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 3

Silencing RPL12 enhances F508del-CFTR functional expression in primary HBE.

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Silencing RPL12 enhances F508del-CFTR functional expression in primary H...
(A) Following RPL12 knockdown, endogenous expression of mature, fully glycosylated F508del protein (band C, white arrowhead) is compared with levels of immature glycoform (band B, black arrowhead). Amounts of band B, band C, and RPL12 are quantified on right. Data are represented as mean ± SEM obtained from siRPL12-treated cells normalized to NS siRNA controls (dotted line set to 100%) (n = 3). Asterisks represent statistical comparison between siRPL12 and NS siRNA. *P < 0.0167; **P < 0.01 (unequal variance t test on log2-transformed data with post hoc Bonferroni’s correction; α = 0.0167). (B) Conversion ratio of band C to bands B and C (i.e., test of maturation efficiency) from A. Data include mean ± SEM (n = 3). ***P < 0.001 (2-sample t test). (C) F508del-mediated transepithelial ion transport (ISC) is augmented by RPL12 depletion (n = 3). Asterisks represent statistical comparison of forskolin+genistein stimulation (i.e., total constitutive plus acutely potentiated CFTR function). ****P < 0.0001, 2-sample t test. Fsk, forskolin; Gen, genistein. 48, deidentified patient code. (D) siRPL12 application enhances levels of cAMP-dependent CFTR ion transport (i.e., ratio of forskolin- to forskolin+genistein). Data are represented as mean ± SEM (n = 3). **P < 0.01, 2-sample t test. NS, NS siRNA; siRPL12, RPL12-targeting siRNA; Forskolin (5 μM), activator of PKA; genistein (50 μM), stimulator of CFTR-gating activity (i.e., potentiator); Inh172, 10 μM, inhibitor of CFTR. HBE were isolated from a CF individual with CFTRF508del/F508del genotype, cultured at air-liquid interface, and transfected twice per week with siRPL12 or NS siRNA (100 nM) for 3 weeks.