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1.
Cell ; 181(4): 818-831.e19, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32359423

RESUMO

Cells sense elevated temperatures and mount an adaptive heat shock response that involves changes in gene expression, but the underlying mechanisms, particularly on the level of translation, remain unknown. Here we report that, in budding yeast, the essential translation initiation factor Ded1p undergoes heat-induced phase separation into gel-like condensates. Using ribosome profiling and an in vitro translation assay, we reveal that condensate formation inactivates Ded1p and represses translation of housekeeping mRNAs while promoting translation of stress mRNAs. Testing a variant of Ded1p with altered phase behavior as well as Ded1p homologs from diverse species, we demonstrate that Ded1p condensation is adaptive and fine-tuned to the maximum growth temperature of the respective organism. We conclude that Ded1p condensation is an integral part of an extended heat shock response that selectively represses translation of housekeeping mRNAs to promote survival under conditions of severe heat stress.


Assuntos
RNA Helicases DEAD-box/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Biossíntese de Proteínas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , RNA Helicases DEAD-box/fisiologia , Expressão Gênica/genética , Genes Essenciais/genética , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia
2.
Cell ; 183(3): 771-785.e12, 2020 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-33125892

RESUMO

Trained innate immunity, induced via modulation of mature myeloid cells or their bone marrow progenitors, mediates sustained increased responsiveness to secondary challenges. Here, we investigated whether anti-tumor immunity can be enhanced through induction of trained immunity. Pre-treatment of mice with ß-glucan, a fungal-derived prototypical agonist of trained immunity, resulted in diminished tumor growth. The anti-tumor effect of ß-glucan-induced trained immunity was associated with transcriptomic and epigenetic rewiring of granulopoiesis and neutrophil reprogramming toward an anti-tumor phenotype; this process required type I interferon signaling irrespective of adaptive immunity in the host. Adoptive transfer of neutrophils from ß-glucan-trained mice to naive recipients suppressed tumor growth in the latter in a ROS-dependent manner. Moreover, the anti-tumor effect of ß-glucan-induced trained granulopoiesis was transmissible by bone marrow transplantation to recipient naive mice. Our findings identify a novel and therapeutically relevant anti-tumor facet of trained immunity involving appropriate rewiring of granulopoiesis.


Assuntos
Granulócitos/imunologia , Imunidade Inata , Neoplasias/imunologia , Imunidade Adaptativa , Transferência Adotiva , Animais , Epigênese Genética , Interferon Tipo I/metabolismo , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Neoplasias/patologia , Neutrófilos/metabolismo , Fenótipo , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/metabolismo , Transcrição Gênica , Transcriptoma/genética , beta-Glucanas/metabolismo
3.
EMBO J ; 42(24): e114072, 2023 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-37984437

RESUMO

Piwi-interacting RNAs (piRNAs) direct PIWI proteins to transposons to silence them, thereby preserving genome integrity and fertility. The piRNA population can be expanded in the ping-pong amplification loop. Within this process, piRNA-associated PIWI proteins (piRISC) enter a membraneless organelle called nuage to cleave their target RNA, which is stimulated by Gtsf proteins. The resulting cleavage product gets loaded into an empty PIWI protein to form a new piRISC complex. However, for piRNA amplification to occur, the new RNA substrates, Gtsf-piRISC, and empty PIWI proteins have to be in physical proximity. In this study, we show that in silkworm cells, the Gtsf1 homolog BmGtsf1L binds to piRNA-loaded BmAgo3 and localizes to granules positive for BmAgo3 and BmVreteno. Biochemical assays further revealed that conserved residues within the unstructured tail of BmGtsf1L directly interact with BmVreteno. Using a combination of AlphaFold modeling, atomistic molecular dynamics simulations, and in vitro assays, we identified a novel binding interface on the BmVreteno-eTudor domain, which is required for BmGtsf1L binding. Our study reveals that a single eTudor domain within BmVreteno provides two binding interfaces and thereby interconnects piRNA-loaded BmAgo3 and BmGtsf1L.


