Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 10.063
Filtrar
1.
Cell Syst ; 11(1): 102-108.e3, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32673562

RESUMO

SARS-CoV-2 genomic and subgenomic RNA (sgRNA) transcripts hijack the host cell's machinery. Subcellular localization of its viral RNA could, thus, play important roles in viral replication and host antiviral immune response. We perform computational modeling of SARS-CoV-2 viral RNA subcellular residency across eight subcellular neighborhoods. We compare hundreds of SARS-CoV-2 genomes with the human transcriptome and other coronaviruses. We predict the SARS-CoV-2 RNA genome and sgRNAs to be enriched toward the host mitochondrial matrix and nucleolus, and that the 5' and 3' viral untranslated regions contain the strongest, most distinct localization signals. We interpret the mitochondrial residency signal as an indicator of intracellular RNA trafficking with respect to double-membrane vesicles, a critical stage in the coronavirus life cycle. Our computational analysis serves as a hypothesis generation tool to suggest models for SARS-CoV-2 biology and inform experimental efforts to combat the virus. A record of this paper's Transparent Peer Review process is included in the Supplemental Information.


Assuntos
Betacoronavirus/genética , Nucléolo Celular/virologia , Infecções por Coronavirus/virologia , Mitocôndrias/virologia , Pneumonia Viral/virologia , RNA Viral/metabolismo , Betacoronavirus/metabolismo , Nucléolo Celular/metabolismo , Bases de Dados Genéticas , Genoma Viral , Humanos , Aprendizado de Máquina , Mitocôndrias/metabolismo , Modelos Genéticos , Pandemias , RNA Viral/genética
2.
Anticancer Res ; 40(6): 3505-3512, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32487651

RESUMO

AIM: To assess the prognostic significance of nucleolar morphological parameters in a large cohort of patients with uveal melanoma. PATIENTS AND METHODS: The presence, size and number of nucleoli of cancer cells were assessed in haematoxylin and eosin (HE)-stained slides of 164 formalin-fixed paraffin-embedded primary uveal melanoma tissue specimens. The results were correlated with clinicopathological features and patient survival. RESULTS: The presence of macronucleoli and multiple nucleoli significantly correlated with the epithelioid type of uveal melanoma, high mitotic rate, and marked pleomorphism. There was a positive correlation between the presence of macronucleoli as well as the number of nucleoli and the largest tumour basal diameter. The increased nucleolus count in tumour cells positively correlated with primary tumour (pT) staging. The presence of both prominent and multiple nucleoli was associated with significantly reduced overall and disease-free survival. CONCLUSION: Histological assessment of nucleolar morphology in routine HE staining would be a helpful low-cost method to obtain reliable prognostic information.


Assuntos
Nucléolo Celular/patologia , Melanoma/patologia , Neoplasias Uveais/patologia , Idoso , Nucléolo Celular/ultraestrutura , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Melanoma/mortalidade , Melanoma/ultraestrutura , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Carga Tumoral , Neoplasias Uveais/mortalidade , Neoplasias Uveais/ultraestrutura
4.
Nat Commun ; 11(1): 2907, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32518300

RESUMO

The three-dimensional architecture of the genome affects genomic functions. Multiple genome architectures at different length scales, including chromatin loops, domains, compartments, and lamina- and nucleolus-associated regions, have been discovered. However, how these structures are arranged in the same cell and how they are mutually correlated in different cell types in mammalian tissue are largely unknown. Here, we develop Multiplexed Imaging of Nucleome Architectures that measures multiscale chromatin folding, copy numbers of numerous RNA species, and associations of numerous genomic regions with nuclear lamina, nucleoli and surface of chromosomes in the same, single cells. We apply this method in mouse fetal liver, and identify de novo cell-type-specific chromatin architectures associated with gene expression, as well as cell-type-independent principles of chromatin organization. Polymer simulation shows that both intra-chromosomal self-associating interactions and extra-chromosomal interactions are necessary to establish the observed organization. Our results illustrate a multi-faceted picture and physical principles of chromatin organization.


