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1.
J Cell Sci ; 136(10)2023 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-37218462

RESUMO

Translation of mRNAs containing premature termination codons (PTCs) results in truncated protein products with deleterious effects. Nonsense-mediated decay (NMD) is a surveillance pathway responsible for detecting PTC containing transcripts. Although the molecular mechanisms governing mRNA degradation have been extensively studied, the fate of the nascent protein product remains largely uncharacterized. Here, we use a fluorescent reporter system in mammalian cells to reveal a selective degradation pathway specifically targeting the protein product of an NMD mRNA. We show that this process is post-translational and dependent on the ubiquitin proteasome system. To systematically uncover factors involved in NMD-linked protein quality control, we conducted genome-wide flow cytometry-based screens. Our screens recovered known NMD factors but suggested that protein degradation did not depend on the canonical ribosome-quality control (RQC) pathway. A subsequent arrayed screen demonstrated that protein and mRNA branches of NMD rely on a shared recognition event. Our results establish the existence of a targeted pathway for nascent protein degradation from PTC containing mRNAs, and provide a reference for the field to identify and characterize required factors.


Assuntos
Mamíferos , Degradação do RNAm Mediada por Códon sem Sentido , Animais , Degradação do RNAm Mediada por Códon sem Sentido/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Mamíferos/metabolismo
2.
Proc Natl Acad Sci U S A ; 112(36): 11306-11, 2015 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-26240355

RESUMO

Identifying the mechanisms that create and maintain biodiversity is a central challenge in biology. Stable diversification of microbial populations often requires the evolution of differences in resource utilization. Alternatively, coexistence can be maintained by specialization to exploit spatial heterogeneity in the environment. Here, we report spontaneous diversification maintained by a related but distinct mechanism: crowding avoidance. During experimental evolution of laboratory Saccharomyces cerevisiae populations, we observed the repeated appearance of "adherent" (A) lineages able to grow as a dispersed film, in contrast to their crowded "bottom-dweller" (B) ancestors. These two types stably coexist because dispersal reduces interference competition for nutrients among kin, at the cost of a slower maximum growth rate. This tradeoff causes the frequencies of the two types to oscillate around equilibrium over the course of repeated cycles of growth, crowding, and dispersal. However, further coevolution of the A and B types can perturb and eventually destroy their coexistence over longer time scales. We introduce a simple mathematical model of this "semistable" coexistence, which explains the interplay between ecological and evolutionary dynamics. Because crowded growth generally limits nutrient access in biofilms, the mechanism we report here may be broadly important in maintaining diversity in these natural environments.


Assuntos
Biodiversidade , Evolução Biológica , Meio Ambiente , Saccharomyces cerevisiae/crescimento & desenvolvimento , Algoritmos , Antifúngicos/farmacologia , Ecossistema , Fluconazol/farmacologia , Miconazol/farmacologia , Modelos Biológicos , Densidade Demográfica , Dinâmica Populacional , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/efeitos dos fármacos , Imagem com Lapso de Tempo
3.
bioRxiv ; 2024 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-39091747

RESUMO

Cells must adapt to environmental changes to maintain homeostasis. One of the most striking environmental adaptations is entry into hibernation during which core body temperature can decrease from 37°C to as low at 4°C. How mammalian cells, which evolved to optimally function within a narrow range of temperatures, adapt to this profound decrease in temperature remains poorly understood. In this study, we conducted the first genome-scale CRISPR-Cas9 screen in cells derived from Syrian hamster, a facultative hibernator, as well as human cells to investigate the genetic basis of cold tolerance in a hibernator and a non-hibernator in an unbiased manner. Both screens independently revealed glutathione peroxidase 4 (GPX4), a selenium-containing enzyme, and associated proteins as critical for cold tolerance. We utilized genetic and pharmacological approaches to demonstrate that GPX4 is active in the cold and its catalytic activity is required for cold tolerance. Furthermore, we show that the role of GPX4 as a suppressor of cold-induced cell death extends across hibernating species, including 13-lined ground squirrels and greater horseshoe bats, highlighting the evolutionary conservation of this mechanism of cold tolerance. This study identifies GPX4 as a central modulator of mammalian cold tolerance and advances our understanding of the evolved mechanisms by which cells mitigate cold-associated damage-one of the most common challenges faced by cells and organisms in nature.

