The goal of this proposal is to understand some surprising results concerning the mechanism by which tRNA is spliced in eukaryotes. tRNA splicing is essential for the survival of some, if not all, eukaryotes, including the yeast Saccharomyces cerevisiae and humans. The mechanism of tRNA splicing is highly conserved, and best understood in yeast. Superficially, tRNA splicing appears simple: an endonuclease excises the intron; a ligase joins the two half-molecules while generating a 2'-phosphate at the splice junction; and then the 2'-phosphate is removed. Yet the mechanism of splicing, particularly of the last step, is much more complicated than one would have guessed. Removal of the splice junction 2'-phosphate, rather than being catalyzed by a phosphatase, is catalyzed by an NAD- dependent, 2'-phosphate-specific phosphotransferase. Moreover, the phosphate is transferred to NAD to form a novel cellular metabolite: ADP- ribose I""""""""-2"""""""" cyclic phosphate (Appr>p). This means that removal of the splice junction 2'-phosphate is at least four chemical steps: two to form Appr>p and at least two to return this molecule to a normal cellular metabolite. This constitutes a new metabolic pathway that takes place on a relatively large scale in yeast and other organisms. Both the mechanism by which Appr>p is formed and its metabolic fate are unknown. Thus, identifying the protein that catalyzes the phosphotransferase reaction, deducing how this unusual NAD derivative is made, and following its metabolic fate is a high priority of this proposal. A second goal of this proposal is to understand a perplexing fact about tRNA splicing in vertebrates. Although the three splicing enzymes in yeast are absolutely required in yeast and highly conserved in plants and vertebrates, vertebrates have another ligase, which does not generate a 2 -phosphate splice junction and which has been implicated in tRNA splicing in vivo in Xenopus oocytes. Thus, for vertebrates the yeast-like pathway appears to be redundant, despite the fact that all of its enzymes are still there. This suggests that the yeast pathway is required in vertebrates either to ensure a supply of Appr>p for other purposes, or to splice tRNA under certain conditions of growth or differentiation. To approach this question, the vertebrate form of the yeast ligase or phosphotransferase will be cloned to examine its expression in different conditions.
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