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 phenocopies the ΔF508-CFTR folding and trafficking defects. Genome-wide phenotypic (phenomic) analysis of the Yor1-ΔF670 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 ΔF508-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 ΔF508-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^{ΔF508/ΔF508} human bronchial epithelia. These results and the observation that silencing of other ribosomal stalk proteins partially rescue the loss-of-function phenotype of ΔF508-CFTR suggest that the ribosomal stalk modulates the folding efficiency of the mutant and is a potential therapeutic target for correction of the ΔF508-CFTR folding defect. Cystic fibrosis (CF) is one of the most common autosomal recessive diseases in Caucasians. It is caused by mutations in the CF transmembrane conductance regulator (CFTR), which functions as an anion channel at the apical plasma membrane of secretory epithelia. The most common CF mutation, a deletion of the phenylalanine residue at position 508 (ΔF508), results in the channel misfolding and subsequent intracellular degradation. Our previous genome-wide phenotypic screens, using a yeast variant, have predicted modifier genes for ΔF508-CFTR biogenesis. Here, we show that silencing of one of these candidate genes, RPL12, a component of the ribosomal stalk, increased the folding and stabilization of ΔF508-CFTR, resulting in its increased plasma membrane expression and function. Our data suggest that reducing the translational elongation rate via RPL12 silencing can partially reverse the ΔF508-CFTR folding defect. Importantly, RPL12 silencing in combination with the corrector drug VX-809 (lumacaftor), increased the mutant function to 50% of the wild-type CFTR channel, suggesting that the ribosomal stalk perturbation may represent a therapeutic target for rescuing the ΔF508-CFTR biogenesis defect.

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.

Epistasis refers to the dependence of a mutation on other mutation(s) and the genetic context in general. In the context of human disorders, epistasis complicates the spectrum of disease symptoms and has been proposed as a major contributor to variations in disease outcome. The nonadditive relationship between mutations and the lack of complete understanding of the underlying physiological effects limit our ability to predict phenotypic outcome. Here, we report positive epistasis between intragenic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR)—the gene responsible for cystic fibrosis (CF) pathology. We identified a synonymous single-nucleotide polymorphism (sSNP) that is invariant for the CFTR amino acid sequence but inverts translation speed at the affected codon. This sSNP in cis exhibits positive epistatic effects on some CF disease–causing missense mutations. Individually, both mutations alter CFTR structure and function, yet when combined, they lead to enhanced protein expression and activity. The most robust effect was observed when the sSNP was present in combination with missense mutations that, along with the primary amino acid change, also alter the speed of translation at the affected codon. Functional studies revealed that synergistic alteration in ribosomal velocity is the underlying mechanism; alteration of translation speed likely increases the time window for establishing crucial domain–domain interactions that are otherwise perturbed by each individual mutation. Significance The same mutation can have different effects among individuals, and one reason for this is the presence of additional mutations, a relationship known as epistasis. Epistatic effects are difficult to predict. We report a rare example of positive epistasis between a silent mutation (that changes nucleotide but not the encoded amino acid) and missense mutation(s) (that change amino acid and cause cystic fibrosis disease). Individually, each mutation has a detrimental effect, but they are beneficial when present together. The underlying molecular effect is in their ability to markedly modulate the ribosomal speed on mRNA in a synergistic fashion. In a broader setting of human diseases, our results suggest that epistasis with silent mutation broadens the spectrum of disease outcome.

Patients with cystic fibrosis (CF) harboring the P67L variant in the CF transmembrane conductance regulator (CFTR) often exhibit a typical CF phenotype, including severe respiratory compromise. This rare mutation (reported in <300 patients worldwide) responds robustly to CFTR correctors such as lumacaftor and tezacaftor, with rescue in model systems that far exceeds what can be achieved for the archetypical CFTR mutant F508del. However, the specific molecular consequences of the P67L mutation are poorly characterized. In this study, we conducted biochemical measurements following low temperature growth and/or intragenic suppression which suggest a mechanism underlying P67L that: 1) shares key pathogenic features with F508del, including off-pathway (non-native) folding intermediates, 2) is linked to folding stability of nucleotide binding domains (NBDs) 1 and 2, and 3) demonstrates pharmacologic rescue that requires domains in the carboxyl half of the protein. We also investigated the "lasso" helices 1 (Lh1) and 2 (Lh2), which occur immediately upstream of P67. Based on limited proteolysis, pulse chase, and molecular dynamics analysis of full-length CFTR and a series of deletion constructs, we argue that P67L and other maturational processing (class 2) defects impair the integrity of the lasso motif and confer misfolding of downstream domains. Thus, amino terminal missense variants elicit a conformational change throughout CFTR that abrogates maturation while providing a robust substrate for pharmacologic repair.

By definition, effect of synonymous single-nucleotide variants (SNVs) on protein folding and function are neutral, as they alter the codon and not the encoded amino acid. Recent examples indicate tissue-specific and transfer RNA (tRNA)-dependent effects of some genetic variations arguing against neutrality of synonymous SNVs for protein biogenesis. We performed systematic analysis of tRNA abunandance across in various models used in cystic fibrosis (CF) research and drug development, including Fischer rat thyroid (FRT) cells, patient-derived primary human bronchial epithelia (HBE) from lung biopsies, primary human nasal epithelia (HNE) from nasal curettage, intestinal organoids, and airway progenitor-directed differentiation of human induced pluripotent stem cells (iPSCs). These were compared to an immortalized CF bronchial cell model (CFBE41o−) and two widely used laboratory cell lines, HeLa and HEK293. We discovered that specific synonymous SNVs exhibited differential effects which correlated with variable concentrations of cognate tRNAs. Our results highlight ways in which the presence of synonymous SNVs may alter local kinetics of mRNA translation; and thus, impact protein biogenesis and function. This effect is likely to influence results from mechansistic analysis and/or drug screeining efforts, and establishes importance of cereful model system selection based on genetic variation profile.