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1.
Nat Commun ; 11(1): 4593, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32929070

RESUMO

Gene-targeted animal models that are generated by injecting Cas9 and sgRNAs into zygotes are often accompanied by undesired double-strand break (DSB)-induced byproducts and random biallelic targeting due to uncontrollable Cas9 targeting activity. Here, we establish a parental allele-specific gene-targeting (Past-CRISPR) method, based on the detailed observation that pronuclear transfer-mediated cytoplasmic dilution can effectively terminate Cas9 activity. We apply this method in embryos to efficiently target the given parental alleles of a gene of interest and observed little genomic mosaicism because of the spatiotemporal control of Cas9 activity. This method allows us to rapidly explore the function of individual parent-of-origin effects and to construct animal models with a single genomic change. More importantly, Past-CRISPR could also be used for therapeutic applications or disease model construction.


Assuntos
Alelos , Sistemas CRISPR-Cas/genética , Núcleo Celular/genética , Edição de Genes , Terapia de Substituição Mitocondrial , Animais , Sequência de Bases , Modelos Animais de Doenças , Nanismo/genética , Perda do Embrião/genética , Feminino , Marcação de Genes , Genes Dominantes , Impressão Genômica , Heterozigoto , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Reprodutibilidade dos Testes , Fatores de Tempo
2.
Molecules ; 25(18)2020 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-32899754

RESUMO

The emergence of the Coronavirus Disease 2019 (COVID-19) caused by the SARS-CoV-2 virus has led to an unprecedented pandemic, which demands urgent development of antiviral drugs and antibodies; as well as prophylactic approaches, namely vaccines. Algae biotechnology has much to offer in this scenario given the diversity of such organisms, which are a valuable source of antiviral and anti-inflammatory compounds that can also be used to produce vaccines and antibodies. Antivirals with possible activity against SARS-CoV-2 are summarized, based on previously reported activity against Coronaviruses or other enveloped or respiratory viruses. Moreover, the potential of algae-derived anti-inflammatory compounds to treat severe cases of COVID-19 is contemplated. The scenario of producing biopharmaceuticals in recombinant algae is presented and the cases of algae-made vaccines targeting viral diseases is highlighted as valuable references for the development of anti-SARS-CoV-2 vaccines. Successful cases in the production of functional antibodies are described. Perspectives on how specific algae species and genetic engineering techniques can be applied for the production of anti-viral compounds antibodies and vaccines against SARS-CoV-2 are provided.


Assuntos
Antivirais/farmacologia , Produtos Biológicos/farmacologia , Chlamydomonas reinhardtii/genética , Infecções por Coronavirus/tratamento farmacológico , Lectinas/farmacologia , Pneumonia Viral/tratamento farmacológico , Polifenóis/farmacologia , Polissacarídeos/farmacologia , Antivirais/química , Antivirais/isolamento & purificação , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/patogenicidade , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Chlamydomonas reinhardtii/química , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/química , Cloroplastos/genética , Cloroplastos/metabolismo , Infecções por Coronavirus/prevenção & controle , Engenharia Genética/métodos , Humanos , Lectinas/química , Lectinas/isolamento & purificação , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Pandemias , Polifenóis/química , Polifenóis/isolamento & purificação , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/patogenicidade , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Vacinas Virais/biossíntese , Vacinas Virais/farmacologia
3.
Nat Commun ; 11(1): 4868, 2020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-32985517

RESUMO

DNA damage sensors DDB2 and XPC initiate global genome nucleotide excision repair (NER) to protect DNA from mutagenesis caused by helix-distorting lesions. XPC recognizes helical distortions by binding to unpaired ssDNA opposite DNA lesions. DDB2 binds to UV-induced lesions directly and facilitates efficient recognition by XPC. We show that not only lesion-binding but also timely DDB2 dissociation is required for DNA damage handover to XPC and swift progression of the multistep repair reaction. DNA-binding-induced DDB2 ubiquitylation and ensuing degradation regulate its homeostasis to prevent excessive lesion (re)binding. Additionally, damage handover from DDB2 to XPC coincides with the arrival of the TFIIH complex, which further promotes DDB2 dissociation and formation of a stable XPC-TFIIH damage verification complex. Our results reveal a reciprocal coordination between DNA damage recognition and verification within NER and illustrate that timely repair factor dissociation is vital for correct spatiotemporal control of a multistep repair process.


Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição TFIIH/metabolismo , Ubiquitina/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Humanos , Fator de Transcrição TFIIH/genética , Ubiquitinação
4.
Anticancer Res ; 40(10): 5659-5666, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988890

RESUMO

BACKGROUND/AIM: Long interspersed nuclear element-1 (LINE-1) methylation status is a marker for global DNA methylation. However, the relationship between LINE-1 methylation and the biology of lung adenocarcinoma remains unclear. Here, we aimed to examine the role of LINE-1 in lung cancer. MATERIALS AND METHODS: LINE-1 methylation levels were quantified by bisulfite pyrosequencing of resected tumor specimens from 162 patients with lung adenocarcinoma. The relationships of LINE-1 methylation with clinicopathological factors, gene mutations, and Ki-67 immunoreactivity were investigated. RESULTS: LINE-1 hypomethylation was associated with tumor invasion and advanced stage. TP53 mutations were more frequently detected in the LINE-1 hypomethylation group than in the hypermethylation group. LINE-1 hypomethylation was associated with poor recurrence-free survival, high maximum standardized uptake value in positron-emission tomography, and high Ki-67 expression in tumors. CONCLUSION: LINE-1 hypomethylation was associated with high-grade malignancy and poor prognosis in lung adenocarcinoma, but was not related to driver mutations.


Assuntos
Adenocarcinoma de Pulmão/genética , Biomarcadores Tumorais/genética , Metilação de DNA/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Adenocarcinoma de Pulmão/patologia , Adulto , Idoso , Núcleo Celular/genética , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Antígeno Ki-67/genética , Masculino , Pessoa de Meia-Idade , Mutação/genética , Prognóstico , Proteína Supressora de Tumor p53/genética
5.
Virology ; 548: 31-38, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32838944

RESUMO

Entering the nucleus is important for Porcine circovirus type 2 (PCV2) replication. Karyopherins (KPNs) mediate the nuclear import of many cytoplasmic proteins. Our previous study showed that KPNA3 is involved in interferon production during PCV2 infection induced by Poly I:C and ISD (Interferon stimulatory DNA). However, it remains unclear whether PCV2 replication is associated with KPNA3. In the present study, knockdown of KPNA3 promoted the replication of PCV2, whereas overexpression of KPNA3 inhibited PCV2 replication in PK-15 cells. Furthermore, KPNA3 knockdown inhibited IRF3 and reduced the expression of antiviral genes including IFN-ß, ISG54, Mx1 and ISG56, while the opposite results were obtained after KPNA3 overexpression. KPNA3 knockdown also promoted p65 nuclear translocation and increased the mRNA expression of IL-10 and IL-1ß. These results suggested that KPNA3 facilitates IRF3 entry into the nucleus and the production of an antiviral response, resulting in PCV2 replication inhibition and blockage of NF-κB signal activation.


Assuntos
Núcleo Celular/metabolismo , Infecções por Circoviridae/veterinária , Circovirus/fisiologia , Doenças dos Suínos/metabolismo , alfa Carioferinas/metabolismo , Animais , Núcleo Celular/genética , Infecções por Circoviridae/genética , Infecções por Circoviridae/metabolismo , Infecções por Circoviridae/virologia , Circovirus/genética , Interações Hospedeiro-Patógeno , Interleucina-10/genética , Interleucina-10/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Transporte Proteico , Transdução de Sinais , Suínos , Doenças dos Suínos/genética , Doenças dos Suínos/virologia , Replicação Viral , alfa Carioferinas/genética
6.
PLoS Pathog ; 16(8): e1008752, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32760121

