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1.
Mol Biol (Mosk) ; 55(3): 460-467, 2021.
Artigo em Russo | MEDLINE | ID: mdl-34097680

RESUMO

Cytoskeletal protein ß-actin is abundant both in the cytoplasm and the nucleus, its mRNA is commonly utilized an internal control for gene expression analysis. Recent reports demostrated that hypoxia influences the levels of ß-actin in a variety of cells. The mechanism underlying this change are not yet elucidated. In this work, we show that the changes in the levels of hypoxia-induced Nuclear respiratory factor-1 (NRF-1) lead to the change in expression of ß-actin. We compared the protein levels of NRF-1 and ß-actin in gastric cancer and adjacent tissues and found their significantly upregulation in cancer (33% patitents). When gastric cancer cells and normal gastric cells were treated with 1% O2 for 48 h, the trends in expression levels of NRF-1 and ß-actin were similar. When NRF-1 expression was modified by its overexpressing or silencing, the levels of ß-actin changed accordingly. In ß-actin gene (ACTB), three binding sites for NRF-1 were found. These sites are conserved in human, mouse and rat genomes. In ChIP experiments, we showed that NRF-1 directly binds to human ACTB and mouse Actb coding regions. Its seems that the transcription of ß-actin encoding gene is NRF-1 dependent.


Assuntos
Actinas , Fator 1 Nuclear Respiratório , Actinas/genética , Animais , Núcleo Celular/genética , Hipóxia/genética , Camundongos , Fator 1 Nuclear Respiratório/genética , Ratos , Ativação Transcricional
2.
Int J Mol Sci ; 22(11)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072343

RESUMO

In this work, we put forward a hypothesis about the decisive role of multivalent nonspecific interactions in the early stages of PML body formation. Our analysis of the PML isoform sequences showed that some of the PML isoforms, primarily PML-II, are prone to phase separation due to their polyampholytic properties and the disordered structure of their C-terminal domains. The similarity of the charge properties of the C-terminal domains of PML-II and PML-VI isoforms made it possible for the first time to detect migration of PML-VI from PML bodies to the periphery of the cell nucleus, similar to the migration of PML-II isoforms. We found a population of "small" (area less than 1 µm2) spherical PML bodies with high dynamics of PML isoforms exchange with nucleoplasm and a low fraction of immobilized proteins, which indicates their liquid state properties. Such structures can act as "seeds" of functionally active PML bodies, providing the necessary concentration of PML isoforms for the formation of intermolecular disulfide bonds between PML monomers. FRAP analysis of larger bodies of toroidal topology showed the existence of an insoluble scaffold in their structure. The hypothesis about the role of nonspecific multiple weak interactions in the formation of PML bodies is further supported by the change in the composition of the scaffold proteins of PML bodies, but not their solidification, under conditions of induction of dimerization of PML isoforms under oxidative stress. Using the colocalization of ALT-associated PML bodies (APBs) with TRF1, we identified APBs and showed the difference in the dynamic properties of APBs and canonical PML bodies.


Assuntos
Corpos de Inclusão Intranuclear/metabolismo , Proteína da Leucemia Promielocítica/metabolismo , Telômero/genética , Telômero/metabolismo , Sequência de Aminoácidos , Biomarcadores , Núcleo Celular/metabolismo , Imunofluorescência , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Imagem Molecular , Estresse Oxidativo , Proteína da Leucemia Promielocítica/química , Proteína da Leucemia Promielocítica/genética , Ligação Proteica , Isoformas de Proteínas , Transporte Proteico , Homeostase do Telômero
3.
Talanta ; 232: 122442, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34074427

RESUMO

The anti-tumor effects of metformin hydrochloride (MH), an initial pharmacologic agent for type 2 diabetes, were reexamined by surface-enhanced Raman scattering (SERS) spectroscopy. A SERS immuno-tag fabricated by decorating silver nanoparticles (AgNPs) with a specific antibody was employed to trace the dynamic expression of the tumor metastasis-related N-cadherin. With the MH action, the N-cadherin expression on the cell membranes decreases, proving that MH has a pharmacological effect on prohibiting cancer cell metastasis. Another AgNP-based nucleus targeting nanoprobe was adopted to culture with the MH acted cells, which can help the label-free SERS collection of the cell nuclei to explore the MH influences on intranuclear genes and proteins. By analyzing the intranuclear SERS spectra, the find is that MH has impacts on the transcription and translation of genes, thus regulates the expression of tumor metastasis-related proteins (N-cadherins). This study presents a proof-of-concept for MH as a potential drug for diabetes patients associated with tumors. The developed plasmonic immune analytical platform can be extended to assess other substances of the cell membrane and applicable for the SERS-based screening of membrane receptor-related drugs at the cellular level.


