Tuning translation efficiency to overcome refractory defects in CFTR

PROJECT SUMMARY (ABSTRACT) Approximately 100,000 people worldwide are living with cystic fibrosis (CF), a lethal autosomal recessive disorder caused by mutation of the CF transmembrane conductance regulator (CFTR). More than 2,100 disease-associated CFTR variants have been identified to date. Although deletion of phenylalanine 508 (F508del) remains the most commonly reported defect, ~13% of people with CF harbor premature termination codons (PTCs) in CFTR. These variants result in little-to-no protein expression, as well as unstable mRNA susceptible to degradation by nonsense mediated decay. CFTR PTCs are associated with more severe clinical manifestations and are unresponsive to clinically approved therapies. An overarching goal of our work is to identify cellular targets that might ameliorate disease by correcting the basic defect resulting from these abnormalities. Our previous yeast phenomic analyses led to discovery of specific ribosomal proteins (RPs) as effectors of F508del-CFTR trafficking. With K99/R00 support, we established that depletion of RPL12 (uL11) rescues F508del by diminishing rates of translation initiation and elongation, thereby allowing the ribosome and/or associated chaperones to promote a functional protein conformation. Findings outlined in the present R01 demonstrate that suppression of RPL12 and other RPs such as RPL8 (uL2) also correct two rare PTCs, W1282X- and G542X-CFTR. Thus, we hypothesize that ribosomal protein depletion alters translational velocity, ribosome fidelity, and/or mRNA utilization to partially rescue synthesis and assembly of CFTR nonsense variants. We will investigate this hypothesis according to three specific aims: (1) ascertain effect(s) of ribosomal protein inhibition on biogenesis of CFTR PTCs; (2) define mechanism(s) by which silencing ribosomal proteins alters translation kinetics and/or mRNA surveillance pathways to rescue CFTR nonsense alleles; and (3) determine outcomes of ribosomal protein disruption in humanized CF mice. We will employ multidisciplinary expertise directed towards mammalian physiology, biochemistry, molecular genetics, and computational biology to mechanistically address a fundamental question regarding new ways the ribosome influences mRNA utilization and protein structure. The studies are intended to establish translational control as a novel and critical checkpoint during CFTR PTC processing, and identify specific ribosomal proteins in addition to RPL12 and RPL8 that mediate this pathway. Such results will improve understanding of CF disease mechanism, establish safety of repressing ribosomal proteins in CF animals, and provide a basis for testing relevance of the strategy in other inherited human disease states. Emory University provides a rich environment for support of early-stage investigators and leverages state-of-the-art facilities in a highly collaborative academic research center. NIH support of the studies proposed in the current application would catalyze the PI (Dr. Oliver) and her efforts towards strengthening academic research independence.