Assuntos
Nucléolo Celular/metabolismo , Fígado/embriologia , RNA/metabolismo , Animais , Cromatina/metabolismo , Cromossomos/metabolismo , Simulação por Computador , Elementos Facilitadores Genéticos , Dosagem de Genes , Camundongos , Camundongos Endogâmicos C57BL , Hibridização de Ácido Nucleico , Nucleossomos/metabolismo , Sondas de Oligonucleotídeos/química , Polímeros/química , Regiões Promotoras Genéticas
5.
Nature ; 581(7807): 209-214, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32405004

RESUMO

Intracellular bodies such as nucleoli, Cajal bodies and various signalling assemblies represent membraneless organelles, or condensates, that form via liquid-liquid phase separation (LLPS)1,2. Biomolecular interactions-particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions-are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems3-6 have led to the concept that a single fixed saturation concentration is a defining feature of endogenous LLPS7-9, and has been suggested as a mechanism for intracellular concentration buffering2,7,8,10. However, the assumption of a fixed saturation concentration remains largely untested within living cells, in which the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed saturation concentration. As the concentration of individual components is varied, their partition coefficients change in a manner that can be used to determine the thermodynamic free energies that underlie LLPS. We find that heterotypic interactions among protein and RNA components stabilize various archetypal intracellular condensates-including the nucleolus, Cajal bodies, stress granules and P-bodies-implying that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA-processing condensates such as the nucleolus, this manifests in the selective exclusion of fully assembled ribonucleoprotein complexes, providing a thermodynamic basis for vectorial ribosomal RNA flux out of the nucleolus. This methodology is conceptually straightforward and readily implemented, and can be broadly used to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deeper understanding of the thermodynamics of multicomponent intracellular phase behaviour and its interplay with the nonequilibrium activity that is characteristic of endogenous condensates.


Assuntos
Espaço Intracelular/química , Espaço Intracelular/metabolismo , Organelas/química , Organelas/metabolismo , Termodinâmica , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Nucléolo Celular/química , Nucléolo Celular/metabolismo , Corpos Enovelados/química , Corpos Enovelados/metabolismo , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/deficiência , Células HeLa , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Transição de Fase , Proteínas de Ligação a Poli-ADP-Ribose/deficiência , RNA Helicases/deficiência , Proteínas com Motivo de Reconhecimento de RNA/deficiência , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA , Ribossomos/química , Ribossomos/metabolismo
6.
Nucleic Acids Res ; 48(11): 5891-5906, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32421830

RESUMO

Originally identified as an RNA polymerase II interactor, Che-1/AATF (Che-1) has now been recognized as a multifunctional protein involved in cell-cycle regulation and cancer progression, as well as apoptosis inhibition and response to stress. This protein displays a peculiar nucleolar localization and it has recently been implicated in pre-rRNA processing and ribosome biogenesis. Here, we report the identification of a novel function of Che-1 in the regulation of ribosomal RNA (rRNA) synthesis, in both cancer and normal cells. We demonstrate that Che-1 interacts with RNA polymerase I and nucleolar upstream binding factor (UBF) and promotes RNA polymerase I-dependent transcription. Furthermore, this protein binds to the rRNA gene (rDNA) promoter and modulates its epigenetic state by contrasting the recruitment of HDAC1. Che-1 downregulation affects RNA polymerase I and UBF recruitment on rDNA and leads to reducing rDNA promoter activity and 47S pre-rRNA production. Interestingly, Che-1 depletion induces abnormal nucleolar morphology associated with re-distribution of nucleolar proteins. Finally, we show that upon DNA damage Che-1 re-localizes from rDNA to TP53 gene promoter to induce cell-cycle arrest. This previously uncharacterized function of Che-1 confirms the important role of this protein in the regulation of ribosome biogenesis, cellular proliferation and response to stress.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , DNA Ribossômico/genética , Genes de RNAr/genética , RNA Polimerase I/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Genética , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Nucléolo Celular/metabolismo , Nucléolo Celular/patologia , Dano ao DNA , DNA Ribossômico/metabolismo , Homeostase , Humanos , Fosforilação , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Ribossomos/metabolismo
7.
Nucleic Acids Res ; 48(11): 6210-6222, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32365182

RESUMO

The ribotoxin α-sarcin belongs to a family of ribonucleases that cleave the sarcin/ricin loop (SRL), a critical functional rRNA element within the large ribosomal subunit (60S), thereby abolishing translation. Whether α-sarcin targets the SRL only in mature 60S subunits remains unresolved. Here, we show that, in yeast, α-sarcin can cleave SRLs within late 60S pre-ribosomes containing mature 25S rRNA but not nucleolar/nuclear 60S pre-ribosomes containing 27S pre-rRNA in vivo. Conditional expression of α-sarcin is lethal, but does not impede early pre-rRNA processing, nuclear export and the cytoplasmic maturation of 60S pre-ribosomes. Thus, SRL-cleaved containing late 60S pre-ribosomes seem to escape cytoplasmic proofreading steps. Polysome analyses revealed that SRL-cleaved 60S ribosomal subunits form 80S initiation complexes, but fail to progress to the step of translation elongation. We suggest that the functional integrity of a α-sarcin cleaved SRL might be assessed only during translation.


