Project Summary This research program focuses on tRNA biology and its subcellular trafficking. tRNAs are small noncoding RNAs that are essential for decoding the genome by delivering amino acids to translating ribosomes according to codon directions in mRNAs. Defects in tRNA biology cause numerous human disorders from metabolic diseases, to neuromuscular diseases, and to cancer. tRNA biology requires a complex set of conserved gene products for post-transcriptional processing, subcellular traffic, and intron turnover. We employ budding yeast and in vivo technologies to discover unknown important aspects of tRNA biology. In Aim 1 of this proposal we will study tRNA nuclear export. It is not completely understood how tRNAs that are transcribed and partially processed in the nucleus are exported to the cytoplasm for their iterative function in translation. One pathway utilizes the conserved b-importin Los1/Exportin that is dedicated to tRNA nuclear export, but it is unessential in all tested organisms. Employing an unbiased genome-wide screen for gene products involved in tRNA biology, we discovered three additional gene products that export tRNA to the cytoplasm: the heterodimer, Mex67-Mtr2 and Crm1. However, Mex67-Mtr2 and Crm1 are not dedicated to tRNA and they have major roles in mRNA and protein nuclear export. Thus, it is not understood how they recognize tRNAs. Moreover, Mex67-Mtr2 appears to be error-prone, delivering tRNA to the cytoplasm prior to removal of leader/trailer sequences. We will identify adapters needed to complex Mex67-Mtr2 and Crm1 with tRNAs and learn how mistakes by the error-prone exporters are dealt with. In Aim2 of this proposal we will study trafficking of tRNAs between the nucleus and the cytoplasm. Although for decades it was thought that tRNA movement is unidirectional, nucleus to cytoplasm, we co-discovered that tRNAs move bi-directionally between the nucleus and the cytoplasm and that the dynamics are conserved between yeast and vertebrate cells. We developed a new methodology, the HCl/aniline assay, that reports tRNA retrograde nuclear import and re-export to the cytoplasm. We will employ this methodology to characterize the gene products that function in the tRNA retrograde pathway and assess whether tRNA retrograde traffic is iterative. Aim 3 addresses tRNA introns. Possession of tRNA introns in subsets of tRNA genes is conserved from Archaea to humans. Although the mechanism to remove introns from pre-tRNAs is understood, the fate and function of tRNA introns is largely mysterious. We discovered one mechanism for tRNA intron turnover; however, there are at least four additional unknown mechanisms to destroy tRNA introns which we propose to characterize. Surprisingly, we also learned that under particular stresses, tRNA introns accumulate to high levels. Furthermore, tRNA introns contain long stretches of complementarity to mRNAs. Our preliminary data support the hypothesis that they regulate gene express throug...