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 December 2, 2019; First 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 6

In FRT epithelia, Rpl12 inhibition is additive with concomitant F508del-CFTR second-site suppression by R555K or R1070W mutations.

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In FRT epithelia, Rpl12 inhibition is additive with concomitant F508del-...
(A) F508del-R555K immature (band B, black arrowhead) and mature (band C, white arrowhead) protein levels are markedly enhanced following approximately 50%–70% knockdown of Rpl12. Representative immunoblot (left panel) is quantified on right (n = 3). (B) Rpl12 siRNAs significantly increase F508del-R555K-HRP trafficking to the PM (n = 9). (C) ISC measurements using F508del-R555K demonstrate an increase following Rpl12 inhibition (n = 4). (D) Augmentation of F508del-R1070W-CFTR band B and band C expression following an approximately 50%–60% reduction in Rpl12 protein levels. Representative immunoblot (left panel) is quantified on right (n = 3). (E) Rpl12 knockdown significantly increases F508del-R1070W-HRP at the cell surface (n = 12). (F) ISC measurements using F508del-R1070W demonstrate an increase in CFTR-dependent ion transport following Rpl12 suppression (n = 4). Data in A and D are represented as mean ± SEM obtained from siRPL12-treated cells normalized to NS siRNA (dotted line set to 100%). Asterisks represent statistical comparison between siRPL12 and NS control. *P < 0.0167; **P < 0.01, unequal variance t test on log2-transformed data with post-hoc Bonferroni’s correction; α = 0.0167. Data in B and E data are represented as mean ± SEM. *P < 0.0083; **P < 0.001; ****P < 0.00001, unequal variance t test with post-hoc Bonferroni’s correction; α = 0.00833. Data in C and F data are represented as mean ± SEM. Asterisks represent statistical comparison of forskolin+VX-770 stimulation (i.e., total constitutive plus activated CFTR function). *P < 0.0125; **P < 0.001; ***P < 0.0001; ****P < 0.00001, unequal variance t test with post-hoc Bonferroni’s correction; α = 0.0125. RPL12_6, _7, _9, and _11 denote RPL12 siRNAs with crossreactivity against rat mRNA; forskolin, 5 μM; VX-770, 5 μM; Inh172, 10 μM.