Assuntos
Endorribonucleases/metabolismo , Proteínas Fúngicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/química , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Ricina/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Endorribonucleases/farmacologia , Proteínas Fúngicas/farmacologia , Biossíntese de Proteínas , RNA Ribossômico/metabolismo , Ricina/química , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento
8.
Proc Natl Acad Sci U S A ; 117(19): 10368-10377, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32332163

RESUMO

Nucleoli, the sites of ribosome biogenesis and the largest structures in human nuclei, form around nucleolar organizer regions (NORs) comprising ribosomal DNA (rDNA) arrays. NORs are located on the p-arms of the five human acrocentric chromosomes. Defining the rules of engagement between these p-arms and nucleoli takes on added significance as describing the three-dimensional organization of the human genome represents a major research goal. Here we used fluorescent in situ hybridization (FISH) and immuno-FISH on metaphase chromosomes from karyotypically normal primary and hTERT-immortalized human cell lines to catalog NORs in terms of their relative rDNA content and activity status. We demonstrate that a proportion of acrocentric p-arms in cell lines and from normal human donors have no detectable rDNA. Surprisingly, we found that all NORs with detectable rDNA are active, as defined by upstream binding factor loading. We determined the nucleolar association status of all NORs during interphase, and found that nucleolar association of acrocentric p-arms can occur independently of rDNA content, suggesting that sequences elsewhere on these chromosome arms drive nucleolar association. In established cancer lines, we characterize a variety of chromosomal rearrangements involving acrocentric p-arms and observe silent, rDNA-containing NORs that are dissociated from nucleoli. In conclusion, our findings indicate that within human nuclei, positioning of all 10 acrocentric chromosomes is dictated by nucleolar association. Furthermore, these nucleolar associations are buffered against interindividual variation in the distribution of rDNA.


Assuntos
DNA Ribossômico/genética , Região Organizadora do Nucléolo/metabolismo , Região Organizadora do Nucléolo/fisiologia , Linhagem Celular , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Centrômero/fisiologia , Cromossomos Humanos/metabolismo , DNA Ribossômico/metabolismo , Genoma Humano/genética , Genoma Humano/fisiologia , Humanos , Hibridização in Situ Fluorescente/métodos , Região Organizadora do Nucléolo/genética , Ribossomos/metabolismo
9.
PLoS One ; 15(3): e0223030, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32119673

RESUMO

Numerous studies show that various genes in all kinds of organisms are transcribed discontinuously, i.e. in short bursts or pulses with periods of inactivity between them. But it remains unclear whether ribosomal DNA (rDNA), represented by multiple copies in every cell, is also expressed in such manner. In this work, we synchronized the pol I activity in the populations of tumour derived as well as normal human cells by cold block and release. Our experiments with 5-fluorouridine (FU) and BrUTP confirmed that the nucleolar transcription can be efficiently and reversibly arrested at +4°C. Then using special software for analysis of the microscopic images, we measured the intensity of transcription signal (incorporated FU) in the nucleoli at different time points after the release. We found that the ribosomal genes in the human cells are transcribed discontinuously with periods ranging from 45 min to 75 min. Our data indicate that the dynamics of rDNA transcription follows the undulating pattern, in which the bursts are alternated by periods of rare transcription events.


