Project Summary This research program focuses on tRNA biosynthesis and its subcellular trafficking. In addition to their essential role in protein synthesis, tRNAs are required for nutrient signaling, regulation of apoptosis, protein degradation, and priming retroviral reverse transcription. tRNA biogenesis requires a complex set of conserved gene products for post-transcriptional processing and subcellular traffic. 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. tRNA dynamics consist of 3 steps: “primary export” of tRNA from the nucleus to the cytoplasm, “retrograde nuclear import” of cytoplasmic tRNA into the nucleus, and “re-export” of the imported tRNAs back to the cytoplasm. The mechanisms by which tRNAs move between the nucleus and the cytoplasm are not completely understood. Because tRNA nuclear export is essential and the known exporters are unessential, we conducted an unbiased genome-wide screen in yeast to search for the missing tRNA nuclear exporter(s). We discovered that mutations of two pathways utilized for protein (Crm1) and mRNA (Mex67-Mtr2) nuclear export also cause defective tRNA nuclear export; the data support the model that Crm1 and Mex67- Mtr2 function in tRNA nuclear export. Aim 1 employs in vivo biochemical analyses to test the hypothesis that tRNAs directly interact with the Crm1 and/or Mex67-Mtr2 nuclear export machinery and to learn how these alternative nuclear export pathways recognize tRNA substrates. Aim 1 also seeks to test whether a candidate tRNA nuclear importer physically interacts with tRNA. Aim 2 addresses the biological function for tRNA bi- directional traffic between the nucleus and cytoplasm. We discovered that one function is for tRNA quality control that prevents aberrant tRNA from interacting with the protein synthesis machinery. Aberrant tRNAs reach the cytoplasm, in part, due to error-prone nuclear export. We will analyze the fidelity of the parallel tRNA nuclear export pathways and determine whether aberrant tRNAs are corrected and/or destroyed upon retrograde import into the nucleus. Aim 3 addresses RNA processing steps that occur on the mitochondrial membrane. Pre-tRNA splicing in yeast and piRNA processing in metazoans occur on the mitochondrial surface. We identified proteins that likely function in directing the tRNA splicing endonuclease and/or tRNAs to mitochondria and we propose to test these roles. The information gained should inform how and why the mitochondrial surface functions as a “warehouse” for RNA processing. Thus, the proposed research program impacts upon multiple facets of gene expression, quality control, and issues important to human health.