Assuntos
Bombyx , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Bombyx/genética , Bombyx/metabolismo , RNA de Interação com Piwi , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Domínio Tudor
4.
Genes Dev ; 33(13-14): 857-870, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31147388

RESUMO

Piwi proteins are important for germ cell development in most animals. These proteins are guided to specific targets by small guide RNAs, referred to as piRNAs or 21U RNAs in Caenorhabditis elegans In this organism, even though genetic screens have uncovered 21U RNA biogenesis factors, little is known about how these factors interact or what they do. Based on the previously identified 21U biogenesis factor PID-1 (piRNA-induced silencing-defective 1), we here define a novel protein complex, PETISCO (PID-3, ERH-2, TOFU-6, and IFE-3 small RNA complex), that is required for 21U RNA biogenesis. PETISCO contains both potential 5' cap and 5' phosphate RNA-binding domains and interacts with capped 21U precursor RNA. We resolved the architecture of PETISCO and revealed a second function for PETISCO in embryonic development. This essential function of PETISCO is mediated not by PID-1 but by the novel protein TOST-1 (twenty-one U pathway antagonist). In contrast, TOST-1 is not essential for 21U RNA biogenesis. Both PID-1 and TOST-1 interact directly with ERH-2 using a conserved sequence motif. Finally, our data suggest a role for TOST-1:PETISCO in SL1 homeostasis in the early embryo. Our work describes a key complex for 21U RNA processing in C. elegans and strengthens the view that 21U RNA biogenesis is built on an snRNA-related pathway.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Embrião não Mamífero/fisiologia , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , RNA Nucleolar Pequeno/biossíntese , Animais , RNA Nuclear Pequeno/metabolismo
5.
EMBO J ; 40(3): e105280, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33231880

RESUMO

In Caenorhabditis elegans, the piRNA (21U RNA) pathway is required to establish proper gene regulation and an immortal germline. To achieve this, PRG-1-bound 21U RNAs trigger silencing mechanisms mediated by RNA-dependent RNA polymerase (RdRP)-synthetized 22G RNAs. This silencing can become PRG-1-independent and heritable over many generations, a state termed RNA-induced epigenetic gene silencing (RNAe). How and when RNAe is established, and how it is maintained, is not known. We show that maternally provided 21U RNAs can be sufficient for triggering RNAe in embryos. Additionally, we identify PID-2, a protein containing intrinsically disordered regions (IDRs), as a factor required for establishing and maintaining RNAe. PID-2 interacts with two newly identified and partially redundant eTudor domain-containing proteins, PID-4 and PID-5. PID-5 has an additional domain related to the X-prolyl aminopeptidase APP-1, and binds APP-1, implicating potential N-terminal proteolysis in RNAe. All three proteins are required for germline immortality, localize to perinuclear foci, affect size and appearance of RNA inheritance-linked Z granules, and are required for balancing of 22G RNA populations. Overall, our study identifies three new proteins with crucial functions in C. elegans small RNA silencing.


Assuntos
Caenorhabditis elegans/embriologia , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , RNA Interferente Pequeno/genética , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Proteínas Intrinsicamente Desordenadas/química , Ligação Proteica , Domínios Proteicos
6.
Development ; 148(2)2021 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-33298460

RESUMO

Primordial germ cells (PGCs) are the precursors of germ cells, which migrate to the genital ridge during early development. Relatively little is known about PGCs after their migration. We studied this post-migratory stage using microscopy and sequencing techniques, and found that many PGC-specific genes, including genes known to induce PGC fate in the mouse, are only activated several days after migration. At this same time point, PGC nuclei become extremely gyrated, displaying general broad opening of chromatin and high levels of intergenic transcription. This is accompanied by changes in nuage morphology, expression of large loci (PGC-expressed non-coding RNA loci, PERLs) that are enriched for retro-transposons and piRNAs, and a rise in piRNA biogenesis signatures. Interestingly, no nuclear Piwi protein could be detected at any time point, indicating that the zebrafish piRNA pathway is fully cytoplasmic. Our data show that the post-migratory stage of zebrafish PGCs holds many cues to both germ cell fate establishment and piRNA pathway activation.