Assuntos
DNA Ribossômico/genética , Ribossomos/genética , Transcrição Genética , Idoso , Cadáver , Nucléolo Celular/genética , Temperatura Baixa , Células Epiteliais/metabolismo , Células HeLa , Humanos , Cinética , Limbo da Córnea/citologia , Pessoa de Meia-Idade , RNA Ribossômico/genética , Software , Transfecção , Uridina/análogos & derivados , Uridina/imunologia , Uridina/metabolismo , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/imunologia , Uridina Trifosfato/metabolismo
10.
Biochem J ; 477(4): 773-786, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32011671

RESUMO

NF-κB repressing factor (NKRF) was recently identified as an RNA binding protein that together with its associated proteins, the 5'-3' exonuclease XRN2 and the helicase DHX15, is required to process the precursor ribosomal RNA. XRN2 is a multi-functional ribonuclease that is also involved in processing mRNAs, tRNAs and lncRNAs. The activity and stability of XRN2 are controlled by its binding partners, PAXT-1, CDKN2AIP and CDKN2AIPNL. In each case, these proteins interact with XRN2 via an XRN2 binding domain (XTBD), the structure and mode of action of which is highly conserved. Rather surprisingly, although NKRF interacts directly with XRN2, it was not predicted to contain such a domain, and NKRF's interaction with XRN2 was therefore unexplained. We have identified an alternative upstream AUG start codon within the transcript that encodes NKRF and demonstrate that the full-length form of NKRF contains an XTBD that is conserved across species. Our data suggest that NKRF is tethered in the nucleolus by binding directly to rRNA and that the XTBD in the N-terminal extension of NKRF is essential for the retention of XRN2 in this sub-organelle. Thus, we propose NKRF regulates the early steps of pre-rRNA processing during ribosome biogenesis by controlling the spatial distribution of XRN2 and our data provide further support for the XTBD as an XRN2 interacting motif.


Assuntos
Nucléolo Celular/metabolismo , Exorribonucleases/metabolismo , Domínios e Motivos de Interação entre Proteínas , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Exorribonucleases/genética , Células HeLa , Humanos , Ligação Proteica , Proteínas Repressoras/genética , Homologia de Sequência
11.
Proc Natl Acad Sci U S A ; 117(8): 4300-4309, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32047031

RESUMO

V(D)J recombination assembles and diversifies Ig and T cell receptor genes in developing B and T lymphocytes. The reaction is initiated by the RAG1-RAG2 protein complex which binds and cleaves at discrete gene segments in the antigen receptor loci. To identify mechanisms that regulate V(D)J recombination, we used proximity-dependent biotin identification to analyze the interactomes of full-length and truncated forms of RAG1 in pre-B cells. This revealed an association of RAG1 with numerous nucleolar proteins in a manner dependent on amino acids 216 to 383 and allowed identification of a motif required for nucleolar localization. Experiments in transformed pre-B cell lines and cultured primary pre-B cells reveal a strong correlation between disruption of nucleoli, reduced association of RAG1 with a nucleolar marker, and increased V(D)J recombination activity. Mutation of the RAG1 nucleolar localization motif boosts recombination while removal of the first 215 amino acids of RAG1, required for efficient egress from nucleoli, reduces recombination activity. Our findings indicate that nucleolar sequestration of RAG1 is a negative regulatory mechanism in V(D)J recombination and identify regions of the RAG1 N-terminal region that control nucleolar association and egress.


Assuntos
Nucléolo Celular/metabolismo , Proteínas de Homeodomínio/metabolismo , Recombinação V(D)J , Motivos de Aminoácidos , Animais , Nucléolo Celular/genética , Células Cultivadas , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Camundongos , Células Precursoras de Linfócitos B/metabolismo , Transporte Proteico
12.
Nat Commun ; 11(1): 156, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31919354

RESUMO

Technical problems intrinsic to the purification of preribosome intermediates have limited our understanding of ribosome biosynthesis in humans. Addressing this issue is important given the implication of this biological process in human disease. Here we report a preribosome purification and tagging strategy that overcomes some of the existing technical difficulties. Using these tools, we find that the pre-40S precursors go through two distinct maturation phases inside the nucleolus and follow a regulatory step that precedes late maturation in the cytoplasm. This regulatory step entails the intertwined actions of both PARN (a metazoan-specific ribonuclease) and RRP12 (a phylogenetically conserved 40S biogenesis factor that has acquired additional functional features in higher eukaryotes). Together, these results demonstrate the usefulness of this purification method for the dissection of ribosome biogenesis in human cells. They also identify distinct maturation stages and metazoan-specific regulatory mechanisms involved in the generation of the human 40S ribosomal subunit.