RESUMO

Members of the family of pyrin and HIN domain containing (PYHIN) proteins play an emerging role in innate immunity. While absent in melanoma 2 (AIM2) acts a cytosolic sensor of non-self DNA and plays a key role in inflammasome assembly, the γ-interferon-inducible protein 16 (IFI16) restricts retroviral gene expression by sequestering the transcription factor Sp1. Here, we show that the remaining two human PYHIN proteins, i.e. myeloid cell nuclear differentiation antigen (MNDA) and pyrin and HIN domain family member 1 (PYHIN1 or IFIX) share this antiretroviral function of IFI16. On average, knock-down of each of these three nuclear PYHIN proteins increased infectious HIV-1 yield from human macrophages by more than an order of magnitude. Similarly, knock-down of IFI16 strongly increased virus transcription and production in primary CD4+ T cells. The N-terminal pyrin domain (PYD) plus linker region containing a nuclear localization signal (NLS) were generally required and sufficient for Sp1 sequestration and anti-HIV-1 activity of IFI16, MNDA and PYHIN1. Replacement of the linker region of AIM2 by the NLS-containing linker of IFI16 resulted in a predominantly nuclear localization and conferred direct antiviral activity to AIM2 while attenuating its ability to form inflammasomes. The reverse change caused nuclear-to-cytoplasmic relocalization of IFI16 and impaired its antiretroviral activity but did not result in inflammasome assembly. We further show that the Zn-finger domain of Sp1 is critical for the interaction with IFI16 supporting that pyrin domains compete with DNA for Sp1 binding. Finally, we found that human PYHIN proteins also inhibit Hepatitis B virus and simian vacuolating virus 40 as well as the LINE-1 retrotransposon. Altogether, our data show that IFI16, PYHIN1 and MNDA restrict HIV-1 and other viral pathogens by interfering with Sp1-dependent gene expression and support an important role of nuclear PYHIN proteins in innate antiviral immunity.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Núcleo Celular/metabolismo , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Macrófagos/imunologia , Proteínas Nucleares/metabolismo , Fator de Transcrição Sp1/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Núcleo Celular/genética , DNA Viral/genética , Células HEK293 , Infecções por HIV/imunologia , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/isolamento & purificação , Células Hep G2 , Humanos , Imunidade Inata/imunologia , Inflamassomos/genética , Inflamassomos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Proteínas Nucleares/genética , Fator de Transcrição Sp1/genética , Replicação Viral
7.
Nat Commun ; 11(1): 4028, 2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32788591

RESUMO

Changes in atmospheric CO2 concentration have played a central role in algal and plant adaptation and evolution. The commercially important red algal genus, Pyropia (Bangiales) appears to have responded to inorganic carbon (Ci) availability by evolving alternating heteromorphic generations that occupy distinct habitats. The leafy gametophyte inhabits the intertidal zone that undergoes frequent emersion, whereas the sporophyte conchocelis bores into mollusk shells. Here, we analyze a high-quality genome assembly of Pyropia yezoensis to elucidate the interplay between Ci availability and life cycle evolution. We find horizontal gene transfers from bacteria and expansion of gene families (e.g. carbonic anhydrase, anti-oxidative related genes), many of which show gametophyte-specific expression or significant up-regulation in gametophyte in response to dehydration. In conchocelis, the release of HCO3- from shell promoted by carbonic anhydrase provides a source of Ci. This hypothesis is supported by the incorporation of 13C isotope by conchocelis when co-cultured with 13C-labeled CaCO3.


Assuntos
Carbono/metabolismo , Genoma , Rodófitas/genética , Rodófitas/metabolismo , Movimentos da Água , Exoesqueleto/química , Animais , Antioxidantes/farmacologia , Composição de Bases/genética , Evolução Biológica , Carbonato de Cálcio/metabolismo , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Núcleo Celular/genética , Dosagem de Genes , Perfilação da Expressão Gênica , Transferência Genética Horizontal/genética , Moluscos , Fotossíntese/efeitos dos fármacos , Ploidias , Rodófitas/efeitos dos fármacos , Superóxido Dismutase/genética , Transcrição Genética/efeitos dos fármacos
8.
Nat Cell Biol ; 22(7): 767-778, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32601371

