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1.
Artigo em Inglês | MEDLINE | ID: mdl-38972781

RESUMO

The presence of membrane-bound organelles with specific functions is one of the main hallmarks of eukaryotic cells. Organelle membranes are composed of specific lipids that govern their function and interorganelle communication. Discoveries in cell biology using imaging and omic technologies have revealed the mechanisms that drive membrane remodeling, organelle contact sites, and metabolite exchange. The interplay between multiple organelles and their interdependence is emerging as the next frontier for discovery using 3D reconstruction of volume electron microscopy (vEM) datasets. We discuss recent findings on the links between organelles that underlie common functions and cellular pathways. Specifically, we focus on the metabolism of ether glycerophospholipids that mediate organelle dynamics and their communication with each other, and the new imaging techniques that are powering these discoveries.

2.
Nat Cell Biol ; 26(1): 57-71, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38129691

RESUMO

The structures and functions of organelles in cells depend on each other but have not been systematically explored. We established stable knockout cell lines of peroxisomal, Golgi and endoplasmic reticulum genes identified in a whole-genome CRISPR knockout screen for inducers of mitochondrial biogenesis stress, showing that defects in peroxisome, Golgi and endoplasmic reticulum metabolism disrupt mitochondrial structure and function. Our quantitative total-organelle profiling approach for focussed ion beam scanning electron microscopy revealed in unprecedented detail that specific organelle dysfunctions precipitate multi-organelle biogenesis defects, impair mitochondrial morphology and reduce respiration. Multi-omics profiling showed a unified proteome response and global shifts in lipid and glycoprotein homeostasis that are elicited when organelle biogenesis is compromised, and that the resulting mitochondrial dysfunction can be rescued with precursors for ether-glycerophospholipid metabolic pathways. This work defines metabolic and morphological interactions between organelles and how their perturbation can cause disease.


Assuntos
Biogênese de Organelas , Organelas , Organelas/metabolismo , Peroxissomos/metabolismo , Complexo de Golgi/metabolismo , Mitocôndrias/metabolismo , Lipídeos
3.
Sci Adv ; 9(38): eadh8228, 2023 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-37738349

RESUMO

Breakdown of mitochondrial proteostasis activates quality control pathways including the mitochondrial unfolded protein response (UPRmt) and PINK1/Parkin mitophagy. However, beyond the up-regulation of chaperones and proteases, we have a limited understanding of how the UPRmt remodels and restores damaged mitochondrial proteomes. Here, we have developed a functional proteomics framework, termed MitoPQ (Mitochondrial Proteostasis Quantification), to dissect the UPRmt's role in maintaining proteostasis during stress. We find essential roles for the UPRmt in both protecting and repairing proteostasis, with oxidative phosphorylation metabolism being a central target of the UPRmt. Transcriptome analyses together with MitoPQ reveal that UPRmt transcription factors drive independent signaling arms that act in concert to maintain proteostasis. Unidirectional interplay between the UPRmt and PINK1/Parkin mitophagy was found to promote oxidative phosphorylation recovery when the UPRmt failed. Collectively, this study defines the network of proteostasis mediated by the UPRmt and highlights the value of functional proteomics in decoding stressed proteomes.


Assuntos
Proteoma , Proteostase , Fosforilação Oxidativa , Ubiquitina-Proteína Ligases/genética , Proteínas Quinases
4.
Methods Mol Biol ; 2661: 317-328, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37166645

RESUMO

RNA-binding proteins and mitochondrial ribosomes have been found to be linchpins of mitochondrial gene expression in health and disease. The expanding repertoire of proteins that bind and regulate the mitochondrial transcriptome has necessitated the development of new tools and methods to examine their molecular functions. Next-generation sequencing technologies have advanced the RNA biology field through application of high-throughput methods to study RNA-protein interactions. Here we describe a digital RNase footprinting method to analyze protein and ribosome interactions with mitochondrially encoded transcripts that provides insight into their mechanisms and minimal binding sites. We provide details on RNase digestion and next-generation sequencing, along with computational analyses and visualization of the binding targets within the mitochondrial transcriptome.


