tRNAs are exquisitely designed for their role in translation, since their overall structures are uniform enough for comparable use by the translation apparatus, yet different enough for specific aminoacylation and specific decoding during translation. tRNA is by far the most common RNA species found in cells, and its maturation requires extensive processing and a large number of modifications. In the yeast Saccharomyces cerevisiae, the average cytoplasmic tRNA bears 13 modified residues, and there are a total of 25 different chemical modifications in different tRNAs. These tRNA modifications are very highly conserved in different organisms, including humans. However, understanding the roles of many modifications has been elusive, particularly for the large number that are remote from the anticodon, because the corresponding mutants have no obvious phenotype. The long term goal of this project is to define the roles of tRNA modifications found in yeast. This laboratory has shown that yeast mutants lacking m7G in their tRNA (trm8 or trm82 mutants) and lacking any one of seven other modifications remote from the anticodon, have severe growth defects at elevated temperature, whereas cells that lack other combinations of these modifications have no defect. In particular, trm8 trm4 mutants, which lack m7G and m5C, die at elevated temperature because their tRNAVal(AAC) is rapidly degraded by a previously undescribed quality control pathway that acts on mature tRNA. This is the first description of a pathway that removes mature tRNA in the cell, and this pathway differs from the only other previously described tRNA quality control pathway, which acts instead on pre-tRNA in the nucleus through the poly(A) polymerase Trf4 and the nuclear exosome. This laboratory has also examined formation of the unique extra guanine nucleotide residue found at the -1 position (G-1) of the 5'end of tRNAHis in virtually all organisms, a position that is normally unoccupied in the vast majority of tRNAs. The essential tRNAHis guanylyltransferase (Thg1) that adds G-1 to tRNAHis was identified, shown to recognize tRNAHis through its anticodon, and shown to catalyze an unusual reverse 3'-5'polymerase activity in this laboratory, and implicated by others in cell cycle progression. This proposal has four broad aims. (1) To identify the components of the tRNAVal(AAC) degradation pathway in trm8 trm4 mutants (2) To determine the mechanism of tRNAVal(AAC) degradation. (3) To examine the essential roles of Thg1 in the cell and the importance of G-1 for tRNAHis function, and (4) To identify the roles of other modifications. Recently, several modifications and tRNA processing enzymes have been implicated in conditions that affect human health. The proposed studies should cast light on these processes in humans.PROJECT NARRATIVE Synthesis of all proteins in cells requires decoding of the messenger RNA by transfer RNA (tRNA) in the ribosome. Recent studies have shown that a number of human health conditions and diseases are associated with defects in enzymes that process tRNA and catalyze the formation of tRNA modifications that are important for its function in the cell. This project is directed toward study of the role of tRNA modifications in the model eukaryotic organism Saccharomyces cerevisiae, in which it is possible to study tRNA processing and modifications in great detail, for subsequent application to human conditions.