RESUMO

Following fertilization in mammals, the gametes are reprogrammed to create a totipotent zygote, a process that involves de novo establishment of chromatin domains. A major feature occurring during preimplantation development is the dramatic remodelling of constitutive heterochromatin, although the functional relevance of this is unknown. Here, we show that heterochromatin establishment relies on the stepwise expression and regulated activity of SUV39H enzymes. Enforcing precocious acquisition of constitutive heterochromatin results in compromised development and epigenetic reprogramming, which demonstrates that heterochromatin remodelling is essential for natural reprogramming at fertilization. We find that de novo H3K9 trimethylation (H3K9me3) in the paternal pronucleus after fertilization is catalysed by SUV39H2 and that pericentromeric RNAs inhibit SUV39H2 activity and reduce H3K9me3. De novo H3K9me3 is initially non-repressive for gene expression, but instead bookmarks promoters for compaction. Overall, we uncover the functional importance for the restricted transmission of constitutive heterochromatin during reprogramming and a non-repressive role for H3K9me3.


Assuntos
Centrômero/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Heterocromatina/metabolismo , Histonas/metabolismo , RNA/metabolismo , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Epigênese Genética , Feminino , Heterocromatina/genética , Histonas/genética , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , RNA/genética
9.
Nat Cell Biol ; 22(7): 856-867, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32601372

RESUMO

The ESCRT-III membrane fission machinery maintains the integrity of the nuclear envelope. Although primary nuclei resealing takes minutes, micronuclear envelope ruptures seem to be irreversible. Instead, micronuclear ruptures result in catastrophic membrane collapse and are associated with chromosome fragmentation and chromothripsis, complex chromosome rearrangements thought to be a major driving force in cancer development. Here we use a combination of live microscopy and electron tomography, as well as computer simulations, to uncover the mechanism underlying micronuclear collapse. We show that, due to their small size, micronuclei inherently lack the capacity of primary nuclei to restrict the accumulation of CHMP7-LEMD2, a compartmentalization sensor that detects loss of nuclear integrity. This causes unrestrained ESCRT-III accumulation, which drives extensive membrane deformation, DNA damage and chromosome fragmentation. Thus, the nuclear-integrity surveillance machinery is a double-edged sword, as its sensitivity ensures rapid repair at primary nuclei while causing unrestrained activity at ruptured micronuclei, with catastrophic consequences for genome stability.


Assuntos
Núcleo Celular/patologia , Cromatina/metabolismo , Aberrações Cromossômicas , Dano ao DNA , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Instabilidade Genômica , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Células HeLa , Humanos
10.
Nucleic Acids Res ; 48(15): 8626-8644, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32621609

RESUMO

The exon junction complex (EJC) is an essential constituent and regulator of spliced messenger ribonucleoprotein particles (mRNPs) in metazoans. As a core component of the EJC, CASC3 was described to be pivotal for EJC-dependent nuclear and cytoplasmic processes. However, recent evidence suggests that CASC3 functions differently from other EJC core proteins. Here, we have established human CASC3 knockout cell lines to elucidate the cellular role of CASC3. In the knockout cells, overall EJC composition and EJC-dependent splicing are unchanged. A transcriptome-wide analysis reveals that hundreds of mRNA isoforms targeted by nonsense-mediated decay (NMD) are upregulated. Mechanistically, recruiting CASC3 to reporter mRNAs by direct tethering or via binding to the EJC stimulates mRNA decay and endonucleolytic cleavage at the termination codon. Building on existing EJC-NMD models, we propose that CASC3 equips the EJC with the persisting ability to communicate with the NMD machinery in the cytoplasm. Collectively, our results characterize CASC3 as a peripheral EJC protein that tailors the transcriptome by promoting the degradation of EJC-dependent NMD substrates.


Assuntos
Proteínas de Neoplasias/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , Processamento de RNA/genética , Proteínas de Ligação a RNA/genética , Transcriptoma/genética , Sequência de Aminoácidos/genética , Núcleo Celular/genética , Éxons/genética , Técnicas de Inativação de Genes , Humanos , RNA Mensageiro/genética , Ribonucleoproteínas/genética
11.
Nucleic Acids Res ; 48(15): 8332-8348, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32633757