Assuntos
Núcleo Celular/genética , Células Germinativas/metabolismo , Transcrição Gênica , Peixe-Zebra/genética , Animais , Núcleo Celular/ultraestrutura , Elementos de DNA Transponíveis/genética , DNA Intergênico/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Fertilização , Regulação da Expressão Gênica no Desenvolvimento , Loci Gênicos , Células Germinativas/ultraestrutura , Mutação/genética , RNA Interferente Pequeno/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Regulação para Cima/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Zigoto/metabolismo
7.
Genes Dev ; 30(5): 553-66, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26944680

RESUMO

Nuclear export factor 1 (NXF1) exports mRNA to the cytoplasm after recruitment to mRNA by specific adaptor proteins. How and why cells use numerous different export adaptors is poorly understood. Here we critically evaluate members of the SR protein family (SRSF1-7) for their potential to act as NXF1 adaptors that couple pre-mRNA processing to mRNA export. Consistent with this proposal, >1000 endogenous mRNAs required individual SR proteins for nuclear export in vivo. To address the mechanism, transcriptome-wide RNA-binding profiles of NXF1 and SRSF1-7 were determined in parallel by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP). Quantitative comparisons of RNA-binding sites showed that NXF1 and SR proteins bind mRNA targets at adjacent sites, indicative of cobinding. SRSF3 emerged as the most potent NXF1 adaptor, conferring sequence specificity to RNA binding by NXF1 in last exons. Interestingly, SRSF3 and SRSF7 were shown to bind different sites in last exons and regulate 3' untranslated region length in an opposing manner. Both SRSF3 and SRSF7 promoted NXF1 recruitment to mRNA. Thus, SRSF3 and SRSF7 couple alternative splicing and polyadenylation to NXF1-mediated mRNA export, thereby controlling the cytoplasmic abundance of transcripts with alternative 3' ends.


Assuntos
Processamento Alternativo/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas , Transporte Ativo do Núcleo Celular/genética , Animais , Linhagem Celular , Camundongos , Proteínas Nucleares/metabolismo , Ligação Proteica , Reprodutibilidade dos Testes , Ribonucleoproteínas/metabolismo , Fatores de Processamento de Serina-Arginina
8.
Hepatology ; 75(4): 881-897, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34519101

RESUMO

BACKGROUND AND AIMS: NAFLD is initiated by steatosis and can progress through fibrosis and cirrhosis to HCC. The RNA binding protein human antigen R (HuR) controls RNAs at the posttranscriptional level; hepatocyte HuR has been implicated in the regulation of diet-induced hepatic steatosis. The present study aimed to understand the role of hepatocyte HuR in NAFLD development and progression to fibrosis and HCC. APPROACH AND RESULTS: Hepatocyte-specific, HuR-deficient mice and control HuR-sufficient mice were fed either a normal diet or an NAFLD-inducing diet. Hepatic lipid accumulation, inflammation, fibrosis, and HCC development were studied by histology, flow cytometry, quantitative PCR, and RNA sequencing. The liver lipidome was characterized by lipidomics analysis, and the HuR-RNA interactions in the liver were mapped by RNA immunoprecipitation sequencing. Hepatocyte-specific, HuR-deficient mice displayed spontaneous hepatic steatosis and fibrosis predisposition compared to control HuR-sufficient mice. On an NAFLD-inducing diet, hepatocyte-specific HuR deficiency resulted in exacerbated inflammation, fibrosis, and HCC-like tumor development. A multi-omic approach, including lipidomics, transcriptomics, and RNA immunoprecipitation sequencing revealed that HuR orchestrates a protective network of hepatic-metabolic and lipid homeostasis-maintaining pathways. Consistently, HuR-deficient livers accumulated, already at steady state, a triglyceride signature resembling that of NAFLD livers. Moreover, up-regulation of secreted phosphoprotein 1 expression mediated, at least partially, fibrosis development in hepatocyte-specific HuR deficiency on an NAFLD-inducing diet, as shown by experiments using antibody blockade of osteopontin. CONCLUSIONS: HuR is a gatekeeper of liver homeostasis, preventing NAFLD-related fibrosis and HCC, suggesting that the HuR-dependent network could be exploited therapeutically.


