Slowing ribosome velocity restores folding and function of mutant CFTR

• Type of Material: Article
• Authors: Kathryn E. Oliver, Emory University; Robert Rauscher, University of Hamburg; Marjolein Mijnders, Utrecht University; Wei Wang, University of Alabama Birmingham; Matthew J. Wolpert, University of Alabama Birmingham; Jessica Maya, University of Alabama Birmingham; Carleen M. Sabusap, University of Alabama Birmingham; Robert A. Kesterson, University of Alabama Birmingham; Kevin L. Kirk, University of Alabama Birmingham; Andras Rab, Emory University; Ineke Braakman, Utrecht University; Jeong Hong, Emory University; John L. Hartman, IV, University of Alabama Birmingham; Zoya Ignatova, University of Hamburg; Eric Sorscher, Emory University
• Language: English
• Date: 2019-12-02
• Publisher: AMER SOC CLINICAL INVESTIGATION INC
• Publication Version: Final Published Version
• Copyright Statement: © 2019, American Society for Clinical Investigation.
• Final Published Version (URL): http://dx.doi.org/10.1172/JCI124282
• Title of Journal or Parent Work: JOURNAL OF CLINICAL INVESTIGATION
• Volume: 129
• Issue: 12
• Start Page: 5236
• End Page: 5253
• Grant/Funding Information: Funding was provided by the NIH (F31HL131231, R01HL136414, P30DK072482), the Cystic Fibrosis Foundation (OLIVER17F0, BRAAKM14XXO, HARTMA15G0, IGNATO17XXO, SORSCH13XXO, SORSCH14XXO), the Burroughs Wellcome Fund (Collaborative Research Travel Grant, 1018774), Deutsche Forschungsgemeinschaft FOR1805 (IG74/14-1, IG74/14-2), the German Cystic Fibrosis Foundation (muko e.V. 1603), and the Netherlands Science Organization (graduate school program grant).
• Keywords: PROTEIN; Medicine, Research & Experimental; MUTATION; IN-VIVO; TRANSMEMBRANE CONDUCTANCE REGULATOR; TRANSLATION; DELTA-F508 CFTR; CYSTIC-FIBROSIS; Life Sciences & Biomedicine; RESCUE; DOMAIN; MESSENGER-RNA; Research & Experimental Medicine; Science & Technology
• Research Categories: Chemistry, Biochemistry; Biology, Genetics

Abstract

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), with approximately 90% of patients harboring at least one copy of the disease-associated variant F508del. We utilized a yeast phenomic system to identify genetic modifiers of F508del-CFTR biogenesis, from which ribosomal protein L12 (RPL12/uL11) emerged as a molecular target. In the present study, we investigated mechanism(s) by which suppression of RPL12 rescues F508del protein synthesis and activity. Using ribosome profiling, we found that rates of translation initiation and elongation were markedly slowed by RPL12 silencing. However, proteolytic stability and patch-clamp assays revealed RPL12 depletion significantly increased F508del-CFTR steady-state expression, interdomain assembly, and baseline open-channel probability. We next evaluated whether Rpl12-corrected F508del-CFTR could be further enhanced with concomitant pharmacologic repair (e.g., using clinically approved modulators lumacaftor and tezacaftor) and demonstrated additivity of these treatments. Rpl12 knockdown also partially restored maturation of specific CFTR variants in addition to F508del, and WT Cftr biogenesis was enhanced in the pancreas, colon, and ileum of Rpl12 haplosufficient mice. Modulation of ribosome velocity therefore represents a robust method for understanding both CF pathogenesis and therapeutic response.