokaryotic proteins. The lack of N-terminal acetylation of at least some proteins leads to the loss or diminution of their functions. Our long term goal is the identification and characterization of N-terminal acetyltransferases (NATs), their substrates, their physical structures, and the functions of N-terminal acetylation of specific proteins. Yeast contains five NATs, designated NatA, NatB, NatC, NatD and NatE, with each having a different catalytic subunit and with all, but NatD, having auxiliary subunits. We propose to further investigate the subunit composition of NatE and to define minimal amino acid sequence required for acetylation by NatD. Studies of the physical structure of the NATs will be continued. We will determine if human and yeast NATs differ in their substrate specificity. We recently discovered a novel means of gene regulation that occurs by degrading a subset of normal mRNAs in the nucleus by a pathway designated DRN. Our working hypothesis is that DRN acts on all normal mRNAs, with the degree of degradation dependent on the degree of their nuclear retention. The major components of DRN are a subunit of the nuclear cap binding complex, Cbdp, and a nuclear exosome component, Rrp6p. Furthermore, DRN activity is regulated by the level of Cbdp; deletion of Cbdp diminishes DRN, whereas overproduction enhances DRN. We further suggested that these special class of normal mRNAs are differentially retained in the nucleus because of difference in their structures. We propose to continue our studies on the further characterization of DRN by identifying unknown components of the system and unknown elements in mRNAs causing susceptibility to DRN. Our studies with yeast have direct bearings to normal and pathological conditions of humans, including, for example, diseases such as polymyositis and scleroderma, which have diminutions in Rrp6p. Human NAT subunits are essential for development and are over-expressed in thyroid and hepatocellular carcinomas. ? ? ?
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