Assuntos
Carcinoma Hepatocelular , Proteína Semelhante a ELAV 1 , Neoplasias Hepáticas , Hepatopatia Gordurosa não Alcoólica , Animais , Carcinoma Hepatocelular/patologia , Proteína Semelhante a ELAV 1/metabolismo , Homeostase , Inflamação/metabolismo , Fígado/patologia , Cirrose Hepática/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/patologia , RNA , Triglicerídeos/metabolismo
9.
PLoS Genet ; 15(2): e1007784, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30759082

RESUMO

Endogenous small RNAs (sRNAs) and Argonaute proteins are ubiquitous regulators of gene expression in germline and somatic tissues. sRNA-Argonaute complexes are often expressed in gametes and are consequently inherited by the next generation upon fertilization. In Caenorhabditis elegans, 26G-RNAs are primary endogenous sRNAs that trigger the expression of downstream secondary sRNAs. Two subpopulations of 26G-RNAs exist, each of which displaying strongly compartmentalized expression: one is expressed in the spermatogenic gonad and associates with the Argonautes ALG-3/4; plus another expressed in oocytes and in embryos, which associates with the Argonaute ERGO-1. The determinants and dynamics of gene silencing elicited by 26G-RNAs are largely unknown. Here, we provide diverse new insights into these endogenous sRNA pathways of C. elegans. Using genetics and deep sequencing, we dissect a maternal effect of the ERGO-1 branch of the 26G-RNA pathway. We find that maternal primary sRNAs can trigger the production of zygotic secondary sRNAs that are able to silence targets, even in the absence of zygotic primary triggers. Thus, the interaction of maternal and zygotic sRNA populations, assures target gene silencing throughout animal development. Furthermore, we explore other facets of 26G-RNA biology related to the ALG-3/4 branch. We find that sRNA abundance, sRNA pattern of origin and the 3' UTR length of target transcripts are predictors of the regulatory outcome by the Argonautes ALG-3/4. Lastly, we provide evidence suggesting that ALG-3 and ALG-4 regulate their own mRNAs in a negative feedback loop. Altogether, we provide several new regulatory insights on the dynamics, target regulation and self-regulation of the endogenous RNAi pathways of C. elegans.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Reguladores/genética , Interferência de RNA/fisiologia , Zigoto/fisiologia , Regiões 3' não Traduzidas/genética , Animais , Proteínas Argonautas/genética , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Inativação Gênica/fisiologia , Células Germinativas/fisiologia , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética
10.
Int J Mol Sci ; 23(3)2022 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-35163219

RESUMO

Prostate cancer (PCa) is the most frequent malignancy in older men with a high propensity for bone metastases. Characteristically, PCa causes osteosclerotic lesions as a result of disrupted bone remodeling. Extracellular vesicles (EVs) participate in PCa progression by conditioning the pre-metastatic niche. However, how EVs mediate the cross-talk between PCa cells and osteoprogenitors in the bone microenvironment remains poorly understood. We found that EVs derived from murine PCa cell line RM1-BM increased metabolic activity, vitality, and cell proliferation of osteoblast precursors by >60%, while significantly impairing mineral deposition (-37%). The latter was further confirmed in two complementary in vivo models of ossification. Accordingly, gene and protein set enrichments of osteoprogenitors exposed to EVs displayed significant downregulation of osteogenic markers and upregulation of proinflammatory factors. Additionally, transcriptomic profiling of PCa-EVs revealed the abundance of three microRNAs, miR-26a-5p, miR-27a-3p, and miR-30e-5p involved in the suppression of BMP-2-induced osteogenesis in vivo, suggesting the critical role of these EV-derived miRNAs in PCa-mediated suppression of osteoblast activity. Taken together, our results indicate the importance of EV cargo in cancer-bone cross-talk in vitro and in vivo and suggest that exosomal miRNAs may contribute to the onset of osteosclerotic bone lesions in PCa.


