Project Summary The equipment supplement funds will facilitate parent grant GM122448 (A.K. Hopper, PI) by supporting purchase of an Integra Mini 96-channel pipetter which will greatly facilitate original proposed work in Aims 2 and 3. GM122448 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. The research program impacts upon multiple facets of gene expression, quality control, and issues important to human health. We employ budding yeast and in vivo technologies to discover unknown important aspects of tRNA biology. In Aim2 of GM122884 we 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 (Shaheen & Hopper 2005) and that the dynamics are conserved between yeast and vertebrate cells (Shaheen et al. 2007). We developed a new methodology, the HCl/aniline assay (Nostramo et al. 2020), that reports tRNA retrograde nuclear import and re-export to the cytoplasm. We are employing this methodology in a genome-wide screen of ~6000 yeast genes to identify and characterize the proteins functioning in the tRNA retrograde pathway. Aim 3 of GM122884 investigates tRNA introns. Possession of tRNA introns in subsets of tRNA genes has been conserved from Archaea to humans. We recently made the exciting discovery (Nostramo et al., Mol. Cell, in revision) that introns spliced from pre-tRNAs provide a novel complementary-dependent mechanism to fine-tune basal mRNA levels and to assist cellular responses to stress. We also learned that under particular stresses, subsets of tRNA introns accumulate to high levels due to increased stability. We discovered one mechanism for tRNA intron turnover (Wu & Hopper 2014); however, there are at least four additional unknown mechanisms to destroy tRNA introns. None of the yeast annotated RNases appear to function in the unknown turnover pathways. Thus, we are conducting a genome-wide screen for mutants that accumulate each of the 8 tRNA intron families whose turnover remains unknown. Although we are able to grow yeast mutants in the deep 96-well plates, we are unable to extract RNAs in this format due to the opaqueness of the microtiter plates; therefore, we must conduct the biochemical steps in labor-intensive analyses of single mutants. The Integra Mini 96-channel pipettes will allow us to conduct all steps preceding northern gel analyses for 96 strains simultaneously and thereby greatly reduce the time and...