Assuntos
Ribonucleases , Ribossomos , Ribonucleases/metabolismo , Ribossomos/metabolismo , RNA/química , Ribossomos Mitocondriais/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Endorribonucleases/metabolismo , Ribonuclease Pancreático/metabolismo
5.
Nat Commun ; 14(1): 2210, 2023 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-37072429

RESUMO

The number of tRNA isodecoders has increased dramatically in mammals, but the specific molecular and physiological reasons for this expansion remain elusive. To address this fundamental question we used CRISPR editing to knockout the seven-membered phenylalanine tRNA gene family in mice, both individually and combinatorially. Using ATAC-Seq, RNA-seq, ribo-profiling and proteomics we observed distinct molecular consequences of single tRNA deletions. We show that tRNA-Phe-1-1 is required for neuronal function and its loss is partially compensated by increased expression of other tRNAs but results in mistranslation. In contrast, the other tRNA-Phe isodecoder genes buffer the loss of each of the remaining six tRNA-Phe genes. In the tRNA-Phe gene family, the expression of at least six tRNA-Phe alleles is required for embryonic viability and tRNA-Phe-1-1 is most important for development and survival. Our results reveal that the multi-copy configuration of tRNA genes is required to buffer translation and viability in mammals.


Assuntos
Variações do Número de Cópias de DNA , RNA de Transferência , Camundongos , Animais , RNA de Transferência/genética , RNA de Transferência/metabolismo , Mamíferos/genética
6.
EMBO Mol Med ; 15(6): e17463, 2023 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-37093546

RESUMO

Prostate cancer is the most commonly diagnosed malignancy and the third leading cause of cancer deaths. GWAS have identified variants associated with prostate cancer susceptibility; however, mechanistic and functional validation of these mutations is lacking. We used CRISPR-Cas9 genome editing to introduce a missense variant identified in the ELAC2 gene, which encodes a dually localised nuclear and mitochondrial RNA processing enzyme, into the mouse Elac2 gene as well as to generate a prostate-specific knockout of Elac2. These mutations caused enlargement and inflammation of the prostate and nodule formation. The Elac2 variant or knockout mice on the background of the transgenic adenocarcinoma of the mouse prostate (TRAMP) model show that Elac2 mutation with a secondary genetic insult exacerbated the onset and progression of prostate cancer. Multiomic profiling revealed defects in energy metabolism that activated proinflammatory and tumorigenic pathways as a consequence of impaired noncoding RNA processing and reduced protein synthesis. Our physiologically relevant models show that the ELAC2 variant is a predisposing factor for prostate cancer and identify changes that underlie the pathogenesis of this cancer.


Assuntos
Multiômica , Neoplasias da Próstata , Humanos , Masculino , Camundongos , Animais , Processamento Pós-Transcricional do RNA , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Mutação , Mutação de Sentido Incorreto
7.
PLoS Genet ; 17(11): e1009873, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34748562

RESUMO

Transcription of the human mitochondrial genome and correct processing of the two long polycistronic transcripts are crucial for oxidative phosphorylation. According to the tRNA punctuation model, nucleolytic processing of these large precursor transcripts occurs mainly through the excision of the tRNAs that flank most rRNAs and mRNAs. However, some mRNAs are not punctuated by tRNAs, and it remains largely unknown how these non-canonical junctions are resolved. The FASTK family proteins are emerging as key players in non-canonical RNA processing. Here, we have generated human cell lines carrying single or combined knockouts of several FASTK family members to investigate their roles in non-canonical RNA processing. The most striking phenotypes were obtained with loss of FASTKD4 and FASTKD5 and with their combined double knockout. Comprehensive mitochondrial transcriptome analyses of these cell lines revealed a defect in processing at several canonical and non-canonical RNA junctions, accompanied by an increase in specific antisense transcripts. Loss of FASTKD5 led to the most severe phenotype with marked defects in mitochondrial translation of key components of the electron transport chain complexes and in oxidative phosphorylation. We reveal that the FASTK protein family members are crucial regulators of non-canonical junction and non-coding mitochondrial RNA processing.


Assuntos
Proteínas Mitocondriais/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mitocondrial/metabolismo , Proteínas de Ligação a RNA/metabolismo , Linhagem Celular , Técnicas de Inativação de Genes , Humanos , Proteínas Mitocondriais/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Transcriptoma
8.
J Physiol ; 599(14): 3449-3462, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-32710561

RESUMO

The evolutionary acquisition of mitochondria has given rise to the diversity of eukaryotic life. Mitochondria have retained their ancestral α-proteobacterial traits through the maintenance of double membranes and their own circular genome. Their genome varies in size from very large in plants to the smallest in animals and their parasites. The mitochondrial genome encodes essential genes for protein synthesis and has to coordinate its expression with the nuclear genome from which it sources most of the proteins required for mitochondrial biogenesis and function. The mitochondrial protein synthesis machinery is unique because it is encoded by both the nuclear and mitochondrial genomes thereby requiring tight regulation to produce the respiratory complexes that drive oxidative phosphorylation for energy production. The fidelity and coordination of mitochondrial protein synthesis are essential for ATP production. Here we compare and contrast the mitochondrial translation mechanisms in mammals and fungi to bacteria and reveal that their diverse regulation can have unusual impacts on the health and disease of these organisms. We highlight that in mammals the rate of protein synthesis is more important than the fidelity of translation, enabling coordinated biogenesis of the mitochondrial respiratory chain with respiratory chain proteins synthesised by cytoplasmic ribosomes. Changes in mitochondrial protein fidelity can trigger the activation of the diverse cellular signalling networks in fungi and mammals to combat dysfunction in energy conservation. The physiological consequences of altered fidelity of protein synthesis can range from liver regeneration to the onset and development of cardiomyopathy.