Assuntos
Diabetes Mellitus Tipo 2 , Nanopartículas Metálicas , Metformina , Caderinas/genética , Membrana Celular , Núcleo Celular , Humanos , Metformina/farmacologia , Prata , Análise Espectral Raman
4.
Braz J Biol ; 82: e242403, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34133565

RESUMO

Artemisia is one of the biggest genera in the family Asteraceae, with around 500-600 taxa at specific and sub-specific levels and organised in 5 subgenera. Due to the high number of taxa, a lot taxonomists are trying to solve the problem of its classification and phylogeny but its natural classification still hasn't been achieved. In this research, 60 individuals belonging to 4 taxa of the subgenus Dracunculus of Artemisia L. in Turkey were examined. For all the examined individuals from both the same and different populations belonging to the taxa of the subgenus Dracunculus, the sequences of the regions both psbA-trnH of chloroplast DNA and ITS of nuclear DNA were determined. Also, the gene regions obtained were recorded in the NCBI GenBank database and an accession number was taken. It was found that there was no gene flow and hybridization between the four studied taxa of the subgenus Dracunculus, and these 4 taxa also completed their speciation. According to the results of this molecular study, A. campestris var. campestris, A. campestris var. marschalliana and A. campestris var. araratica were proposed to be raised from the variety level to the species level. This research is important as it is the first molecular based study relating with the subgenus Dracunculus growing in Turkey.


Assuntos
Artemisia , Artemisia/genética , Núcleo Celular , Cloroplastos , Humanos , Filogenia , Turquia
5.
Int J Mol Sci ; 22(9)2021 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-34067160

RESUMO

Puccinia striiformis f. sp. tritici (Pst) is an important pathogen of wheat (Triticum aestivum L.) stripe rust, and the effector protein secreted by haustoria is a very important component involved in the pathogenic process. Although the candidate effector proteins secreted by Pst haustoria have been predicted to be abundant, few have been functionally validated. Our study confirmed that chitin and flg22 could be used as elicitors of the pathogenic-associated molecular pattern-triggered immune (PTI) reaction in wheat leaves and that TaPr-1-14 could be used as a marker gene to detect the PTI reaction. In addition, the experimental results were consistent in wheat protoplasts. A rapid and efficient method for screening and identifying the effector proteins of Pst was established by using the wheat protoplast transient expression system. Thirty-nine Pst haustorial effector genes were successfully cloned and screened for expression in the protoplast. We identified three haustorial effector proteins, PSEC2, PSEC17, and PSEC45, that may inhibit the response of wheat to PTI. These proteins are localized in the somatic cytoplasm and nucleus of wheat protoplasts and are highly expressed during the infection and parasitism of wheat.


Assuntos
Proteínas Fúngicas/metabolismo , Imunidade , Padrões Moleculares Associados a Patógenos/metabolismo , Protoplastos/microbiologia , Puccinia/fisiologia , Triticum/imunologia , Triticum/microbiologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Quitina/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Imunidade/efeitos dos fármacos , Doenças das Plantas/microbiologia , Imunidade Vegetal/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/imunologia , Folhas de Planta/microbiologia , Protoplastos/efeitos dos fármacos , Puccinia/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Reprodutibilidade dos Testes , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Transcrição Genética/efeitos dos fármacos , Triticum/efeitos dos fármacos , Triticum/genética
6.
Int J Mol Sci ; 22(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34065983

RESUMO

Dysregulation of messenger RNA (mRNA) processing-in particular mRNA splicing-is a hallmark of cancer. Compared to normal cells, cancer cells frequently present aberrant mRNA splicing, which promotes cancer progression and treatment resistance. This hallmark provides opportunities for developing new targeted cancer treatments. Splicing of precursor mRNA into mature mRNA is executed by a dynamic complex of proteins and small RNAs called the spliceosome. Spliceosomes are part of the supraspliceosome, a macromolecular structure where all co-transcriptional mRNA processing activities in the cell nucleus are coordinated. Here we review the biology of the mRNA splicing machinery in the context of other mRNA processing activities in the supraspliceosome and present current knowledge of its dysregulation in lung cancer. In addition, we review investigations to discover therapeutic targets in the spliceosome and give an overview of inhibitors and modulators of the mRNA splicing process identified so far. Together, this provides insight into the value of targeting the spliceosome as a possible new treatment for lung cancer.


