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 5

RPL12 depletion stabilizes CFTR interdomain assembly.

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RPL12 depletion stabilizes CFTR interdomain assembly. (A) WT-CFTR with d...
(A) WT-CFTR with domains annotated: TMD1 (dotted); NBD1 (black); regulatory domain (R) (white); TMD2 (light gray); NBD2 (dark gray). N, amino; C, carboxy. (B) CFBE expressing WT- or F508del-CFTR and treated with siRPL12 or NS siRNA were pulse labeled for 15 minutes and chased for 0–2 hours. RPL12 silencing was confirmed by immunoblotting (with tubulin-loading control). Lysates were digested with proteinase K and protease-resistant CFTR domain fragments immunoprecipitated/resolved by SDS-PAGE. Immature, ER-resident CFTR (band B, black arrowhead), fully glycosylated, mature CFTR (band C, white arrowhead), early domain fragments (T1a-c, N1a, T2a-b, N2a), and late domain fragments (T1d-f, T2b-c) are designated (see Methods). V, empty vector; asterisk indicates background. Brief chase applied here shows F508del-CFTR exits the ER slowly (i.e., band B at 2 hours remains significantly elevated following siRPL12 treatment), yet is stabilized in post-ER compartments, as evidenced by moderate band C accumulation (see also ref. 59). Increased proteolytic fragments predominantly reflect the ER-localized glycoform. (C) Quantification of RPL12 knockdown (n = 3–4). (D) Comparison of total radiolabeled CFTR (bands B and C) following RPL12 repression (n = 4–5). (E and F) Fold increase of domain-specific fragment intensities for WT- (E) or F508del-CFTR (F). Bands are corrected for total radiolabeling (n = 4–5). Note NS-treated F508del signal in the 2-hour chase is less than 2-fold over background in D and F. (C–F) Data are shown as mean ± SEM from siRPL12-treated cells normalized to NS siRNA (dotted line set to 100% or 1 as shown). Asterisks represent statistical comparison between siRPL12 and NS. Unequal variance t test on log2-transformed data with post-hoc Bonferroni’s correction. α = 0.025 (C and D); α = 0.0125 (E and F). **P < 0.01; ***P < 0.001 (C and D). *P < 0.0125; **P < 0.001; ***P < 0.0001 (E and F).