4.
Nat Med ; 26(2): 244-251, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31959991

RESUMO

Mucociliary clearance, the physiological process by which mammalian conducting airways expel pathogens and unwanted surface materials from the respiratory tract, depends on the coordinated function of multiple specialized cell types, including basal stem cells, mucus-secreting goblet cells, motile ciliated cells, cystic fibrosis transmembrane conductance regulator (CFTR)-rich ionocytes, and immune cells1,2. Bronchiectasis, a syndrome of pathological airway dilation associated with impaired mucociliary clearance, may occur sporadically or as a consequence of Mendelian inheritance, for example in cystic fibrosis, primary ciliary dyskinesia (PCD), and select immunodeficiencies3. Previous studies have identified mutations that affect ciliary structure and nucleation in PCD4, but the regulation of mucociliary transport remains incompletely understood, and therapeutic targets for its modulation are lacking. Here we identify a bronchiectasis syndrome caused by mutations that inactivate NIMA-related kinase 10 (NEK10), a protein kinase with previously unknown in vivo functions in mammals. Genetically modified primary human airway cultures establish NEK10 as a ciliated-cell-specific kinase whose activity regulates the motile ciliary proteome to promote ciliary length and mucociliary transport but which is dispensable for normal ciliary number, radial structure, and beat frequency. Together, these data identify a novel and likely targetable signaling axis that controls motile ciliary function in humans and has potential implications for other respiratory disorders that are characterized by impaired mucociliary clearance.


Assuntos
Ciliopatias/imunologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Depuração Mucociliar , Quinases Relacionadas a NIMA/metabolismo , Adolescente , Adulto , Separação Celular , Criança , Ciliopatias/metabolismo , Células Epiteliais/metabolismo , Exoma , Feminino , Citometria de Fluxo , Células HEK293 , Homozigoto , Humanos , Microscopia de Contraste de Fase , Microscopia de Vídeo , Mutação , Fenótipo , Proteoma , Sistema Respiratório , Tomografia Computadorizada por Raios X , Microtomografia por Raio-X , Adulto Jovem
6.
Science ; 360(6390): 751-758, 2018 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-29700228

RESUMO

The lysosome degrades and recycles macromolecules, signals to the master growth regulator mTORC1 [mechanistic target of rapamycin (mTOR) complex 1], and is associated with human disease. We performed quantitative proteomic analyses of rapidly isolated lysosomes and found that nutrient levels and mTOR dynamically modulate the lysosomal proteome. Upon mTORC1 inhibition, NUFIP1 (nuclear fragile X mental retardation-interacting protein 1) redistributes from the nucleus to autophagosomes and lysosomes. Upon these conditions, NUFIP1 interacts with ribosomes and delivers them to autophagosomes by directly binding to microtubule-associated proteins 1A/1B light chain 3B (LC3B). The starvation-induced degradation of ribosomes via autophagy (ribophagy) depends on the capacity of NUFIP1 to bind LC3B and promotes cell survival. We propose that NUFIP1 is a receptor for the selective autophagy of ribosomes.


Assuntos
Aminoácidos/deficiência , Autofagossomos/metabolismo , Autofagia , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Ribossomos/metabolismo , Animais , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/genética , Proteoma/metabolismo , Proteômica , Proteínas de Ligação a RNA/genética , Receptores Citoplasmáticos e Nucleares/genética , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição
7.
Genetics ; 196(4): 1217-26, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24514901

RESUMO

The outcomes of evolution are determined by which mutations occur and fix. In rapidly adapting microbial populations, this process is particularly hard to predict because lineages with different beneficial mutations often spread simultaneously and interfere with one another's fixation. Hence to predict the fate of any individual variant, we must know the rate at which new mutations create competing lineages of higher fitness. Here, we directly measured the effect of this interference on the fates of specific adaptive variants in laboratory Saccharomyces cerevisiae populations and used these measurements to infer the distribution of fitness effects of new beneficial mutations. To do so, we seeded marked lineages with different fitness advantages into replicate populations and tracked their subsequent frequencies for hundreds of generations. Our results illustrate the transition between strongly advantageous lineages that decisively sweep to fixation and more moderately advantageous lineages that are often outcompeted by new mutations arising during the course of the experiment. We developed an approximate likelihood framework to compare our data to simulations and found that the effects of these competing beneficial mutations were best approximated by an exponential distribution, rather than one with a single effect size. We then used this inferred distribution of fitness effects to predict the rate of adaptation in a set of independent control populations. Finally, we discuss how our experimental design can serve as a screen for rare, large-effect beneficial mutations.


Assuntos
Aptidão Genética , Mutação , Saccharomyces cerevisiae/genética , Adaptação Biológica , Evolução Molecular , Genoma Fúngico , Funções Verossimilhança , Modelos Genéticos
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