Assuntos
Neoplasias Pulmonares/genética , Splicing de RNA , Spliceossomos/metabolismo , Processamento Alternativo , Núcleo Celular/metabolismo , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/metabolismo
7.
Nat Commun ; 12(1): 3308, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083519

RESUMO

The spatial partitioning of the transcriptome in the cell is an important form of gene-expression regulation. Here, we address how intron retention influences the spatio-temporal dynamics of transcripts from two clinically relevant genes: TERT (Telomerase Reverse Transcriptase) pre-mRNA and TUG1 (Taurine-Upregulated Gene 1) lncRNA. Single molecule RNA FISH reveals that nuclear TERT transcripts uniformly and robustly retain specific introns. Our data suggest that the splicing of TERT retained introns occurs during mitosis. In contrast, TUG1 has a bimodal distribution of fully spliced cytoplasmic and intron-retained nuclear transcripts. We further test the functionality of intron-retention events using RNA-targeting thiomorpholino antisense oligonucleotides to block intron excision. We show that intron retention is the driving force for the nuclear compartmentalization of these RNAs. For both RNAs, altering this splicing-driven subcellular distribution has significant effects on cell viability. Together, these findings show that stable retention of specific introns can orchestrate spatial compartmentalization of these RNAs within the cell. This process reveals that modulating RNA localization via targeted intron retention can be utilized for RNA-based therapies.


Assuntos
Núcleo Celular/genética , Núcleo Celular/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Telomerase/genética , Animais , Compartimento Celular , Linhagem Celular , Linhagem Celular Tumoral , Células HCT116 , Células HEK293 , Células HeLa , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Hibridização in Situ Fluorescente , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Íntrons , Camundongos , Mitose , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , Estabilidade de RNA , Especificidade da Espécie
8.
Nat Commun ; 12(1): 2981, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016985

RESUMO

The spatial folding of chromosomes inside the nucleus has regulatory effects on gene expression, yet the impact of genome reshuffling on this organization remains unclear. Here, we take advantage of chromosome conformation capture in combination with single-nucleotide polymorphism (SNP) genotyping and analysis of crossover events to study how the higher-order chromatin organization and recombination landscapes are affected by chromosomal fusions in the mammalian germ line. We demonstrate that chromosomal fusions alter the nuclear architecture during meiosis, including an increased rate of heterologous interactions in primary spermatocytes, and alterations in both chromosome synapsis and axis length. These disturbances in topology were associated with changes in genomic landscapes of recombination, resulting in detectable genomic footprints. Overall, we show that chromosomal fusions impact the dynamic genome topology of germ cells in two ways: (i) altering chromosomal nuclear occupancy and synapsis, and (ii) reshaping landscapes of recombination.


Assuntos
Cromatina/metabolismo , Cromossomos/metabolismo , Recombinação Genética , Espermatócitos/metabolismo , Animais , Evolução Biológica , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Cromatina/genética , Pareamento Cromossômico/genética , Segregação de Cromossomos , Cromossomos/genética , Europa (Continente) , Fertilidade/genética , Técnicas de Genotipagem/métodos , Masculino , Camundongos , Polimorfismo de Nucleotídeo Único , Cultura Primária de Células , Análise do Sêmen , Espermatócitos/citologia
9.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33947021

