Discovery of novel mechanisms that impact CFTR translation and contribute to cystic fibrosis pathogenesis Abstract This project is intended to advance high throughput yeast phenomics technology and provide new mechanistic information regarding a model genetic disease, cystic fibrosis (CF). When clinically significant mutations in the CF transmembrane conductance regulator (CFTR) are engineered in a yeast homolog (YOR1), genome-wide phenomic analysis using the yeast deletion strain library (YDSL) identifies novel pathways that impact CF molecular phenotype. New experimental targets for CF therapy also result from studies of this type. Under Specific Aim 1 of the renewal, we show ways in which genome-wide yeast studies can advance understanding of CFTR variants including nonsense (Type 1) and severe folding (Type 2) defects for which no “personalized” modulator treatment currently exists. We also demonstrate phenomics can be applied to elucidate pathways necessary for pharmacologic rescue. Specific Aim 2 engages leading-edge technology (ribosomal profiling, deep-sequencing based on modification mapping (Psi-Seq)) to delve deeply into novel (and unexpected) mediators of CFTR biogenesis based on analysis of mammalian translational efficiency. In particular, we examine the role of ribosomal “collisions” or “queueing” described by phenomic analysis, and mRNA pseudouridinylation for effects on CFTR translation and protein synthesis. Specific Aim 3, applies innovative CF cell models to establish clinical significance of findings from Aims 1 and 2. The proposed studies will test important hypotheses relevant to CF pathogenesis, provide new knowledge regarding translational velocity and mRNA utilization during CFTR expression, and show that ribosomal collisions and pseudouridinylation contribute to cystic fibrosis. Our findings will also help advance new targets for treating refractory forms of CF. Lessons learned from the studies can be applied to many other inherited and serious human illnesses.