Assuntos
Nucléolo Celular/metabolismo , Proteínas Ribossômicas/biossíntese , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Linhagem Celular Tumoral , Exorribonucleases/metabolismo , Células HCT116 , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Precursores de RNA/biossíntese , Precursores de RNA/metabolismo , RNA Ribossômico/biossíntese , Subunidades Ribossômicas Menores de Eucariotos/genética , Coloração e Rotulagem/métodos
13.
Chem Commun (Camb) ; 56(12): 1859-1862, 2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-31950954

RESUMO

We present the design and synthesis of water-soluble quinoline-indole-based derivatives (IM-1, IM-2, and IM-3) with three-photon absorption activity. IM-3 can specifically target DNA and RNA accompanied by an obvious three-photon fluorescence enhancement in the second near-infrared window (1000-1700 nm). The in vitro experiments demonstrate that IM-3 can simultaneously stain mitochondria and the nucleolus both in living and fixed cells. The organelle-specific targeting behaviour is successfully visualized under stimulated emission depletion (STED) nanoscopy.


Assuntos
DNA Mitocondrial/análise , Corantes Fluorescentes/química , Indóis/química , Fótons , Quinolinas/química , RNA Neoplásico/análise , Nucléolo Celular/química , Células Hep G2 , Humanos , Estrutura Molecular , Imagem Óptica , Solubilidade , Água/química
14.
Nat Commun ; 11(1): 123, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31913317

RESUMO

Induction of DNA double-strand breaks (DSBs) in ribosomal DNA (rDNA) repeats is associated with ATM-dependent repression of ribosomal RNA synthesis and large-scale reorganization of nucleolar architecture, but the signaling events that regulate these responses are largely elusive. Here we show that the nucleolar response to rDNA breaks is dependent on both ATM and ATR activity. We further demonstrate that ATM- and NBS1-dependent recruitment of TOPBP1 in the nucleoli is required for inhibition of ribosomal RNA synthesis and nucleolar segregation in response to rDNA breaks. Mechanistically, TOPBP1 recruitment is mediated by phosphorylation-dependent interactions between three of its BRCT domains and conserved phosphorylated Ser/Thr residues at the C-terminus of the nucleolar phosphoprotein Treacle. Our data thus reveal an important cooperation between TOPBP1 and Treacle in the signaling cascade that triggers transcriptional inhibition and nucleolar segregation in response to rDNA breaks.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Transporte/metabolismo , Nucléolo Celular/genética , DNA Ribossômico/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Motivos de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Nucléolo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Ribossômico/metabolismo , Proteínas de Ligação a DNA/genética , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Ligação Proteica , RNA Ribossômico/genética , RNA Ribossômico/metabolismo
15.
J Virol ; 94(7)2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-31941785

RESUMO

Biological macromolecule condensates formed by liquid-liquid phase separation (LLPS) have been discovered in recent years to be prevalent in biology. These condensates are involved in diverse processes, including the regulation of gene expression. LLPS of proteins have been found in animal, plant, and bacterial species but have scarcely been identified in viral proteins. Here, we discovered that Epstein-Barr virus (EBV) EBNA2 and EBNALP form nuclear puncta that exhibit properties of liquid-like condensates (or droplets), which are enriched in superenhancers of MYC and Runx3. EBNA2 and EBNALP are transcription factors, and the expression of their target genes is suppressed by chemicals that perturb LLPS. Intrinsically disordered regions (IDRs) of EBNA2 and EBNALP can form phase-separated droplets, and specific proline residues of EBNA2 and EBNALP contribute to droplet formation. These findings offer a foundation for understanding the mechanism by which LLPS, previously determined to be related to the organization of P bodies, membraneless organelles, nucleolus homeostasis, and cell signaling, plays a key role in EBV-host interactions and is involved in regulating host gene expression. This work suggests a novel anti-EBV strategy where developing appropriate drugs of interfering LLPS can be used to destroy the function of the EBV's transcription factors.IMPORTANCE Protein condensates can be assembled via liquid-liquid phase separation (LLPS), a process involving the concentration of molecules in a confined liquid-like compartment. LLPS allows for the compartmentalization and sequestration of materials and can be harnessed as a sensitive strategy for responding to small changes in the environment. This study identified the Epstein-Barr virus (EBV) proteins EBNA2 and EBNALP, which mediate virus and cellular gene transcription, as transcription factors that can form liquid-like condensates at superenhancer sites of MYC and Runx3. This study discovered the first identified LLPS of EBV proteins and emphasized the importance of LLPS in controlling host gene expression.