RESUMO

Despite increasing reports on the function of CCCH zinc finger proteins in plant development and stress response, the functions and molecular aspects of many non-tandem CCCH zinc finger (non-TZF) proteins remain uncharacterized. AtC3H59/ZFWD3 is an Arabidopsis non-TZF protein and belongs to the ZFWD subfamily harboring a CCCH zinc finger motif and a WD40 domain. In this study, we characterized the biological and molecular functions of AtC3H59, which is subcellularly localized in the nucleus. The seeds of AtC3H59-overexpressing transgenic plants (OXs) germinated faster than those of wild type (WT), whereas atc3h59 mutant seeds germinated slower than WT seeds. AtC3H59 OX seedlings were larger and heavier than WT seedlings, whereas atc3h59 mutant seedlings were smaller and lighter than WT seedlings. Moreover, AtC3H59 OX seedlings had longer primary root length than WT seedlings, whereas atc3h59 mutant seedlings had shorter primary root length than WT seedlings, owing to altered cell division activity in the root meristem. During seed development, AtC3H59 OXs formed larger and heavier seeds than WT. Using yeast two-hybrid screening, we isolated Desi1, a PPPDE family protein, as an interacting partner of AtC3H59. AtC3H59 and Desi1 interacted via their WD40 domain and C-terminal region, respectively, in the nucleus. Taken together, our results indicate that AtC3H59 has pleiotropic effects on seed germination, seedling development, and seed development, and interacts with Desi1 in the nucleus via its entire WD40 domain. To our knowledge, this is the first report to describe the biological functions of the ZFWD protein and Desi1 in Arabidopsis.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Sementes/metabolismo , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Contagem de Células , Núcleo Celular/metabolismo , Sequência Consenso , Germinação , Meristema/citologia , Família Multigênica , Raízes de Plantas/crescimento & desenvolvimento , Brotos de Planta/crescimento & desenvolvimento , Mapeamento de Interação de Proteínas , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
10.
BMC Bioinformatics ; 22(1): 256, 2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34011275

RESUMO

BACKGROUND: Pseudogenes are non-functional copies of protein coding genes that typically follow a different molecular evolutionary path as compared to functional genes. The inclusion of pseudogene sequences in DNA barcoding and metabarcoding analysis can lead to misleading results. None of the most widely used bioinformatic pipelines used to process marker gene (metabarcode) high throughput sequencing data specifically accounts for the presence of pseudogenes in protein-coding marker genes. The purpose of this study is to develop a method to screen for nuclear mitochondrial DNA segments (nuMTs) in large COI datasets. We do this by: (1) describing gene and nuMT characteristics from an artificial COI barcode dataset, (2) show the impact of two different pseudogene removal methods on perturbed community datasets with simulated nuMTs, and (3) incorporate a pseudogene filtering step in a bioinformatic pipeline that can be used to process Illumina paired-end COI metabarcode sequences. Open reading frame length and sequence bit scores from hidden Markov model (HMM) profile analysis were used to detect pseudogenes. RESULTS: Our simulations showed that it was more difficult to identify nuMTs from shorter amplicon sequences such as those typically used in metabarcoding compared with full length DNA barcodes that are used in the construction of barcode libraries. It was also more difficult to identify nuMTs in datasets where there is a high percentage of nuMTs. Existing bioinformatic pipelines used to process metabarcode sequences already remove some nuMTs, especially in the rare sequence removal step, but the addition of a pseudogene filtering step can remove up to 5% of sequences even when other filtering steps are in place. CONCLUSIONS: Open reading frame length filtering alone or combined with hidden Markov model profile analysis can be used to effectively screen out apparent pseudogenes from large datasets. There is more to learn from COI nuMTs such as their frequency in DNA barcoding and metabarcoding studies, their taxonomic distribution, and evolution. Thus, we encourage the submission of verified COI nuMTs to public databases to facilitate future studies.


Assuntos
Código de Barras de DNA Taxonômico , Pseudogenes , Núcleo Celular , DNA Mitocondrial , Mitocôndrias/genética , Filogenia , Pseudogenes/genética , Análise de Sequência de DNA
11.
Nat Commun ; 12(1): 2812, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990570

