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/metabolismoRESUMO
A subset of human neocortical neurons harbors complex karyotypes wherein megabase-scale copy-number variants (CNVs) alter allelic diversity. Divergent levels of neurons with complex karyotypes (CNV neurons) are reported in different individuals, yet genome-wide and familial studies implicitly assume a single brain genome when assessing the genetic risk architecture of neurological disease. We assembled a brain CNV atlas using a robust computational approach applied to a new dataset (>800 neurons from 5 neurotypical individuals) and to published data from 10 additional neurotypical individuals. The atlas reveals that the frequency of neocortical neurons with complex karyotypes varies widely among individuals, but this variability is not readily accounted for by tissue quality or CNV detection approach. Rather, the age of the individual is anti-correlated with CNV neuron frequency. Fewer CNV neurons are observed in aged individuals than in young individuals.