RESUMO

Negative cofactor 2 (NC2), including two subunits NC2α and NC2ß, is a conserved positive/negative regulator of class II gene transcription in eukaryotes. It is known that NC2 functions by regulating the assembly of the transcription preinitiation complex. However, the exact role of NC2 in transcriptional regulation is still unclear. Here, we reveal that, in Neurospora crassa, NC2 activates catalase-3 (cat-3) gene transcription in the form of heterodimer mediated by histone fold (HF) domains of two subunits. Deletion of HF domain in either of two subunits disrupts the NC2α-NC2ß interaction and the binding of intact NC2 heterodimer to cat-3 locus. Loss of NC2 dramatically increases histone variant H2A.Z deposition at cat-3 locus. Further studies show that NC2 recruits chromatin remodeling complex INO80C to remove H2A.Z from the nucleosomes around cat-3 locus, resulting in transcriptional activation of cat-3. Besides HF domains of two subunits, interestingly, C-terminal repression domain of NC2ß is required not only for NC2 binding to cat-3 locus, but also for the recruitment of INO80C to cat-3 locus and removal of H2A.Z from the nucleosomes. Collectively, our findings reveal a novel mechanism of NC2 in transcription activation through recruiting INO80C to remove H2A.Z from special H2A.Z-containing nucleosomes.


Assuntos
Catalase/genética , Fosfoproteínas/genética , Fatores de Transcrição/genética , Transcrição Genética , Núcleo Celular/genética , Montagem e Desmontagem da Cromatina/genética , Regulação da Expressão Gênica/genética , Genes MHC da Classe II/genética , Histonas/genética , Neurospora crassa/genética , Nucleossomos/genética , Nucleossomos/ultraestrutura , Fosfoproteínas/ultraestrutura , Ligação Proteica/genética , Fatores de Transcrição/ultraestrutura , Ativação Transcricional/genética
12.
Gene ; 758: 144962, 2020 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-32687946

RESUMO

Multiple sclerosis (MS) is a chronic disease of the central nervous system characterized by the autoimmune inflammation, demyelination, and neurodegeneration. This complex disease develops in genetically predisposed individuals under adverse environmental factors. To date, a large number of MS-associated polymorphic loci of the nuclear genome have been identified; however, their total variability can explain only about 48% of the observed inheritance of MS. Polymorphic variants of the mitochondrial genome and interactions of mitochondrial and nuclear genes (mitonuclear interactions) may be the possible sources of the "missing heritability". We analyzed the association with MS of 10 mitochondrial DNA polymorphisms (m.1719, m.4216, m.4580, m.4917, m.7028, m.9055, m.10398, m.12308, m.13368, m.13708) in DNA of 540 MS patients and 406 healthy individuals. The allele m.9055*G was the only mitochondrial variant associated with MS (Pf = 0.027). To evaluate interactions of mitochondrial and nuclear genomes, we searched for biallelic combinations containing one of 10 mitochondrial variants and one of 35 variants of immune-related nuclear genes. Carriership of mitochondrial variants m.4216, m.4580, or m.13708 in biallelic combinations with variants of nuclear genes IL7R, CLEC16A, CD6, CD86 or PVT1 was associated with MS (Pf = 0.0036-0.00030). We identified epistatic interaction between components of a combination (m.13708*A + PVT1 rs4410871*T). The existence of epistatic biallelic combination can reflect the genuine mitonuclear epistasis.


Assuntos
Núcleo Celular/genética , DNA Mitocondrial/genética , Predisposição Genética para Doença/genética , Genoma Mitocondrial/genética , Esclerose Múltipla/genética , Adulto , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/genética , Antígeno B7-2/genética , Feminino , Estudos de Associação Genética , Humanos , Subunidade alfa de Receptor de Interleucina-7/genética , Lectinas Tipo C/genética , Masculino , Mitocôndrias/genética , Proteínas de Transporte de Monossacarídeos/genética , Polimorfismo de Nucleotídeo Único/genética , RNA Longo não Codificante/genética
13.
PLoS Genet ; 16(7): e1008949, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32702045