RESUMO

Trastuzumab is the backbone of HER2-directed gastric cancer therapy, but poor patient response due to insufficient cell sensitivity and drug resistance remains a clinical challenge. Here, we report that HER2 is involved in cell mitotic promotion for tumorigenesis by hyperactivating a crucial HER2-SHCBP1-PLK1 axis that drives trastuzumab sensitivity and is targeted therapeutically. SHCBP1 is an Shc1-binding protein but is detached from scaffold protein Shc1 following HER2 activation. Released SHCBP1 responds to HER2 cascade by translocating into the nucleus following Ser273 phosphorylation, and then contributing to cell mitosis regulation through binding with PLK1 to promote the phosphorylation of the mitotic interactor MISP. Meanwhile, Shc1 is recruited to HER2 for MAPK or PI3K pathways activation. Also, clinical evidence shows that increased SHCBP1 prognosticates a poor response of patients to trastuzumab therapy. Theaflavine-3, 3'-digallate (TFBG) is identified as an inhibitor of the SHCBP1-PLK1 interaction, which is a potential trastuzumab sensitizing agent and, in combination with trastuzumab, is highly efficacious in suppressing HER2-positive gastric cancer growth. These findings suggest an aberrant mitotic HER2-SHCBP1-PLK1 axis underlies trastuzumab sensitivity and offer a new strategy to combat gastric cancer.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptor ErbB-2/metabolismo , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Trastuzumab/farmacologia , Animais , Antineoplásicos Imunológicos/farmacologia , Biflavonoides/farmacologia , Catequina/análogos & derivados , Catequina/farmacologia , Proteínas de Ciclo Celular/química , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Camundongos , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Mitose/efeitos dos fármacos , Modelos Biológicos , Modelos Moleculares , Fosfoproteínas/metabolismo , Prognóstico , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/química , Proteínas Proto-Oncogênicas/química , Receptor ErbB-2/antagonistas & inibidores , Proteínas Adaptadoras da Sinalização Shc/antagonistas & inibidores , Proteínas Adaptadoras da Sinalização Shc/química , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Nat Commun ; 12(1): 2650, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976192

RESUMO

Live cell imaging using fluorescent DNA markers are an indispensable molecular tool in various biological and biomedical fields. It is a challenge to develop DNA probes that avoid UV light photo-excitation, have high specificity for DNA, are cell-permeable and are compatible with cutting-edge imaging techniques such as super-resolution microscopy. Herein, we present N-aryl pyrido cyanine (N-aryl-PC) derivatives as a class of long absorption DNA markers with absorption in the wide range of visible light. The high DNA specificity and membrane permeability allow the staining of both organelle DNA as well as nuclear DNA, in various cell types, including plant tissues, without the need for washing post-staining. N-aryl-PC dyes are also highly compatible with a separation of photon by lifetime tuning method in stimulated emission depletion microscopy (SPLIT-STED) for super-resolution imaging as well as two-photon microscopy for deep tissue imaging, making it a powerful tool in the life sciences.


Assuntos
Núcleo Celular/química , DNA/química , Corantes Fluorescentes/química , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Organelas/química , Animais , Arabidopsis/citologia , Benzimidazóis/química , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Células Cultivadas , DNA/genética , DNA/metabolismo , Fluorescência , Células HeLa , Humanos , Camundongos , Microscopia Confocal/métodos , Estrutura Molecular , Células NIH 3T3 , Organelas/metabolismo , Coloração e Rotulagem/métodos , Imagem com Lapso de Tempo/métodos
13.
BMC Plant Biol ; 21(1): 207, 2021 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-33941091

RESUMO

BACKGROUND: Artificial synthesis of octoploid rapeseed double haploid (DH) induction lines Y3380 and Y3560 was made possible by interspecific hybridization and genome doubling techniques. Production of pure lines by DH induction provides a new way to achieve homozygosity earlier in B.napus. Previously, the mechanism of induction, and whether the induction has obvious maternal genotypic differences or not, are not known so far. RESULTS: In this study, different karyogene and cytoplasmic genotype of B.napus were pollinated with the previously reported DH inducers e.g. Y3380 and Y3560. Our study presents a fine comparison of different cytoplasmic genotypes hybridization to unravel the mechanism of DH induction. Ploidy identification, fertility and SSR marker analysis of induced F1 generation, revealed that ploidy and phenotype of the induced F1 plants were consistent with that type of maternal, rather than paternal parent. The SNP chip analysis revealed that induction efficiency of DH inducers were affected by the karyogene when the maternal cytoplasmic genotypes were the same. However, DH induction efficiency was also affected by cytoplasmic genotype when the karyogenes were same, and the offspring of the ogura cytoplasm showed high frequency inducer gene hybridization or low-frequency infiltration. CONCLUSION: The induction effect is influenced by the interaction between maternal karyogene and cytoplasmic genotype, and the results from the partial hybridization of progeny chromosomes indicate that the induction process may be attributed to the selective elimination of paternal chromosome. This study provides a basis for exploring the mechanism of DH inducer in B.napus, and provides new insights for utilization of inducers in molecular breeding.