Assuntos
Genoma Mitocondrial , Biossíntese de Proteínas , Animais , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Ribossomos/metabolismo
9.
Aging (Albany NY) ; 12(19): 19677-19700, 2020 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024056

RESUMO

The contribution of dysregulated mitochondrial gene expression and consequent imbalance in biogenesis is not well understood in metabolic disorders such as insulin resistance and obesity. The ribosomal RNA maturation protein PTCD1 is essential for mitochondrial protein synthesis and its reduction causes adult-onset obesity and liver steatosis. We used haploinsufficient Ptcd1 mice fed normal or high fat diets to understand how changes in mitochondrial biogenesis can lead to metabolic dysfunction. We show that Akt-stimulated reduction in lipid content and upregulation of mitochondrial biogenesis effectively protected mice with reduced mitochondrial protein synthesis from excessive weight gain on a high fat diet, resulting in improved glucose and insulin tolerance and reduced lipid accumulation in the liver. However, inflammation of the white adipose tissue and early signs of fibrosis in skeletal muscle, as a consequence of reduced protein synthesis, were exacerbated with the high fat diet. We identify that reduced mitochondrial protein synthesis and OXPHOS biogenesis can be recovered in a tissue-specific manner via Akt-mediated increase in insulin sensitivity and transcriptional activation of the mitochondrial stress response.

10.
PLoS Genet ; 16(3): e1008604, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32130224

RESUMO

The influence of environmental insults on the onset and progression of mitochondrial diseases is unknown. To evaluate the effects of infection on mitochondrial disease we used a mouse model of Leigh Syndrome, where a missense mutation in the Taco1 gene results in the loss of the translation activator of cytochrome c oxidase subunit I (TACO1) protein. The mutation leads to an isolated complex IV deficiency that mimics the disease pathology observed in human patients with TACO1 mutations. We infected Taco1 mutant and wild-type mice with a murine cytomegalovirus and show that a common viral infection exacerbates the complex IV deficiency in a tissue-specific manner. We identified changes in neuromuscular morphology and tissue-specific regulation of the mammalian target of rapamycin pathway in response to viral infection. Taken together, we report for the first time that a common stress condition, such as viral infection, can exacerbate mitochondrial dysfunction in a genetic model of mitochondrial disease.


Assuntos
Deficiência de Citocromo-c Oxidase/genética , Infecções por Citomegalovirus/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Muromegalovirus/patogenicidade , Animais , Deficiência de Citocromo-c Oxidase/virologia , Infecções por Citomegalovirus/virologia , Modelos Animais de Doenças , Doença de Leigh/genética , Doença de Leigh/virologia , Camundongos , Camundongos Endogâmicos C57BL , Doenças Mitocondriais/virologia , Mutação/genética , Serina-Treonina Quinases TOR/genética
11.
EMBO J ; 38(24): e102155, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31721250

RESUMO

Translation fidelity is crucial for prokaryotes and eukaryotic nuclear-encoded proteins; however, little is known about the role of mistranslation in mitochondria and its potential effects on metabolism. We generated yeast and mouse models with error-prone and hyper-accurate mitochondrial translation, and found that translation rate is more important than translational accuracy for cell function in mammals. Specifically, we found that mitochondrial mistranslation causes reduced overall mitochondrial translation and respiratory complex assembly rates. In mammals, this effect is compensated for by increased mitochondrial protein stability and upregulation of the citric acid cycle. Moreover, this induced mitochondrial stress signaling, which enables the recovery of mitochondrial translation via mitochondrial biogenesis, telomerase expression, and cell proliferation, and thereby normalizes metabolism. Conversely, we show that increased fidelity of mitochondrial translation reduces the rate of protein synthesis without eliciting a mitochondrial stress response. Consequently, the rate of translation cannot be recovered and this leads to dilated cardiomyopathy in mice. In summary, our findings reveal mammalian-specific signaling pathways that respond to changes in the fidelity of mitochondrial protein synthesis and affect metabolism.