Assuntos
Antígenos Nucleares do Vírus Epstein-Barr/química , Regulação da Expressão Gênica , Proteínas Intrinsicamente Desordenadas/química , Proteínas Virais/química , Linhagem Celular Tumoral , Nucléolo Celular/química , Núcleo Celular , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Genes myc , Células HEK293 , Herpesvirus Humano 4/fisiologia , Humanos , Leucócitos Mononucleares , Microscopia de Fluorescência , Prolina/química , Regiões Promotoras Genéticas , Domínios Proteicos
16.
FASEB J ; 34(1): 1107-1121, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914708

RESUMO

The nucleolus is best known for its cellular role in regulating ribosome production and growth. More recently, an unanticipated role for the nucleolus in innate immunity has recently emerged whereby downregulation of fibrillarin and nucleolar contraction confers pathogen resistance across taxa. The mechanism of this downregulation, however, remains obscure. Here we report that rather than fibrillarin itself being the proximal factor in this pathway, the key player is a fibrillarin-stabilizing deubiquitinylase USP-33. This was discovered by a candidate-gene search of Caenorhabditis elegans in which CED-3 caspase was revealed to execute targeted cleavage of USP-33, thus destabilizing fibrillarin. We also showed that cep-1 and ced-3 mutant worms altered nucleolar size and decreased antimicrobial peptide gene, spp-1, expression rendering susceptibility to bacterial infection. These phenotypes were reversed by usp-33 knockdown, thus linking the CEP-1-CED-3-USP-33 pathway with nucleolar control and resistance to bacterial infection in worms. Parallel experiments with the human analogs of caspases and USP36 revealed similar roles in coordinating these two processes. In summary, our work outlined a conserved cascade that connects cell death signaling to nucleolar control and innate immune response.


Assuntos
Infecções Bacterianas/metabolismo , Caenorhabditis elegans/microbiologia , Nucléolo Celular/metabolismo , Enzimas Desubiquitinantes/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/metabolismo , Animais , Apoptose , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Células HeLa , Humanos , Microscopia de Fluorescência , Infecções por Pseudomonas , Interferência de RNA , Infecções Estafilocócicas , Estaurosporina/farmacologia , Ubiquitina Tiolesterase/metabolismo
17.
Life Sci ; 241: 117100, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31783052

RESUMO

AIMS: The present study aims to define maturation, yield, health, and ease of collection of murine immature oocytes recovered using the conventional method or from mice treated with cilostazol. MAIN METHODS: The conventional method included the superovulation of mice and the recovery of germinal vesicle (GV) or metaphase (MI) oocytes from preovulatory follicles. The cilostazol method included the oral treatment of superovulated mice with 7.5 mg cilostazol once or twice to result in the ovulation of MI or GV oocytes, respectively. KEY FINDINGS: The cilostazol method resulted in >95% of GV or MI oocytes with a diameter range of 60-90 µm or 50.1-70 µm in comparison to <60.0% of GV or MI oocytes resulting from the conventional method, respectively (P < 0.0001). The cilostazol method resulted in GV oocytes having higher levels of co-occurrence of peripheral cortical granules (CG) and chromatin configuration of surrounded nucleolus and MI oocytes having higher levels of co-occurrence of normally organized spindles/chromosomes and peripheral CG with free domains than did the conventional method (P < 0.001). The cilostazol method was more time and labor efficient and resulted in higher oocyte yields of normal morphology than did the conventional method (P < 0.01). SIGNIFICANCE: The presented method provides not only oocytes with uniform size and synchronized developmental maturation but also a technique of oocyte collection that is efficient and resourceful. It is possible that not all immature oocytes resulting from the conventional method are from preovulatory follicles nor have been developed adequately and consequently ovulated as opposed to the presented method.