Assuntos
Brassica napus/genética , Cromossomos de Plantas/genética , Embaralhamento de DNA/métodos , Hibridização Genética , Núcleo Celular/genética , Citoplasma/genética , Genótipo , Haploidia , Fenótipo , Melhoramento Vegetal
14.
Methods Mol Biol ; 2274: 3-14, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050457

RESUMO

The nuclear envelope (NE), a double membrane that separates nuclear components from the cytoplasm, undergoes a breakdown and reformation during cell division. To trace NE dynamics, the NE needs to be labeled with a fluorescent marker, and for this purpose, markers based on inner nuclear membrane (INM) proteins are normally used. However, NE labeling with INM proteins has some limitations. Here, we introduce a protocol for fluorescent labeling and imaging of NE that does not rely on INM proteins, along with protocols for simultaneously imaging two nuclear components and for time-lapse imaging of labeled cells.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Membrana Nuclear/metabolismo , Espectrometria de Fluorescência/métodos , Células HeLa , Humanos
15.
Curr Protoc ; 1(5): e129, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34004049

RESUMO

O-GlcNAc is a common post-translational modification of nuclear, mitochondrial, and cytoplasmic proteins that regulates normal physiology and the cell stress response. Dysregulation of O-GlcNAc cycling is implicated in the etiology of type II diabetes, heart failure, hypertension, and Alzheimer's disease, as well as cardioprotection. These protocols cover simple and comprehensive techniques for detecting proteins modified by O-GlcNAc and studying the enzymes that add or remove O-GlcNAc. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Increasing the stoichiometry of O-GlcNAc on proteins before analysis Basic Protocol 2: Detection of proteins modified by O-GlcNAc using antibodies Basic Protocol 3: Detection of proteins modified by O-GlcNAc using the lectin sWGA Support Protocol 1: Control for O-linked glycosylation Basic Protocol 4: Detection and enrichment of proteins using WGA-agarose Support Protocol 2: Digestion of proteins with hexosaminidase Alternate Protocol: Detection of proteins modified by O-GlcNAc using galactosyltransferase Support Protocol 3: Autogalactosylation of galactosyltransferase Support Protocol 4: Assay of galactosyltransferase activity Basic Protocol 5: Characterization of labeled glycans by ß-elimination and chromatography Basic Protocol 6: Detection of O-GlcNAc in 96-well plates Basic Protocol 7: Assay for OGT activity Support Protocol 5: Desalting of O-GlcNAc transferase Basic Protocol 8: Assay for O-GlcNAcase activity.


Assuntos
Acetilglucosamina , Diabetes Mellitus Tipo 2 , Acetilglucosamina/metabolismo , Núcleo Celular/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glicosilação , Humanos , Processamento de Proteína Pós-Traducional
16.
Curr Protoc ; 1(5): e132, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34043278

RESUMO

Both single-cell RNA sequencing (scRNAseq) and single-nucleus RNA sequencing (snRNAseq) can be used to characterize the transcriptional profile of individual cells, and based on these transcriptional profiles, help define cell type distribution in mixed cell populations. However, scRNAseq analyses are confounded if some of the cells are large (>50 µm) or if some of cells adhere more tightly to some adjacent cells than to others. Further, single cell isolation for scRNAseq requires fresh tissue, which may not be available for human or animal model tissues. Additionally, the current enzymatic and mechanical methods for single-cell dissociation can lead to stress-induced transcriptional artifacts. Nuclei for snRNAseq, on the other hand, can be isolated from any cell, regardless of size, and from either fresh or frozen tissues, and compared to whole cells, they are more resistant to mechanical pressures and appear not to exhibit as many cell isolation-based transcriptional artifacts. Here, we describe a time- and cost-effective procedure to isolate nuclei from mammalian cells and tissues. The protocol incorporates steps to mechanically disrupt samples to release nuclei. Compared to conventional nuclei isolation protocols, the approach described here increases overall efficiency, eliminates risk of contaminant exposure, and reduces volumes of expensive reagents. A series of RNA quality control checks are also incorporated to ensure success and reduce costs of subsequent snRNAseq experiments. Nuclei isolated by this procedure can be separated on the 10× Genomics Chromium system for either snRNAseq and/or Single-Nucleus ATAC-Seq (snATAC-Seq), and is also compatible with other single cell platforms. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Sample preparation and quality control check via RNA Isolation and Analysis Basic Protocol 2: Nuclei Isolation.