Assuntos
Proliferação de Células , Mitocôndrias/metabolismo , Biogênese de Organelas , Transdução de Sinais , Animais , Ciclo do Ácido Cítrico/fisiologia , Escherichia coli/metabolismo , Feminino , Metabolômica , Camundongos , Camundongos Transgênicos , Mitocôndrias/genética , Doenças Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Biossíntese de Proteínas , Proteômica , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
12.
Sci Adv ; 5(12): eaay2118, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31903419

RESUMO

Mammalian mitochondrial ribosomes are unique molecular machines that translate 11 leaderless mRNAs; however, it is not clear how mitoribosomes initiate translation, since mitochondrial mRNAs lack untranslated regions. Mitochondrial translation initiation shares similarities with prokaryotes, such as the formation of a ternary complex of fMet-tRNAMet, mRNA and the 28S subunit, but differs in the requirements for initiation factors. Mitochondria have two initiation factors: MTIF2, which closes the decoding center and stabilizes the binding of the fMet-tRNAMet to the leaderless mRNAs, and MTIF3, whose role is not clear. We show that MTIF3 is essential for survival and that heart- and skeletal muscle-specific loss of MTIF3 causes cardiomyopathy. We identify increased but uncoordinated mitochondrial protein synthesis in mice lacking MTIF3, resulting in loss of specific respiratory complexes. Ribosome profiling shows that MTIF3 is required for recognition and regulation of translation initiation of mitochondrial mRNAs and for coordinated assembly of OXPHOS complexes in vivo.


Assuntos
Fator de Iniciação 3 em Eucariotos/metabolismo , Proteínas Mitocondriais/metabolismo , Fosforilação Oxidativa , Biossíntese de Proteínas/fisiologia , Animais , Cardiomiopatia Dilatada/genética , Fator de Iniciação 3 em Eucariotos/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , RNA de Transferência de Metionina/metabolismo , Ribossomos/metabolismo
13.
Biochem Soc Trans ; 46(5): 1239-1246, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30301847

RESUMO

Mitochondrial biogenesis is intimately dependent on the coordinated expression of the nuclear and mitochondrial genomes that is necessary for the assembly and function of the respiratory complexes to produce most of the energy required by cells. Although highly compacted in animals, the mitochondrial genome and its expression are essential for survival, development, and optimal energy production. The machinery that regulates gene expression within mitochondria is localised within the same compartment and, like in their ancestors, the bacteria, this machinery does not use membrane-based compartmentalisation to order the gene expression pathway. Therefore, the lifecycle of mitochondrial RNAs from transcription through processing, maturation, translation to turnover is mediated by a gamut of RNA-binding proteins (RBPs), all contained within the mitochondrial matrix milieu. Recent discoveries indicate that multiple processes regulating RNA metabolism occur at once but since mitochondria have a new complement of RBPs, many evolved de novo from nuclear genes, we are left wondering how co-ordinated are these processes? Here, we review recently identified examples of the co-ordinated and stochastic processes that govern the mitochondrial transcriptome. These new discoveries reveal the complexity of mitochondrial gene expression and the need for its in-depth exploration to understand how these organelles can respond to the energy demands of the cell.


Assuntos
Regulação da Expressão Gênica , Genes Mitocondriais , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Núcleo Celular/metabolismo , Perfilação da Expressão Gênica , Genoma Mitocondrial , Mitocôndrias/genética , Proteínas Mitocondriais/genética , RNA/análise , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Processos Estocásticos , Fatores de Transcrição/metabolismo , Transcriptoma
14.
Cell Rep ; 23(1): 127-142, 2018 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-29617655

RESUMO

The regulation of mitochondrial RNA life cycles and their roles in ribosome biogenesis and energy metabolism are not fully understood. We used CRISPR/Cas9 to generate heart- and skeletal-muscle-specific knockout mice of the pentatricopeptide repeat domain protein 1, PTCD1, and show that its loss leads to severe cardiomyopathy and premature death. Our detailed transcriptome-wide and functional analyses of these mice enabled us to identify the molecular role of PTCD1 as a 16S rRNA-binding protein essential for its stability, pseudouridylation, and correct biogenesis of the mitochondrial large ribosomal subunit. We show that impaired mitoribosome biogenesis can have retrograde signaling effects on nuclear gene expression through the transcriptional activation of the mTOR pathway and upregulation of cytoplasmic protein synthesis and pro-survival factors in the absence of mitochondrial translation. Taken together, our data show that impaired assembly of the mitoribosome exerts its consequences via differential regulation of mitochondrial and cytoplasmic protein synthesis.


Assuntos
Proteínas Mitocondriais/fisiologia , Ribossomos Mitocondriais/metabolismo , Biogênese de Organelas , RNA Ribossômico 16S/metabolismo , Proteínas de Ligação a RNA/fisiologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/genética , Pseudouridina/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/genética , Serina-Treonina Quinases TOR/metabolismo
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