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Ribosomal Stalk Protein Silencing Partially Corrects the ?F508-CFTR Functional Expression Defect.

ABSTRACT: The most common cystic fibrosis (CF) causing mutation, deletion of phenylalanine 508 (?F508 or Phe508del), results in functional expression defect of the CF transmembrane conductance regulator (CFTR) at the apical plasma membrane (PM) of secretory epithelia, which is attributed to the degradation of the misfolded channel at the endoplasmic reticulum (ER). Deletion of phenylalanine 670 (?F670) in the yeast oligomycin resistance 1 gene (YOR1, an ABC transporter) of Saccharomyces cerevisiae phenocopies the ?F508-CFTR folding and trafficking defects. Genome-wide phenotypic (phenomic) analysis of the Yor1-?F670 biogenesis identified several modifier genes of mRNA processing and translation, which conferred oligomycin resistance to yeast. Silencing of orthologues of these candidate genes enhanced the ?F508-CFTR functional expression at the apical PM in human CF bronchial epithelia. Although knockdown of RPL12, a component of the ribosomal stalk, attenuated the translational elongation rate, it increased the folding efficiency as well as the conformational stability of the ?F508-CFTR, manifesting in 3-fold augmented PM density and function of the mutant. Combination of RPL12 knockdown with the corrector drug, VX-809 (lumacaftor) restored the mutant function to ~50% of the wild-type channel in primary CFTR?F508/?F508 human bronchial epithelia. These results and the observation that silencing of other ribosomal stalk proteins partially rescue the loss-of-function phenotype of ?F508-CFTR suggest that the ribosomal stalk modulates the folding efficiency of the mutant and is a potential therapeutic target for correction of the ?F508-CFTR folding defect.

SUBMITTER: Veit G

PROVIDER: S-EPMC4864299 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Ribosomal Stalk Protein Silencing Partially Corrects the ΔF508-CFTR Functional Expression Defect.

Veit Guido G Oliver Kathryn K Apaja Pirjo M PM Perdomo Doranda D Bidaud-Meynard Aurélien A Lin Sheng-Ting ST Guo Jingyu J Icyuz Mert M Sorscher Eric J EJ Hartman John L JL Lukacs Gergely L GL

PLoS biology 20160511 5

The most common cystic fibrosis (CF) causing mutation, deletion of phenylalanine 508 (ΔF508 or Phe508del), results in functional expression defect of the CF transmembrane conductance regulator (CFTR) at the apical plasma membrane (PM) of secretory epithelia, which is attributed to the degradation of the misfolded channel at the endoplasmic reticulum (ER). Deletion of phenylalanine 670 (ΔF670) in the yeast oligomycin resistance 1 gene (YOR1, an ABC transporter) of Saccharomyces cerevisiae phenoco ...[more]

PMID: 27168400

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Project description:BackgroundWe previously reported that expression of a miR-138 mimic or knockdown of SIN3A in primary cultures of cystic fibrosis (CF) airway epithelia increased ΔF508-CFTR mRNA and protein levels, and partially restored CFTR-dependent chloride transport. Global mRNA transcript profiling in ΔF508-CFBE cells treated with miR-138 mimic or SIN3A siRNA identified two genes, SYVN1 and NEDD8, whose inhibition significantly increased ΔF508-CFTR trafficking, maturation, and function. Little is known regarding the dynamic changes in the CFTR gene network during such rescue events. We hypothesized that analysis of condition-specific gene networks from transcriptomic data characterizing ΔF508-CFTR rescue could help identify dynamic gene modules associated with CFTR biogenesis.MethodsWe applied a computational method, termed M-module, to analyze multiple gene networks, each of which exhibited differential activity compared to a baseline condition. In doing so, we identified both unique and shared gene pathways across multiple differential networks. To construct differential networks, gene expression data from CFBE cells were divided into three groups: (1) siRNA inhibition of NEDD8 and SYVN1; (2) miR-138 mimic and SIN3A siRNA; and (3) temperature (27 °C for 24 h, 40 °C for 24 h, and 27 °C for 24 h followed by 40 °C for 24 h).ResultsInterrogation of individual networks (e.g., NEDD8/SYVN1 network), combinations of two networks (e.g., NEDD8/SYVN1 + temperature networks), and all three networks yielded sets of 1-modules, 2-modules, and 3-modules, respectively. Gene ontology analysis revealed significant enrichment of dynamic modules in pathways including translation, protein metabolic/catabolic processes, protein complex assembly, and endocytosis. Candidate CFTR effectors identified in the analysis included CHURC1, GZF1, and RPL15, and siRNA-mediated knockdown of these genes partially restored CFTR-dependent transepithelial chloride current to ΔF508-CFBE cells.ConclusionsThe ability of the M-module to identify dynamic modules involved in ΔF508 rescue provides a novel approach for studying CFTR biogenesis and identifying candidate suppressors of ΔF508.

Dataset Information

Ribosomal Stalk Protein Silencing Partially Corrects the ?F508-CFTR Functional Expression Defect.

Publications

Ribosomal Stalk Protein Silencing Partially Corrects the ΔF508-CFTR Functional Expression Defect.

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