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 2

RPL12 suppression slows translation rate in CFBE.

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RPL12 suppression slows translation rate in CFBE. (A) Comparison of RD v...
(A) Comparison of RD values for CFBE F508del-CFTR cells treated with RPL12-directed siRNA (siRPL12) or NS siRNA from ribosome-profiling data. Diagonal denotes the position of equivalent RD values. (B) Expression of specific translational machinery proteins assessed by ribosome profiling. RPLs, ribosomal proteins (large subunit); RPSs, ribosomal proteins (small subunit); eIFs, eukaryotic initiation factors. (C) Cumulative metagene profiles of RPFs as a function of position for all protein-coding genes detected over the threshold from ribosome profiling. Each transcript was weighted equally. Zero designates the first nucleotide of the start codon. (D) Ribosome-dwelling occupancy for cells treated with siRPL12 or NS siRNA as determined from ribosome profiling and compared with genome codon usage. Dashed line denotes the upper boundary (90% confidence interval). (E) Difference in ribosome occupancy (y axis) between NS and siRPL12-treated cells quantified from D for single codons (represented as dots) and for all codons (box plot on right). Top 10 most (black) or least (red) utilized codons are annotated. (F) Box plot of 200 most (high RD; P = 3.0 × 10–7) and least (low RD; P = 0.045) expressed genes in NS and siRPL12-treated cells. GC content of high- and low-RD groups is represented in red (right y axis). *P < 0.05; ****P < 0.0001, Wilcoxon’s rank sum test. (G) Profiles of ribosome-dwelling occupancy computed from D for CFTR mRNA in NS and siRPL12-treated cells. Single codons exhibiting an occupancy variance greater than the 95th percentile (*) are considered statistically significant.