Project Summary/Abstract tRNAs are well-known as the physical adaptors of the genetic code, bridging codon- based information with the appropriate amino acids required for protein translation. tRNA function can be regulated by a wide variety of mechanisms, including the modulation of tRNA aminoacylation by cellular amino acid levels. Two previously underappreciated mechanisms have recently emerged as key regulators of tRNA biology – covalent modifications of tRNA nucleotides, and cleavage of tRNAs, which generates stable and potentially functional tRFs. Here, we propose to investigate the regulation of tRNA cleavage, and the functions of resulting tRNA fragments, in budding yeast and in mammalian embryonic stem cells. First, as tRNA nucleotide modifications have been shown to interfere with tRNA cleavage in several model organisms, we will first survey the effects of all known tRNA modifications on tRNA cleavage in yeast. Deep sequencing of small RNAs will be carried out for a large collection of yeast deletion mutants – covering all nonessential tRNA-modifying enzymes known at present – following overexpression of various tRNA nucleases. In parallel, we will develop a comprehensive panel of conditional mutants in tRNA modification machinery in murine ES cells to extend these studies to a mammalian model. A complementary effort will focus on the roles for tRNA fragments in translational control. Again comparing yeast and mammals, we will characterize translation by ribosome footprinting in wild-type and in mutants lacking tRNA cleavage or various tRNA modifications. To allow biochemical access to specific tRNA fragment activities, we will use in vitro translation systems to directly investigate the roles for various purified or synthetic tRNA fragments at well-defined stages of protein translation. Together, these experiments systematically address the roles for tRNA modifications in tRNA cleavage and control of translation by resulting tRNA fragments, providing important insights with relevance to diseases ranging from neurodegeneration to cancer.