Assuntos
Cilostazol/farmacologia , Recuperação de Oócitos/métodos , Oócitos/citologia , Oócitos/fisiologia , Inibidores da Fosfodiesterase 3/farmacologia , Animais , Nucléolo Celular , Núcleo Celular , Cromatina/ultraestrutura , Feminino , Técnicas de Maturação in Vitro de Oócitos/métodos , Metáfase , Camundongos , Ovulação/efeitos dos fármacos , Superovulação/efeitos dos fármacos
18.
Anal Bioanal Chem ; 412(2): 311-319, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31735990

RESUMO

Currently two techniques exist for 3D reconstruction of biological samples by time-of-flight secondary ion mass spectrometry (ToF-SIMS). The first, based on microtomy and combining of successive section images, is successfully applied for tissues, while the second, based on sputter depth profiling, is widely used for cells. In the present work, we report the first successful adaptation of sectioning technique for ToF-SIMS 3D imaging of a single cell-fully grown mouse germinal vesicle (GV) oocyte. In addition, microtomy was combined with sputter depth profiling of individual flat sections for three-dimensional reconstruction of intracellular organelles. GV oocyte sectioning allowed us to obtain molecule-specific 3D maps free from artifacts associated with surface topography and uneven etching depth. Sputter depth profiling of individual flat slices revealed fine structure of specific organelles inside the oocyte. Different oocyte organelles (cytoplasm, germinal vesicle, membranes, cumulus cells) were presented on the ion images. Atypical nucleoli referred to as "nucleolus-like body" (NLB) was detected inside the germinal vesicle in PO3- and CN- ions generated by nucleic acids and proteins respectively. Significant difference in PO3- intensity in the NLB central area and NLB border was found. This difference appears as a bright halo around the center area. The NLB size calculated for PO3- and CN- ion images is 12.9 ± 0.2 µm and 11.9 ± 0.2 µm respectively, which suggests that bright halo of PO3- ions is a chromatin compaction on the NLB surface. Areas of approximately 1.0-2.5 µm size inside nucleoplasm with increased PO3- and CN- signal were registered in germinal vesicle. Observed compartments have different sizes and shapes, and they are likely attributed to chromocenters or chromosomes.


Assuntos
Imageamento Tridimensional/métodos , Oócitos/citologia , Espectrometria de Massa de Íon Secundário/métodos , Animais , Nucléolo Celular/metabolismo , Citoplasma/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL
19.
Anal Chim Acta ; 1096: 148-158, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31883581

RESUMO

Nitric oxide (NO) is a very important signal molecule implicated in numerous physiological and pathological processes, and its detection is the key to understand these processes. For this reason, various fluorescent probes have been developed for detection analysis of NO. However, few rapid-response (<1 min) and ratiometric fluorescent probe are reported for real-time detection of short-time NO in biological systems. In this work, we report a rapid-response (within several seconds) and ratiometric fluorescent probe, RatioTr, which displays selective and sensitive detection of NO in solutions, and detections of exo- and endogenous NO in live RAW 264.7 cells. Unexpectedly, the probe RatioTr and its sensing product (p-Nus) display different cellular localizations, the mitochondria and the nucleus, which were demonstrated by co-stained experiments. The sensing process of RatioTr toward NO from mitochondria to nucleus was observed in live cells by confocal fluorescence images. Furthermore, the subcellular localizations were demonstrated by measurements of pKa and interaction of p-Nus and DNA. In the presence of a natural DNA, calf thymus DNA, RatioTr is more sensitive to NO (LOD = 2.8 nM). Therefore, due to the nucleus localization together with a high fluorescence efficiency in the nucleus, p-Nus is a good candidate of cell-permeant nucleic acid stain or a fluorescent probe for the nucleus.


Assuntos
Nucléolo Celular/química , Corantes Fluorescentes/química , Mitocôndrias/química , Óxido Nítrico/análise , Animais , Limite de Detecção , Camundongos , Microscopia de Fluorescência/economia , Microscopia de Fluorescência/métodos , Modelos Moleculares , Imagem Óptica/economia , Imagem Óptica/métodos , Células RAW 264.7 , Espectrometria de Fluorescência/economia , Espectrometria de Fluorescência/métodos , Fatores de Tempo
20.
Genes Dev ; 33(23-24): 1617-1618, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31792016

RESUMO

Although the nucleolus was first described in the early 19th century from both animal and plant cells, human nucleoli and particularly the five human nucleolus organizers have not been well characterized. In this issue of Genes & Development, van Sluis and colleagues (pp. 1688-1701) present a detailed molecular analysis of these organizers, which occur on the short arms of five human chromosomes. The near identity of these arms suggests extensive interchromosomal exchange during evolutionary history.


Assuntos
Nucléolo Celular , Região Organizadora do Nucléolo , Animais , DNA Ribossômico , Humanos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA