Poster Session Abstracts

Newly synthesized domains of the cystic fibrosis transmembrane conductance regulator (CFTR) fold cotranslationally and then undergo cooperative domain-domain assembly. Folding process during synthesis by the ribosome has biologically evolved to reduce misfolding and maximize native folding outcome. During this process, the rate of translation is controlled by several factors including, codon usage, tRNA availability, mRNA structure, RNA binding proteins, and ribosome cofactors. In this study, we monitored the process of translation using ribosome profiling to obtain a high-resolution and quantitative profile of the location of actively translating ribosomes across the entire cellular transcriptome. Ribosome-protected fragments (“footprints”) were isolated and sequenced by next-generation sequencing from HEK293 cells expressing exogenous CFTR proteins. Footprints were predominantly recovered on protein coding region within mRNA, and showed a three-nucleotide periodicity, indicating that they originated from actively engaged ribosomes. Footprint density, which inversely correlates with the rate of translation, varied across CFTR mRNA. Transmembrane domain 1 (TMD1) was among CFTR domains with the highest ribosome occupancy, whereas nucleotide binding domain 2 (NBD2) had the lowest (TMD1>>TMD2~R~NBD1>NBD2). Assuming that all engaged ribosomes generate a full length CFTR protein, this suggests that TMD1 is most slowly translated, while NBD2 is translated at the fastest rate. Particularly high footprint occupancy was observed at the start codon region, which is mainly caused by bias of cycloheximide used for stabilizing ribosomes. Within TMD1 and TMD2, slower translation was observed for extracellular loop 2 (EL2) and EL5 regions respectively. Considering that the ribosome occludes about 40 amino acid residues within the exit tunnel, the result suggests that newly synthesized intracellular loop 1 (IL1) and IL3 emerge slowly from the ribosome during TMD folding. Few footprints were observed on the 3'UTR, implying efficient termination at the stop codon. Overall ribosome occupancy across CFTR coding region, however, did not correspond well to a predicted relative rate of CFTR translation using an algorithm devised by Spencer et al. (J Mol Biol 2012;422:328-35) that is based on the rate of tRNA abundance and mRNA codon-tRNA anticodon pairings, suggesting that additional factors may be involved. ΔF508 mutation impairs not only NBD1 folding but also interdomain assembly of CFTR. Recent studies on the mechanism of action of VX-809 have shown that it improves NBD1-TMD2 interdomain assembly by acting primarily on cotranslational folding. Ribosome profiling revealed no significant difference in the translation rate between wildtype and ΔF508 CFTR. Furthermore, C18, a VX-809 analog, also had no significant effects on CFTR translation as evidenced by overall ribosome occupancy across CFTR mRNA or location of ribosomes across the entire transcriptome. These results suggest that cotranslational effects of C18 on CFTR folding are not mediated by changes in translation per se, but rather operate at the level of domain folding and/or interdomain assembly. Supported by NIH and CFF. 2w NOVEL MODULATORS OF PROTEIN UBIQUITINATION IMPROVE VX-809-DEPENDENT RESCUE OF F508DEL-CFTR Goeckeler-Fried, J.; Chiang, A.; Larsen, M.; Chung, W.; Denny, R.; Weissman, A.; Watkins, S.C.; Sorscher, E.J.; Frizzell, R.A.; Brodsky, J.L. 1. Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA; 2. Center for Biologic Imaging, University of Pittsburgh, School of Medicine, Pittsburgh, PA, USA; 3. Pediatrics, Emory University School of Medicine, Atlanta, GA, USA; 4. Pfizer Worldwide Medicinal Chemistry, Cambridge, MA, USA; 5. Protein Dynamics and Signaling, National Cancer Institute, Frederick, MD, USA; 6. Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA