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1.
Nat Commun ; 11(1): 5797, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-33199684

RESUMO

ARGONAUTE-2 and associated miRNAs form the RNA-induced silencing complex (RISC), which targets mRNAs for translational silencing and degradation as part of the RNA interference pathway. Despite the essential nature of this process for cellular function, there is little information on the role of RISC components in human development and organ function. We identify 13 heterozygous mutations in AGO2 in 21 patients affected by disturbances in neurological development. Each of the identified single amino acid mutations result in impaired shRNA-mediated silencing. We observe either impaired RISC formation or increased binding of AGO2 to mRNA targets as mutation specific functional consequences. The latter is supported by decreased phosphorylation of a C-terminal serine cluster involved in mRNA target release, increased formation of dendritic P-bodies in neurons and global transcriptome alterations in patient-derived primary fibroblasts. Our data emphasize the importance of gene expression regulation through the dynamic AGO2-RNA association for human neuronal development.


Assuntos
Proteínas Argonauta/genética , Células Germinativas/metabolismo , Mutação/genética , Sistema Nervoso/crescimento & desenvolvimento , Sistema Nervoso/metabolismo , Interferência de RNA , Adolescente , Animais , Proteínas Argonauta/química , Criança , Pré-Escolar , Análise por Conglomerados , Dendritos/metabolismo , Fibroblastos/metabolismo , Inativação Gênica , Células HEK293 , Hipocampo/patologia , Humanos , Camundongos , Simulação de Dinâmica Molecular , Neurônios/metabolismo , Fosforilação , Domínios Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo , Ratos , Transcriptoma/genética
2.
Toxicol Lett ; 334: 102-109, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-33002525

RESUMO

Beauvericin is an ubiquitous mycotoxin with relevant occurrence in food and feed. It causes a high toxicity in several cell lines, but its general mechanism of action is not fully understood and only limited in vivo studies have been performed. We used Caenorhabditis elegans as a model organism to investigate effects of beauvericin. The mycotoxin displays a moderate acute toxicity at 100 µM; at this concentration also reproductive toxicity occurred (reduction of total progeny to 32.1 %), developmental toxicity was detectable at 250 µM. However, even lower concentrations were capable to reduce stress resistance and life span of the nematode: A significant reduction was detected at 10 µM beauvericin (decrease in mean survival time of 4.3 % and reduction in life span of 12.9 %). An increase in lipofuscin fluorescence was demonstrated starting at 10 µM suggesting oxidative stress as a mechanism of beauvericin toxicity. Beauvericin (100 µM) increases the number of apoptotic germ cells comparable to the positive control UV-C (400 J/m2). Conclusion: Low concentrations of beauvericin are capable to cause adverse effects in C. elegans, which may be relevant for hazard identification of this compound.


Assuntos
Apoptose/efeitos dos fármacos , Caenorhabditis elegans/efeitos dos fármacos , Depsipeptídeos/toxicidade , Células Germinativas/efeitos dos fármacos , Lipofuscina/metabolismo , Longevidade/efeitos dos fármacos , Micotoxinas/toxicidade , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Relação Dose-Resposta a Droga , Fertilidade/efeitos dos fármacos , Contaminação de Alimentos , Células Germinativas/patologia , Atividade Motora/efeitos dos fármacos , Testes de Toxicidade Aguda
3.
Nat Commun ; 11(1): 5191, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33060587

RESUMO

In grasses, phased small interfering RNAs (phasiRNAs), 21- or 24-nucleotide (nt) in length, are predominantly expressed in anthers and play a role in regulating male fertility. However, their targets and mode of action on the targets remain unknown. Here we profile phasiRNA expression in premeiotic and meiotic spikelets as well as in purified male meiocytes at early prophase I, tetrads and microspores in rice. We show that 21-nt phasiRNAs are most abundant in meiocytes at early prophase I while 24-nt phasiRNAs are more abundant in tetrads and microspores. By performing highly sensitive degradome sequencing, we find that 21-nt phasiRNAs direct target mRNA cleavage in male germ cells, especially in meiocytes at early prophase I. These targets include 435 protein-coding genes and 71 transposons that show an enrichment for carbohydrate biosynthetic and metabolic pathways. Our study provides strong evidence that 21-nt phasiRNAs act in a target-cleavage mode and may facilitate the progression of meiosis by fine-tuning carbohydrate biosynthesis and metabolism in male germ cells.


Assuntos
Células Germinativas/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Meiose/fisiologia , Nucleotídeos , Oryza/embriologia , Oryza/genética , Proteínas de Plantas/genética , RNA Replicase/metabolismo , RNA de Plantas/genética , RNA Interferente Pequeno/metabolismo , Transcriptoma
4.
Nat Commun ; 11(1): 5397, 2020 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-33106478

RESUMO

The migration of many cell types relies on the formation of actomyosin-dependent protrusions called blebs, but the mechanisms responsible for focusing this kind of protrusive activity to the cell front are largely unknown. Here, we employ zebrafish primordial germ cells (PGCs) as a model to study the role of cell-cell adhesion in bleb-driven single-cell migration in vivo. Utilizing a range of genetic, reverse genetic and mathematical tools, we define a previously unknown role for E-cadherin in confining bleb-type protrusions to the leading edge of the cell. We show that E-cadherin-mediated frictional forces impede the backwards flow of actomyosin-rich structures that define the domain where protrusions are preferentially generated. In this way, E-cadherin confines the bleb-forming region to a restricted area at the cell front and reinforces the front-rear axis of migrating cells. Accordingly, when E-cadherin activity is reduced, the bleb-forming area expands, thus compromising the directional persistence of the cells.


Assuntos
Actinas/metabolismo , Caderinas/metabolismo , Movimento Celular , Células Germinativas/citologia , Pseudópodes/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Actinas/genética , Actomiosina/genética , Actomiosina/metabolismo , Animais , Caderinas/genética , Feminino , Células Germinativas/metabolismo , Masculino , Pseudópodes/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
5.
PLoS One ; 15(9): e0238637, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32903270

RESUMO

Neonicotinoids, a class of insecticides structurally similar to nicotine that target biting and sucking insects, are the most widely used insecticides today, in part due to their supposed low toxicity in other organisms. However, a growing body of research has found that even low doses of neonicotinoids can induce unexpected negative effects on the physiology and survival of a wide range of non-target organisms. Importantly, no work has been done on the commercial formulations of pesticides that include imidacloprid as the active ingredient, but that also contain many other components. The present study examines the sublethal effects of "Tree and Shrub"™ ("T+S"), a commercial insecticide containing the neonicotinoid imidacloprid as its active ingredient, on Caenorhabditis elegans. We discovered that "T+S" significantly stunted the overall growth in wildtype nematodes, an effect that was exacerbated by concurrent exposure to heat stress. "T+S" also negatively impacted fecundity as measured by increased germline apoptosis, a decrease in egg-laying, and fewer viable offspring. Lastly, exposure to "T+S" resulted in degenerative changes in nicotinic cholinergic neurons in wildtype nematodes. As a whole, these findings demonstrate widespread toxic effects of neonicotinoids to critical functions in nematodes.


Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/fisiologia , Inseticidas/toxicidade , Locomoção/efeitos dos fármacos , Neonicotinoides/toxicidade , Animais , Apoptose/efeitos dos fármacos , Caenorhabditis elegans/efeitos dos fármacos , Neurônios Colinérgicos/efeitos dos fármacos , Neurônios Colinérgicos/metabolismo , Aberrações Cromossômicas , Fertilidade/efeitos dos fármacos , Células Germinativas/citologia , Células Germinativas/efeitos dos fármacos , Resposta ao Choque Térmico , Degeneração Neural/patologia , Oviposição/efeitos dos fármacos , Reprodução/efeitos dos fármacos
6.
PLoS One ; 15(9): e0238955, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32997668

RESUMO

Cell shape change is one of the driving forces of animal morphogenesis, and the model organism Caenorhabditis elegans has played a significant role in analyzing the underlying mechanisms involved. The analysis of cell shape change requires quantification of cellular shape descriptors, a method known as cellular morphometry. However, standard C. elegans live imaging methods limit the capability of cellular morphometry in 3D, as spherical aberrations generated by samples and the surrounding medium misalign optical paths. Here, we report a 3D live imaging method for C. elegans embryos that minimized spherical aberrations caused by refractive index (RI) mismatch. We determined the composition of a refractive index matching medium (RIMM) for C. elegans live imaging. The 3D live imaging with the RIMM resulted in a higher signal intensity in the deeper cell layers. We also found that the obtained images improved the 3D cell segmentation quality. Furthermore, our 3D cellular morphometry and 2D cell shape simulation indicated that the germ cell precursor P4 had exceptionally high cortical tension. Our results demonstrate that the RIMM is a cost-effective solution to improve the 3D cellular morphometry of C. elegans. The application of this method should facilitate understanding of C. elegans morphogenesis from the perspective of cell shape changes.


Assuntos
Caenorhabditis elegans/anatomia & histologia , Imageamento Tridimensional/métodos , Refratometria/métodos , Animais , Pesos e Medidas Corporais/métodos , Caenorhabditis elegans/embriologia , Forma Celular/fisiologia , Células Germinativas , Morfogênese
7.
Cell Prolif ; 53(10): e12899, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32896929

RESUMO

OBJECTIVES: Stem cell niche regulated the renewal and differentiation of germline stem cells (GSCs) in Drosophila. Previously, we and others identified a series of genes encoding ribosomal proteins that may contribute to the self-renewal and differentiation of GSCs. However, the mechanisms that maintain and differentiate GSCs in their niches were not well understood. MATERIALS AND METHODS: Flies were used to generate tissue-specific gene knockdown. Small interfering RNAs were used to knockdown genes in S2 cells. qRT-PCR was used to examine the relative mRNA expression level. TUNEL staining or flow cytometry assays were used to detect cell survival. Immunofluorescence was used to determine protein localization and expression pattern. RESULTS: Herein, using a genetic manipulation approach, we investigated the role of ribosomal protein S13 (RpS13) in testes and S2 cells. We reported that RpS13 was required for the self-renewal and differentiation of GSCs. We also demonstrated that RpS13 regulated cell proliferation and apoptosis. Mechanistically, we showed that RpS13 regulated the expression of ribosome subunits and could moderate the expression of the Rho1, DE-cad and Arm proteins. Notably, Rho1 imitated the phenotype of RpS13 in both Drosophila testes and S2 cells, and recruited cell adhesions, which was mediated by the DE-cad and Arm proteins. CONCLUSION: These findings uncover a novel mechanism of RpS13 that mediates Rho1 signals in the stem cell niche of the Drosophila testis.


Assuntos
Proteínas de Drosophila/metabolismo , Proteínas Ribossômicas/metabolismo , Transdução de Sinais , Testículo/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Apoptose , Adesão Celular , Diferenciação Celular , Proliferação de Células , Autorrenovação Celular , Drosophila/metabolismo , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Células Germinativas/citologia , Masculino , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Ribossômicas/antagonistas & inibidores , Proteínas Ribossômicas/genética , Nicho de Células-Tronco , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/genética
8.
Elife ; 92020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32915135

RESUMO

An intricate stem cell niche boundary formed by finger-like extensions generates asymmetry in stem cell divisions.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Células Germinativas , Nicho de Células-Tronco , Células-Tronco
9.
Nat Commun ; 11(1): 4869, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978394

RESUMO

Poly(ADP-ribosyl)ation is a reversible post-translational modification synthetized by ADP-ribose transferases and removed by poly(ADP-ribose) glycohydrolase (PARG), which plays important roles in DNA damage repair. While well-studied in somatic tissues, much less is known about poly(ADP-ribosyl)ation in the germline, where DNA double-strand breaks are introduced by a regulated program and repaired by crossover recombination to establish a tether between homologous chromosomes. The interaction between the parental chromosomes is facilitated by meiotic specific adaptation of the chromosome axes and cohesins, and reinforced by the synaptonemal complex. Here, we uncover an unexpected role for PARG in coordinating the induction of meiotic DNA breaks and their homologous recombination-mediated repair in Caenorhabditis elegans. PARG-1/PARG interacts with both axial and central elements of the synaptonemal complex, REC-8/Rec8 and the MRN/X complex. PARG-1 shapes the recombination landscape and reinforces the tightly regulated control of crossover numbers without requiring its catalytic activity. We unravel roles in regulating meiosis, beyond its enzymatic activity in poly(ADP-ribose) catabolism.


Assuntos
Caenorhabditis elegans/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , DNA/metabolismo , Glicosídeo Hidrolases/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Núcleo Celular/metabolismo , Células Germinativas , Glicosídeo Hidrolases/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poli ADP Ribosilação , Poli Adenosina Difosfato Ribose/metabolismo , Processamento de Proteína Pós-Traducional
11.
PLoS One ; 15(9): e0237792, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32881892

RESUMO

BACKGROUND: Ewing sarcoma (EwS) is a rare, aggressive solid tumor of childhood, adolescence and young adulthood associated with pathognomonic EWSR1-ETS fusion oncoproteins altering transcriptional regulation. Genome-wide association studies (GWAS) have identified 6 common germline susceptibility loci but have not investigated low-frequency inherited variants with minor allele frequencies below 5% due to limited genotyped cases of this rare tumor. METHODS: We investigated the contribution of rare and low-frequency variation to EwS susceptibility in the largest EwS genome-wide association study to date (733 EwS cases and 1,346 unaffected controls of European ancestry). RESULTS: We identified two low-frequency variants, rs112837127 and rs2296730, on chromosome 20 that were associated with EwS risk (OR = 0.186 and 2.038, respectively; P-value < 5×10-8) and located near previously reported common susceptibility loci. After adjusting for the most associated common variant at the locus, only rs112837127 remained a statistically significant independent signal (OR = 0.200, P-value = 5.84×10-8). CONCLUSIONS: These findings suggest rare variation residing on common haplotypes are important contributors to EwS risk. IMPACT: Motivate future targeted sequencing studies for a comprehensive evaluation of low-frequency and rare variation around common EwS susceptibility loci.


Assuntos
Loci Gênicos , Predisposição Genética para Doença , Variação Genética , Células Germinativas/metabolismo , Sarcoma de Ewing/genética , Estudo de Associação Genômica Ampla , Humanos , Desequilíbrio de Ligação/genética , Razão de Chances , Polimorfismo de Nucleotídeo Único/genética
14.
15.
17.
Sci Total Environ ; 745: 140639, 2020 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-32758758

RESUMO

The Deepwater Horizon (DWH) oil spill marked the largest environmental oil spill in human history, where it was estimated a large amount of the polycyclic aromatic hydrocarbons (PAHs) were released with crude oil into the environment. In this study, common PAH compounds were quantitatively determined in crude oil from the DWH spill by gas chromatography-mass spectroscopy (GC-MS). Twelve PAH compounds were identified and quantified from a 100× dilution of DWH crude oil: naphthalene (7800 ng/mL), acenaphthylene (590 ng/mL), acenaphtehen (540 ng/mL), fluorene (2550 ng/mL), phenanthrene (2910 ng/mL), anthracene (840 ng/mL), fluoranthene (490 ng/mL), pyrene (290 ng/mL), benzo(k) fluoranthene (1050 ng/mL), benzo(b)fluoranthene (1360 ng/mL), dibenz(a,h)anthracene (2560 ng/mL), and benzo(g, h, i) perylene (630 ng/mL). Toxicity assays using the nematode, Caenorhabditis elegans (C. elegans), indicated a single PAH compound naphthalene, exposure increased C. elegans germ cell apoptosis which may adversely affect progeny reproduction. The number of apoptotic germ cells significantly increased from 1.4 to 2.5 when worms were treated with 10 µg/mL of naphthalene and from 1.3 to 2.5 and 3.5 cells in presence of 1 µg/mL and 5 µg/mL of benzo(a)pyrene, respectively. Five CYP450 genes (CYP14A3, CYP35A1, CYP35A2, CYP35A5, and CYP35C1) were significantly upregulated following 500× dilution of dispersed crude oil exposure (p < 0.05). These results suggest that CYP450s may play a role in bioactivation of PAHs in crude oil, resulting in DNA damage related germ cell apoptosis.


Assuntos
Petróleo/toxicidade , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Animais , Apoptose , Caenorhabditis elegans , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Células Germinativas , Humanos , Regulação para Cima
19.
Gene ; 761: 145037, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32777526

RESUMO

Primordial germ cells (PGCs) are singled out from somatic cells very early during embryogenesis, then they migrate towards the genital ridge and differentiate into gametes through oogenesis or spermatogenesis. Labeling PGCs with Localized RNAexpression (LRE) technique by fluorescent proteins has been widely applied among teleost species to study the germ cell development and gonad differentiation. In this study, we first cloned and characterized the 3' untranslated regions (3'UTRs) of nanos homolog 1-like (nos1l), dead end (dnd), and vasa in yellow catfish (Pelteobagrus fulvidraco), and then synthesized the GFP-nos1l/dnd/vasa 3'UTR mRNAs. Each of these three 3'UTRs could label PGCs in yellow catfish embryos, of which, vasa 3'UTR exhibited the highest labeling efficiency. To identify the differences in PGCs at embryonic stage, XX all-female and XY all-male yellow catfish embryos were produced and injected with GFP-vasa 3'UTR mRNA. We observed the PGC migration route in these two monosex embryos from 24 hpf to 7 dpf, and found there was no difference between them. Besides, the PGC number was counted at 48 hpf, and the result showed that the average PGC number in XX females (11.3) was significantly larger than that in XY males (8.1).These findings provide an insight into the development of PGCs in yellow catfish embryos and the relationship between embryonicPGCnumberand thelatergonaddifferentiation.


Assuntos
Peixes-Gato/genética , Gametogênese/genética , Células Germinativas/metabolismo , Regiões 3' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Movimento Celular/genética , RNA Helicases DEAD-box/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Proteínas de Peixes/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Gônadas/metabolismo , Masculino , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo
20.
Nucleic Acids Res ; 48(16): 9262-9272, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32766792

RESUMO

LOTUS domains are helix-turn-helix protein folds identified in essential germline proteins and are conserved in prokaryotes and eukaryotes. Despite originally predicted as an RNA binding domain, its molecular binding activity towards RNA and protein is controversial. In particular, the most conserved binding property for the LOTUS domain family remains unknown. Here, we uncovered an unexpected specific interaction of LOTUS domains with G-rich RNA sequences. Intriguingly, LOTUS domains exhibit high affinity to RNA G-quadruplex tertiary structures implicated in diverse cellular processes including piRNA biogenesis. This novel LOTUS domain-RNA interaction is conserved in bacteria, plants and animals, comprising the most ancient binding feature of the LOTUS domain family. By contrast, LOTUS domains do not preferentially interact with DNA G-quadruplexes. We further show that a subset of LOTUS domains display both RNA and protein binding activities. These findings identify the LOTUS domain as a specialized RNA binding domain across phyla and underscore the molecular mechanism underlying the function of LOTUS domain-containing proteins in RNA metabolism and regulation.


Assuntos
Quadruplex G , Conformação Proteica , Proteínas com Motivo de Reconhecimento de RNA/genética , RNA/genética , Sequência de Aminoácidos/genética , Sequência de Bases/genética , Dicroísmo Circular , Células Germinativas , Células HEK293 , Sequências Hélice-Volta-Hélice/genética , Humanos , Estrutura Terciária de Proteína , RNA/metabolismo , RNA/ultraestrutura , Motivos de Ligação ao RNA/genética
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