RESUMO

In Paramecium tetraurelia, a large proportion of the germline genome is reproducibly removed from the somatic genome after sexual events via a process involving small (s)RNA-directed heterochromatin formation and DNA excision and repair. How germline limited DNA sequences are specifically recognized in the context of chromatin remains elusive. Here, we use a reverse genetics approach to identify factors involved in programmed genome rearrangements. We have identified a P. tetraurelia homolog of the highly conserved histone chaperone Spt16 subunit of the FACT complex, Spt16-1, and show its expression is developmentally regulated. A functional GFP-Spt16-1 fusion protein localized exclusively in the nuclei where genome rearrangements take place. Gene silencing of Spt16-1 showed it is required for the elimination of all germline-limited sequences, for the survival of sexual progeny, and for the accumulation of internal eliminated sequence (ies)RNAs, an sRNA population produced when elimination occurs. Normal accumulation of 25 nt scanRNAs and deposition of silent histone marks H3K9me3 and H3K27me3 indicated that Spt16-1 does not regulate the scanRNA-directed heterochromatin pathway involved in the early steps of DNA elimination. We further show that Spt16-1 is required for the correct nuclear localization of the PiggyMac (Pgm) endonuclease, which generates the DNA double-strand breaks required for DNA elimination. Thus, Spt16-1 is essential for Pgm function during programmed genome rearrangements. We propose a model in which Spt16-1 mediates interactions between the excision machinery and chromatin, facilitating endonuclease access to DNA cleavage sites during genome rearrangements.


Assuntos
Núcleo Celular/genética , Chaperonas de Histonas/genética , Paramecium/genética , Transposases/genética , Sequência de Bases/genética , Quebras de DNA de Cadeia Dupla , Clivagem do DNA , DNA de Protozoário/genética , Endonucleases , Rearranjo Gênico/genética , Genoma de Protozoário/genética , Paramecium/crescimento & desenvolvimento
14.
BMC Evol Biol ; 20(1): 92, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32727367

RESUMO

BACKGROUND: Hypotrichia are a group with the most complex morphology and morphogenesis within the ciliated protists. The classification of Gastrostyla-like species, a taxonomically difficult group of hypotrichs with a common ventral cirral pattern but various dorsal and ontogenetic patterns, is poorly understood. Hence, systematic relationships within this group and with other taxa in the subclass Hypotrichia remain unresolved. RESULTS: 18S rRNA gene sequence of a new Gastrostyla-like taxon was obtained. Phylogenetic analyses based on the 18S rRNA gene sequences indicate that this ciliate represents a new genus that is closely related to Heterourosomoida and Kleinstyla within the oxytrichid clade of the Hypotrichia. However, the position of this cluster remains unresolved. All three genera deviate from the typical oxytrichids by their incomplete (or lack of) dorsal kinety fragmentation during morphogenesis. Morphology and morphogenesis of this newly discovered form, Heterogastrostyla salina nov. gen., nov. spec., are described. Heterogastrostyla nov. gen., is characterised as follows: more than 18 fronto-ventral-transverse cirri, cirral anlagen V and VI develop pretransverse cirri, and dorsal ciliature in Urosomoida-like pattern. CONCLUSIONS: Similar to the CEUU-hypothesis about convergent evolution of urostylids and uroleptids, we speculate that the shared ventral cirral patterns of Gastrostyla-like taxa might have resulted from convergent evolution.


Assuntos
Cilióforos/classificação , Classificação , Salinidade , Solo , Animais , Sequência de Bases , Núcleo Celular/genética , DNA Ribossômico/genética , Hypotrichida/classificação , Hypotrichida/genética , Funções Verossimilhança , Morfogênese/genética , Filogenia , Subunidades Ribossômicas Menores/genética , Especificidade da Espécie
15.
Nat Commun ; 11(1): 3439, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651385

RESUMO

Various stress conditions induce the nuclear translocation of cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC), but its nuclear function in plant stress responses remains elusive. Here we show that GAPC interacts with a transcription factor to promote the expression of heat-inducible genes and heat tolerance in Arabidopsis. GAPC accumulates in the nucleus under heat stress. Overexpression of GAPC enhances heat tolerance of seedlings and the expression of heat-inducible genes whereas knockout of GAPCs has opposite effects. Screening of Arabidopsis transcription factors identifies nuclear factor Y subunit C10 (NF-YC10) as a GAPC-binding protein. The effects of GAPC overexpression are abolished when NF-YC10 is deficient, the heat-induced nuclear accumulation of GAPC is suppressed, or the GAPC-NF-YC10 interaction is disrupted. GAPC overexpression also enhances the binding ability of NF-YC10 to its target promoter. The results reveal a cellular and molecular mechanism for the nuclear moonlighting of a glycolytic enzyme in plant response to environmental changes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fator de Ligação a CCAAT/genética , Fator de Ligação a CCAAT/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/fisiologia , Citosol/metabolismo , Citosol/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Gliceraldeído-3-Fosfato Desidrogenases/genética , Resposta ao Choque Térmico/genética , Resposta ao Choque Térmico/fisiologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Plântula/genética , Plântula/metabolismo , Plântula/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
16.
Nat Commun ; 11(1): 3505, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665593

RESUMO

The early steps of HIV-1 infection, such as uncoating, reverse transcription, nuclear import, and transport to integration sites are incompletely understood. Here, we imaged nuclear entry and transport of HIV-1 replication complexes in cell lines, primary monocyte-derived macrophages (MDMs) and CD4+ T cells. We show that viral replication complexes traffic to and accumulate within nuclear speckles and that these steps precede the completion of viral DNA synthesis. HIV-1 transport to nuclear speckles is dependent on the interaction of the capsid proteins with host cleavage and polyadenylation specificity factor 6 (CPSF6), which is also required to stabilize the association of the viral replication complexes with nuclear speckles. Importantly, integration site analyses reveal a strong preference for HIV-1 to integrate into speckle-associated genomic domains. Collectively, our results demonstrate that nuclear speckles provide an architectural basis for nuclear homing of HIV-1 replication complexes and subsequent integration into associated genomic loci.


Assuntos
Infecções por HIV/virologia , HIV-1/patogenicidade , Linfócitos T CD4-Positivos/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Genoma Viral/genética , Células HEK293 , Infecções por HIV/genética , HIV-1/genética , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Microscopia de Fluorescência , Virologia , Integração Viral/genética , Integração Viral/fisiologia , Replicação Viral/genética , Replicação Viral/fisiologia
17.
Mutat Res ; 854-855: 503202, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32660826

RESUMO

Cancer is a genomic disease associated with accumulation of genetic damage. Cancer-initiating events, such as chromosome breakage, loss and rearrangement, can be used as biomarkers to evaluate individual cancer risk. Cytokinesis-block micronucleus cytome (CBMN - Cyt) assay parameters in peripheral blood lymphocytes (PBL) of thirty four patients diagnosed with high-grade squamous intraepithelial lesions (HSIL) and fifteen healthy women were measured. The genomic instability of patients diagnosed with HSIL were investigated in order to compare differences between the two subgroups of HSIL (CIN 2 and CIN 3). The micronucleus (MN) frequencies in PBL, as well as the frequencies of nucleoplasmic bridges (NPB) and nuclear buds (NBUD) were higher in patients than in controls (Mann- Whitney test, p < 0.05). These results provide evidence that CBMN cytome assay in peripheral blood lymphocytes may be used to identify individuals who are at high risk of developing cervical cancer. Since the extent of DNA damage varies between CIN 2 and CIN 3, these findings support the CIN grading system.


Assuntos
Instabilidade Genômica/genética , Linfócitos/patologia , Lesões Intraepiteliais Escamosas/genética , Lesões Intraepiteliais Escamosas/patologia , Adulto , Idoso , Núcleo Celular/genética , Dano ao DNA/genética , Feminino , Humanos , Testes para Micronúcleos/métodos , Pessoa de Meia-Idade , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia
18.
Proc Natl Acad Sci U S A ; 117(32): 19254-19265, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719146

RESUMO

The appropriate arrangement of myonuclei within skeletal muscle myofibers is of critical importance for normal muscle function, and improper myonuclear localization has been linked to a variety of skeletal muscle diseases, such as centronuclear myopathy and muscular dystrophies. However, the molecules that govern myonuclear positioning remain elusive. Here, we report that skeletal muscle-specific CIP (sk-CIP) is a regulator of nuclear positioning. Genetic deletion of sk-CIP in mice results in misalignment of myonuclei along the myofibers and at specialized structures such as neuromuscular junctions (NMJs) and myotendinous junctions (MTJs) in vivo, impairing myonuclear positioning after muscle regeneration, leading to severe muscle dystrophy in mdx mice, a mouse model of Duchenne muscular dystrophy. sk-CIP is localized to the centrosome in myoblasts and relocates to the outer nuclear envelope in myotubes upon differentiation. Mechanistically, we found that sk-CIP interacts with the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex and the centriole Microtubule Organizing Center (MTOC) proteins to coordinately modulate myonuclear positioning and alignment. These findings indicate that sk-CIP may function as a muscle-specific anchoring protein to regulate nuclear position in multinucleated muscle cells.


