How are introns spliced from tRNA molecules in the yeast Saccharomyces cerevisiae? tRNA splicing is essential in yeast. Study of tRNA splicing in yeast is a paradigm for splicing in a wide variety of organisms since the mechanism of splicing is highly conserved in these organisms. Splicing involves excision of the intron by an endonuclease, ligation of the resultant half- molecules by a ligase, and removal of a 2'-phosphate left at the splice junction. Since this phosphate is one base 3' of the tRNA anticodon its removal is likely necessary to generate functional tRNA. Removal of this 2'-phosphate is the subject of this proposal. The PI has recently identified and partially purified a protein from yeast that can efficiently remove the splice junction phosphate from ligated tRNA. This protein is unique: it is specific for 2'-phosphorylated substrates, requires NAD+ and acts as a phosphotransferase. Its activity also appears to be affected by cells carrying a tpd1 mutation. He proposes to determine how NAD+ is used in dephosphorylation of ligated tRNA and to identify the proteins involved in this reaction. He also proposes to determine how TPD1 protein affects dephosphorylation of ligated tRNA in the cell, and to study the interaction of the 2'-phosphotransferase with other components of the tRNA splicing machinery.