Assuntos
Núcleo Celular , Núcleo Solitário , Animais , Separação Celular , Modelos Animais de Doenças , Humanos , Análise de Sequência de RNA
17.
Anal Chim Acta ; 1168: 338609, 2021 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-34051998

RESUMO

As critical players in the regulation of gene expression, RNA-binding proteins (RBPs) play fundamental roles in cellular functions and diseases. In this study, we established an analytical strategy to characterize RBPs from different subcellular regions by combining subcellular fractionation, acidic guanidinium-thiocyanate-phenol-chloroform biphasic extraction, and quantitative mass spectrometry. Using this method, we identified 1775 and 2245 RBPs from the cell nucleus and cytoplasm. The data confirmed a large spectrum of known RBPs, revealed 614 novel ones that have never been reported before, and cataloged their subcellular localizations. Intriguingly, 200 metabolic enzymes from diverse metabolic pathways were observed as RBPs, some of which were further validated through western blotting following UV-mediated crosslinking and biphasic extraction. Furthermore, we characterized 2157 RNA-binding interfaces, providing structural information regarding the complex nature of RNA-protein interactions. Taken together, our data greatly expand the current reservoir of known RBPs and highlight the potential role of RNA-binding in the regulation of cellular metabolism.


Assuntos
Núcleo Celular , Proteínas de Ligação a RNA , Citoplasma
18.
Mol Cell ; 81(9): 1863-1865, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33961775

RESUMO

Using mitochondria-targeted TALENS and ionizing radiation, consequences of mtDNA double-strand (ds) breaks were investigated by Tigano et al. (2021) who uncovered mtRNA as a retrograde second messenger of this form of mtDNA stress that activates the RIG-I/MAVS innate immune signaling pathway.


Assuntos
DNA Mitocondrial , RNA , Núcleo Celular , Imunidade Inata , Mitocôndrias/genética , RNA/genética
19.
Nat Commun ; 12(1): 2876, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001883

RESUMO

Activation of non-shivering thermogenesis is considered a promising approach to lower body weight in obesity. p62 deficiency in adipocytes reduces systemic energy expenditure but its role in sustaining mitochondrial function and thermogenesis remains unresolved. NBR1 shares a remarkable structural similarity with p62 and can interact with p62 through their respective PB1 domains. However, the physiological relevance of NBR1 in metabolism, as compared to that of p62, was not clear. Here we show that whole-body and adipocyte-specific ablation of NBR1 reverts the obesity phenotype induced by p62 deficiency by restoring global energy expenditure and thermogenesis in brown adipose tissue. Impaired adrenergic-induced browning of p62-deficient adipocytes is rescued by NBR1 inactivation, unveiling a negative role of NBR1 in thermogenesis under conditions of p62 loss. We demonstrate that upon p62 inactivation, NBR1 represses the activity of PPARγ, establishing an unexplored p62/NBR1-mediated paradigm in adipocyte thermogenesis that is critical for the control of obesity.


Assuntos
Adipócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , PPAR gama/metabolismo , Proteína Sequestossoma-1/deficiência , Termogênese , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/metabolismo , Animais , Animais Recém-Nascidos , Núcleo Celular/metabolismo , Células Cultivadas , Metabolismo Energético/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , PPAR gama/genética , Ligação Proteica , Receptor X Retinoide alfa/genética , Receptor X Retinoide alfa/metabolismo , Proteína Sequestossoma-1/genética
20.
Nat Commun ; 12(1): 3027, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34021139

RESUMO

Mutations disrupting the nuclear localization of the RNA-binding protein FUS characterize a subset of amyotrophic lateral sclerosis patients (ALS-FUS). FUS regulates nuclear RNAs, but its role at the synapse is poorly understood. Using super-resolution imaging we determined that the localization of FUS within synapses occurs predominantly near the vesicle reserve pool of presynaptic sites. Using CLIP-seq on synaptoneurosomes, we identified synaptic FUS RNA targets, encoding proteins associated with synapse organization and plasticity. Significant increase of synaptic FUS during early disease in a mouse model of ALS was accompanied by alterations in density and size of GABAergic synapses. mRNAs abnormally accumulated at the synapses of 6-month-old ALS-FUS mice were enriched for FUS targets and correlated with those depicting increased short-term mRNA stability via binding primarily on multiple exonic sites. Our study indicates that synaptic FUS accumulation in early disease leads to synaptic impairment, potentially representing an initial trigger of neurodegeneration.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , RNA/metabolismo , Sinapses/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Núcleo Celular/metabolismo , Córtex Cerebral , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , RNA Mensageiro/metabolismo , Proteína FUS de Ligação a RNA/genética
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