Assuntos
Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Miopatias Congênitas Estruturais/fisiopatologia , Proteínas Nucleares/metabolismo , Animais , Proteínas de Transporte/genética , Núcleo Celular/genética , Humanos , Camundongos , Camundongos Endogâmicos mdx , Camundongos Knockout , Músculo Esquelético/fisiopatologia , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/metabolismo , Proteínas Nucleares/genética , Especificidade de Órgãos
19.
PLoS One ; 15(6): e0234011, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32484825

RESUMO

The tomato AGC protein kinase Adi3 is a Ser/Thr kinase that functions as a negative regulator of programmed cell death through cell death suppression (CDS) activity in the nucleus. In this study, to understand the mechanism of Adi3 CDS, peptide microarrays containing random Ser- and Thr-peptide phosphorylation substrates were used to screen for downstream phosphorylation substrates. In the microarray phosphorylation assay, Adi3 showed promiscuous kinase activity more toward Ser-peptides compared to Thr-peptides, and a preference for aromatic and cyclic amino acids on both Ser- and Thr-peptides was seen. The 63 highest phosphorylated peptide sequences from the Ser-peptide microarray were selected as queries for a BLAST search against the tomato proteome. As a result, 294 candidate nuclear Adi3 substrates were selected and categorized based on their functions. Many of these proteins were classified as DNA/RNA polymerases or regulators involved in transcription and translation events. The list of potential Adi3 substrates was narrowed to eleven and four candidates were tested for phosphorylation by Adi3. Two of these candidates, RNA polymerase II 2nd largest subunit (RPB2) and the pathogen defense related transcription factor Pti5, were confirmed as Adi3 phosphorylation substrates by in vitro kinase assays. Using a mutational approach two residues, Thr675 and Thr676, were identified as Adi3 phosphorylation sites on RPB2. This study provides the foundation for understanding Adi3 CDS mechanisms in the nucleus as well as other cellular functions.


Assuntos
Morte Celular/genética , Peptídeos/genética , Células Vegetais/metabolismo , Proteínas de Plantas/genética , Sequência de Aminoácidos , Núcleo Celular/genética , Lycopersicon esculentum/genética , Lycopersicon esculentum/metabolismo , Análise em Microsséries , Mutação/genética , Fosforilação/genética , Proteínas Quinases/genética
20.
Nat Commun ; 11(1): 3122, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561742

RESUMO

During nuclear surveillance in yeast, the RNA exosome functions together with the TRAMP complexes. These include the DEAH-box RNA helicase Mtr4 together with an RNA-binding protein (Air1 or Air2) and a poly(A) polymerase (Trf4 or Trf5). To better determine how RNA substrates are targeted, we analyzed protein and RNA interactions for TRAMP components. Mass spectrometry identified three distinct TRAMP complexes formed in vivo. These complexes preferentially assemble on different classes of transcripts. Unexpectedly, on many substrates, including pre-rRNAs and pre-mRNAs, binding specificity is apparently conferred by Trf4 and Trf5. Clustering of mRNAs by TRAMP association shows co-enrichment for mRNAs with functionally related products, supporting the significance of surveillance in regulating gene expression. We compared binding sites of TRAMP components with multiple nuclear RNA binding proteins, revealing preferential colocalization of subsets of factors. TRF5 deletion reduces Mtr4 recruitment and increases RNA abundance for mRNAs specifically showing high Trf5 binding.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , RNA Helicases DEAD-box/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Espectrometria de Massas , Mutação , Mapeamento de Interação de Proteínas , Precursores de RNA/metabolismo , Estabilidade de RNA , RNA-Seq , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato/genética
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