Assuntos
Complexo Multienzimático de Ribonucleases do Exossomo/genética , Osteoblastos/fisiologia , Neoplasias da Próstata/genética , Animais , Osso e Ossos/metabolismo , Osso e Ossos/fisiologia , Comunicação Celular , Linhagem Celular Tumoral , Proliferação de Células , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Exossomos/genética , Vesículas Extracelulares/metabolismo , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Masculino , Células-Tronco Mesenquimais , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Osteogênese , Transcriptoma/genética , Microambiente Tumoral
11.
EMBO J ; 34(7): 896-910, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25527292

RESUMO

Major efforts are invested to characterize the factors controlling the proliferation of neural stem cells. During mammalian corticogenesis, our group has identified a small pool of genes that are transiently downregulated in the switch of neural stem cells to neurogenic division and reinduced in newborn neurons. Among these switch genes, we found Tox, a transcription factor with hitherto uncharacterized roles in the nervous system. Here, we investigated the role of Tox in corticogenesis by characterizing its expression at the tissue, cellular and temporal level. We found that Tox is regulated by calcineurin/Nfat signalling. Moreover, we combined DNA adenine methyltransferase identification (DamID) with deep sequencing to characterize the chromatin binding properties of Tox including its motif and downstream transcriptional targets including Sox2, Tbr2, Prox1 and other key factors. Finally, we manipulated Tox in the developing brain and validated its multiple roles in promoting neural stem cell proliferation and neurite outgrowth of newborn neurons. Our data provide a valuable resource to study the role of Tox in other tissues and highlight a novel key player in brain development.


Assuntos
Calcineurina/metabolismo , Córtex Cerebral/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição NFATC/metabolismo , Transdução de Sinais/fisiologia , Animais , Calcineurina/genética , Proliferação de Células/fisiologia , Córtex Cerebral/citologia , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Camundongos , Fatores de Transcrição NFATC/genética , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Fatores de Transcrição SOXB1/biossíntese , Fatores de Transcrição SOXB1/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Proteínas com Domínio T/biossíntese , Proteínas com Domínio T/genética , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/genética
12.
RNA Biol ; 14(1): 29-35, 2017 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-27898262

RESUMO

The RNA-binding protein FUS is implicated in transcription, alternative splicing of neuronal genes and DNA repair. Mutations in FUS have been linked to human neurodegenerative diseases such as ALS (amyotrophic lateral sclerosis). We genetically disrupted fus in zebrafish (Danio rerio) using the CRISPR-Cas9 system. The fus knockout animals are fertile and did not show any distinctive phenotype. Mutation of fus induces mild changes in gene expression on the transcriptome and proteome level in the adult brain. We observed a significant influence of genetic background on gene expression and 3'UTR usage, which could mask the effects of loss of Fus. Unlike published fus morphants, maternal zygotic fus mutants do not show motoneuronal degeneration and exhibit normal locomotor activity.


Assuntos
Proteína FUS de Ligação a RNA/genética , Peixe-Zebra/genética , Regiões 3' não Traduzidas , Alelos , Animais , Sequência de Bases , Sítios de Ligação , Encéfalo/metabolismo , Sistemas CRISPR-Cas , Éxons , Técnicas de Inativação de Genes , Marcação de Genes , Patrimônio Genético , Genótipo , Proteoma , RNA Guia de Cinetoplastídeos , Proteína FUS de Ligação a RNA/metabolismo , Transcriptoma , Peixe-Zebra/metabolismo
13.
Nat Cell Biol ; 24(2): 217-229, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35132225

RESUMO

Epigenetic inheritance describes the transmission of gene regulatory information across generations without altering DNA sequences, enabling offspring to adapt to environmental conditions. Small RNAs have been implicated in this, through both the oocyte and the sperm. However, as much of the cellular content is extruded during spermatogenesis, it is unclear whether cytoplasmic small RNAs can contribute to epigenetic inheritance through sperm. Here we identify a sperm-specific germ granule, termed the paternal epigenetic inheritance (PEI) granule, that mediates paternal epigenetic inheritance by retaining the cytoplasmic Argonaute protein WAGO-3 during spermatogenesis in Caenorhabditis elegans. We identify the PEI granule proteins PEI-1 and PEI-2, which have distinct functions in this process: granule formation, Argonaute selectivity and subcellular localization. We show that PEI granule segregation is coupled to the transport of sperm-specific secretory vesicles through PEI-2 in an S-palmitoylation-dependent manner. PEI-like proteins are found in humans, suggesting that the identified mechanism may be conserved.


Assuntos
Proteínas Argonautas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Grânulos Citoplasmáticos/genética , Epigênese Genética , Herança Paterna , Espermatozoides/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas Argonautas/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Grânulos Citoplasmáticos/metabolismo , Humanos , Lipoilação , Masculino , Processamento de Proteína Pós-Traducional , Espermatozoides/metabolismo
14.
J Neurochem ; 117(3): 449-60, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21320125

RESUMO

Functional neurotransmitter receptors are expressed in central white matter, where they mediate ischemic damage to glia and may be involved in cell-cell signalling. In this study, we analysed NMDA receptor NR1, NR2B-C and NR3A-B subunit expression in the brain and optic nerve by molecular cloning. In addition to the canonical forms of NR1 and NR2, four previously unknown NR3B variants, generated by alternative splicing, were identified. The variants encoded for isoforms with deletions of 8/15 amino acids in the N-terminal domain or 200/375 amino acids removing one or three transmembrane domains and part of the C-terminal domain, as compared with the previously characterized NR3B isoform. Co-expression of NR3B isoforms with NR1/NR2A-C modulated the amplitude and Mg(2+)-sensitivity of glutamate responses in a NR2 subunit-dependent fashion, with significant variations in the effects produced by different isoforms. These effects were not the result of reduced surface expression of the receptor complex since all NR3B isoforms reduced surface expression by a similar degree. These data reveal previously uncharacterized regulation of NMDA receptor function by alternative splicing of the NR3B subunit.


Assuntos
Encéfalo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Nervo Óptico/crescimento & desenvolvimento , Nervo Óptico/metabolismo , Isoformas de Proteínas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Processamento Alternativo/fisiologia , Análise de Variância , Animais , Animais Recém-Nascidos , Proteínas de Bactérias/genética , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Cálcio/metabolismo , Clonagem Molecular , Feminino , Citometria de Fluxo/métodos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Ácido Glutâmico/farmacologia , Humanos , Líquido Intracelular/metabolismo , Proteínas Luminescentes/genética , Magnésio/farmacologia , Masculino , Isoformas de Proteínas/genética , RNA Mensageiro/metabolismo , Ratos , Receptores de N-Metil-D-Aspartato/genética , Alinhamento de Sequência/métodos , Transfecção/métodos
15.
Nat Commun ; 12(1): 5671, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34580290

RESUMO

Primary cilia are microtubule based sensory organelles important for receiving and processing cellular signals. Recent studies have shown that cilia also release extracellular vesicles (EVs). Because EVs have been shown to exert various physiological functions, these findings have the potential to alter our understanding of how primary cilia regulate specific signalling pathways. So far the focus has been on lgEVs budding directly from the ciliary membrane. An association between cilia and MVB-derived smEVs has not yet been described. We show that ciliary mutant mammalian cells demonstrate increased secretion of small EVs (smEVs) and a change in EV composition. Characterisation of smEV cargo identified signalling molecules that are differentially loaded upon ciliary dysfunction. Furthermore, we show that these smEVs are biologically active and modulate the WNT response in recipient cells. These results provide us with insights into smEV-dependent ciliary signalling mechanisms which might underly ciliopathy disease pathogenesis.


Assuntos
Síndrome de Bardet-Biedl/patologia , Proteínas de Transporte/metabolismo , Cílios/patologia , Vesículas Extracelulares/metabolismo , Animais , Síndrome de Bardet-Biedl/urina , Proteínas de Transporte/genética , Cílios/metabolismo , Células Epiteliais , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Rim/citologia , Rim/patologia , Camundongos , Cultura Primária de Células , Via de Sinalização Wnt
16.
MethodsX ; 6: 265-272, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30788220

RESUMO

RNA interference was first described in the nematode Caenorhabditis elegans. Ever since, several new endogenous small RNA pathways have been described and characterized to different degrees. The very prominent secondary small interfering RNAs, also called 22G-RNAs, bear a 5' triphosphate group after loading into an Argonaute protein. This creates a technical issue, since 5'PPP groups decrease cloning efficiency for small RNA sequencing. To increase cloning efficiency of these small RNA species, a common practice in the field is the treatment of RNA samples, prior to library preparation, with Tobacco Acid pyrophosphatase (TAP). Recently, TAP production and supply was discontinued, so an alternative must be devised. We turned to RNA 5' pyrophosphohydrolase (RppH), a commercially available pyrophosphatase isolated from E. coli. Here we directly compare TAP and RppH in their use for small RNA library preparation. We show that RppH-treated samples faithfully recapitulate TAP-treated samples. Specifically, there is enrichment for 22G-RNAs and mapped small RNA reads show no small RNA transcriptome-wide differences between RppH and TAP treatment. We propose that RppH can be used as a small RNA pyrophosphatase to enrich for triphosphorylated small RNA species and show that RppH- and TAP-derived datasets can be used in direct comparison. •We show that treatment of small RNA samples with RppH prior to sequencing library preparation increases the cloning efficiency of 5' triphosphorylated small RNAs;•RppH treatment is a valid alternative to TAP treatment.

17.
Dev Cell ; 46(3): 285-301.e9, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-30086300

RESUMO

Phase separation represents an important form of subcellular compartmentalization. However, relatively little is known about how the formation or disassembly of such compartments is regulated. In zebrafish, the Balbiani body (Bb) and the germ plasm (Gp) are intimately linked phase-separated structures essential for germ cell specification and home to many germ cell-specific mRNAs and proteins. Throughout development, these structures occur as a single large aggregate (Bb), which disperses throughout oogenesis and upon fertilization accumulates again into relatively large assemblies (Gp). Formation of the Bb requires Bucky ball (Buc), a protein with prion-like properties. We found that the multi-tudor domain-containing protein Tdrd6a interacts with Buc, affecting its mobility and aggregation properties. Importantly, lack of this regulatory interaction leads to significant defects in germ cell development. Our work presents insights into how prion-like protein aggregations can be regulated and highlights the biological relevance of such regulatory events.


