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1.
Proc Natl Acad Sci U S A ; 118(4)2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33468668

RESUMO

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.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Epistasia Genética , Biossíntese de Proteínas , Sequência de Aminoácidos/genética , Códon/genética , Fibrose Cística/patologia , Humanos , Mutação de Sentido Incorreto/genética , Polimorfismo de Nucleotídeo Único/genética , RNA Mensageiro/genética
2.
J Cyst Fibros ; 19(6): 1021-1026, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32451204

RESUMO

BACKGROUND: Chronic inflammation is a hallmark among patients with cystic fibrosis (CF). We explored whether mutation-induced (F508del) misfolding of the cystic fibrosis transmembrane conductance regulator (CFTR), and/or secondary colonization with opportunistic pathogens, activate tissue remodeling and innate immune response drivers. METHODS: Using RNA-seq to interrogate global gene expression profiles, we analyzed stress response signaling cascades in primary human bronchial epithelia (HBE) and intestinal organoids. RESULTS: Primary HBE acquired from CF patients with advanced disease and prolonged exposure to pathogenic microorganisms display a clear molecular signature of activated tissue remodeling pathways, unfolded protein response (UPR), and chronic inflammation. Furthermore, CFTR misfolding induces inflammatory signaling cascades in F508del patient-derived organoids from both the distal small intestine and colon. CONCLUSION: Despite the small patient cohort size, this proof-of-principle study supports the use of RNA-seq as a means to both identify CF-specific signaling profiles in various tissues and evaluate disease heterogeneity. Our global transcriptomic data is a useful resource for the CF research community for analyzing other gene expression sets influencing CF disease signature but also transcriptionally contributing to CF heterogeneity.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Fibrose Cística/microbiologia , Estresse do Retículo Endoplasmático/genética , Perfilação da Expressão Gênica , Imunidade Inata , Adulto , Brônquios/citologia , Células Cultivadas , Células Epiteliais , Feminino , Humanos , Inflamação , Pessoa de Meia-Idade , Organoides , Estudo de Prova de Conceito , Transdução de Sinais , Exacerbação dos Sintomas , Transcriptoma
3.
J Clin Invest ; 129(12): 5236-5253, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31657788

RESUMO

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.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Mutação , Ribossomos/metabolismo , Aminopiridinas/farmacologia , Animais , Benzodioxóis/farmacologia , Brônquios/metabolismo , Colo/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Epitélio/metabolismo , Feminino , Inativação Gênica , Células HEK293 , Humanos , Íleo/metabolismo , Indóis/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Pâncreas/metabolismo , Técnicas de Patch-Clamp , Conformação Proteica , Dobramento de Proteína , Ratos , Proteínas Ribossômicas/metabolismo
4.
Biochem Soc Trans ; 46(4): 937-944, 2018 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-30065107

RESUMO

Ribosomes translate mRNAs with non-uniform speed. Translation velocity patterns are a conserved feature of mRNA and have evolved to fine-tune protein folding, expression and function. Synonymous single-nucleotide polymorphisms (sSNPs) that alter programmed translational speed affect expression and function of the encoded protein. Synergistic advances in next-generation sequencing have led to the identification of sSNPs associated with disease penetrance. Here, we draw on studies with disease-related proteins to enhance our understanding of mechanistic contributions of sSNPs to functional alterations of the encoded protein. We emphasize the importance of identification of sSNPs along with disease-causing mutations to understand genotype-phenotype relationships.


Assuntos
Mutação , Biossíntese de Proteínas , Dobramento de Proteína , Doença Pulmonar Obstrutiva Crônica/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Receptor alfa de Estrogênio/fisiologia , Humanos , Cinética , Polimorfismo de Nucleotídeo Único , Proteína D Associada a Surfactante Pulmonar/genética , Proteína D Associada a Surfactante Pulmonar/metabolismo , Proteína D Associada a Surfactante Pulmonar/fisiologia , RNA Mensageiro/genética
5.
PLoS Biol ; 15(5): e2000779, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28510592

RESUMO

Synonymous single nucleotide polymorphisms (sSNPs) are considered neutral for protein function, as by definition they exchange only codons, not amino acids. We identified an sSNP that modifies the local translation speed of the cystic fibrosis transmembrane conductance regulator (CFTR), leading to detrimental changes to protein stability and function. This sSNP introduces a codon pairing to a low-abundance tRNA that is particularly rare in human bronchial epithelia, but not in other human tissues, suggesting tissue-specific effects of this sSNP. Up-regulation of the tRNA cognate to the mutated codon counteracts the effects of the sSNP and rescues protein conformation and function. Our results highlight the wide-ranging impact of sSNPs, which invert the programmed local speed of mRNA translation and provide direct evidence for the central role of cellular tRNA levels in mediating the actions of sSNPs in a tissue-specific manner.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , RNA de Transferência/metabolismo , Mutação Silenciosa , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células HEK293 , Células HeLa , Humanos , Polimorfismo de Nucleotídeo Único , Estabilidade Proteica , Relação Estrutura-Atividade
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