Slowing ribosome velocity restores folding and function of mutant CFTR
Kathryn E. Oliver, … , Zoya Ignatova, Eric J. Sorscher
Kathryn E. Oliver, … , Zoya Ignatova, Eric J. Sorscher
Published October 28, 2019
Citation Information: J Clin Invest. 2019;129(12):5236-5253. https://doi.org/10.1172/JCI124282.
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Research Article Cell biology Genetics

Slowing ribosome velocity restores folding and function of mutant CFTR

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.

Authors

Kathryn E. Oliver, Robert Rauscher, Marjolein Mijnders, Wei Wang, Matthew J. Wolpert, Jessica Maya, Carleen M. Sabusap, Robert A. Kesterson, Kevin L. Kirk, Andras Rab, Ineke Braakman, Jeong S. Hong, John L. Hartman IV, Zoya Ignatova, Eric J. Sorscher

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Figure 8

In FRT cells, A455E-CFTR functional expression is partially rescued by siRPL12, whereas P67L-CFTR is unaffected.

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In FRT cells, A455E-CFTR functional expression is partially rescued by s...
(A) Following an approximately 30% knockdown of Rpl12, A455E-CFTR band B (black arrowhead) and band C (open arrowhead) are significantly increased (n = 3). (B) A455E-mediated transepithelial ion transport is augmented by Rpl12 depletion (100 nM siRNA, 4 days) and additive to treatment with small molecule correctors (VX-809 or VX-661, 3 μM, 48 hours) (n = 3). (C) P67L-CFTR band C is significantly enhanced as is band B (to a lesser extent) following addition of VX-809 or VX-661 (n = 3). (D) P67L-CFTR ion transport is markedly increased by either CFTR corrector (3 μM, 48 hours), yet unaltered by siRPL12 (100 nM, 4 days) (n = 3). Representative measurements in left panels are quantified on right. Data shown in A and C are represented as mean ± SEM normalized to NS siRNA without drug treatment (dotted line set to 100%). Asterisks represent statistical comparison versus NS siRNA without pharmacocorrection. *P < 0.0167; **P < 0.01; ***P < 0.001; ****P < 0.0001, unequal variance t test on log2-transformed data with post-hoc Bonferroni’s correction; α = 0.0167. Data shown in B and D are represented as mean ± SEM. Asterisks represent forskolin+VX-770 stimulation (i.e., total constitutive plus activated CFTR function) versus NS siRNA without drug treatment. *P < 0.00625; **P < 0.001; ***P < 0.0001, unequal variance t test with post-hoc Bonferroni’s correction; α = 0.00625. VX-809 or VX-661 augmented A455E-CFTR band C (P < 0.01) and activity (P < 0.001) above siRPL12 alone. Forskolin, 5 μM; genistein, 50 μM; Inh172, 10 μM.