Assuntos
Células Germinativas/metabolismo , Oócitos/metabolismo , Oogênese/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Citoplasma/metabolismo , Organelas/metabolismo , RNA Mensageiro/metabolismo , Peixe-Zebra
18.
Ann N Y Acad Sci ; 1074: 458-65, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17105944

RESUMO

Repeated use of drugs of abuse, namely opiates, has been shown to affect glutamate-releasing neurons. Moreover, blockade of N-methyl-D-aspartate (NMDA) receptors (NMDAR) prevents cell death by apoptosis induced by morphine, a heroin metabolite. Thus, in this article we investigated the involvement of different NMDAR subunits in heroin cytotoxicity. Human embryonic kidney (HEK293) cells, which do not express native NMDAR, were transfected with NR1/NR2A or NR1/NR2B subunits. As a control, cells were transfected with NR1 alone, which does not form functional channels. Incubation with heroin for 24 h induced a dose-dependent decrease in cell viability both in NR1-transfected and nontransfected cells. The loss of membrane integrity induced by heroin was more evident in cells transfected with NR1/NR2B than in cells transfected with NR1 alone or NR1/NR2A. This decrease in cell viability was blocked by MK-801, a selective and noncompetitive antagonist of NMDAR. Nevertheless, no significant changes in intracellular adenosine 5'-triphosphate (ATP) were observed in cells treated with heroin. These data implicate NR2B-composed NMDAR as important mediators of heroin neurotoxicity.


Assuntos
Membrana Celular/fisiologia , Heroína/toxicidade , Receptores de N-Metil-D-Aspartato/fisiologia , Linhagem Celular , Sobrevivência Celular , Humanos , Receptores de N-Metil-D-Aspartato/metabolismo , Transfecção
19.
Genom Data ; 7: 264-8, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26981424

RESUMO

In recent years, DNA adenine methyltransferase identification (DamID) has emerged as a powerful tool to profile protein-DNA interaction on a genome-wide scale. While DamID has been primarily combined with microarray analyses, which limits the spatial resolution and full potential of this technique, our group was the first to combine DamID with sequencing (DamID-Seq) for characterizing the binding loci and properties of a transcription factor (Tox) (sequencing data available at NCBI's Gene Expression Omnibus under the accession number GSE64240). Our approach was based on the combination and optimization of several bioinformatics tools that are here described in detail. Analysis of Tox proximity to transcriptional start sites, profiling on enhancers and binding motif has allowed us to identify this transcription factor as an important new regulator of neural stem cells differentiation and newborn neurons maturation during mouse cortical development. Here we provide a valuable resource to study the role of Tox as a novel key determinant of mammalian somatic stem cells during development of the nervous and lymphatic system, in which this factor is known to be active, and describe a useful pipeline to perform DamID-Seq analyses for any other transcription factor.

20.
Genome Biol ; 17(1): 146, 2016 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-27381023

RESUMO

BACKGROUND: Enhancers, not promoters, are the most dynamic in their DNA methylation status throughout development and differentiation. Generally speaking, enhancers that are primed to or actually drive gene expression are characterized by relatively low levels of DNA methylation (hypo-methylation), while inactive enhancers display hyper-methylation of the underlying DNA. The direct functional significance of the DNA methylation state of enhancers is, however, unclear for most loci. RESULTS: In contrast to conventional epigenetic interactions at enhancers, we find that DNA methylation status and enhancer activity during early zebrafish development display very unusual correlation characteristics: hypo-methylation is a unique feature of primed enhancers whereas active enhancers are generally hyper-methylated. The hypo-methylated enhancers that we identify (hypo-enhancers) are enriched close to important transcription factors that act later in development. Interestingly, hypo-enhancers are de-methylated shortly before the midblastula transition and reside in a unique epigenetic environment. Finally, we demonstrate that hypo-enhancers do become active at later developmental stages and that they are physically associated with the transcriptional start site of target genes, irrespective of target gene activity. CONCLUSIONS: We demonstrate that early development in zebrafish embodies a time window characterized by non-canonical DNA methylation-enhancer relationships, including global DNA hypo-methylation of inactive enhancers and DNA hyper-methylation of active enhancers.


Assuntos
Metilação de DNA/genética , Elementos Facilitadores Genéticos , Epigênese Genética , Peixe-Zebra/genética , Animais , Diferenciação Celular/genética , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Sítio de Iniciação de Transcrição , Peixe-Zebra/crescimento & desenvolvimento
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