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1.
Cells ; 13(1)2023 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-38201217

RESUMO

Conventional dendritic cells (cDCs) are innate immune cells that play a pivotal role in inducing antiviral adaptive immune responses due to their extraordinary ability to prime and polarize naïve T cells into different effector T helper (Th) subsets. The two major subpopulations of cDCs, cDC1 (CD8α+ in mice and CD141+ in human) and cDC2 (CD11b+ in mice and CD1c+ in human), can preferentially polarize T cells toward a Th1 and Th2 phenotype, respectively. During infection with ectromelia virus (ECTV), an orthopoxvirus from the Poxviridae family, the timing and activation of an appropriate Th immune response contributes to the resistance (Th1) or susceptibility (Th2) of inbred mouse strains to the lethal form of mousepox. Due to the high plasticity and diverse properties of cDC subpopulations in regulating the quality of a specific immune response, in the present study we compared the ability of splenic cDC1 and cDC2 originating from different ECTV-infected mouse strains to mature, activate, and polarize the Th immune response during mousepox. Our results demonstrated that during early stages of mousepox, both cDC subsets from resistant C57BL/6 and susceptible BALB/c mice were activated upon in vivo ECTV infection. These cells exhibited elevated levels of surface MHC class I and II, and co-stimulatory molecules and showed enhanced potential to produce cytokines. However, both cDC subsets from BALB/c mice displayed a higher maturation status than that of their counterparts from C57BL/6 mice. Despite their higher activation status, cDC1 and cDC2 from susceptible mice produced low amounts of Th1-polarizing cytokines, including IL-12 and IFN-γ, and the ability of these cells to stimulate the proliferation and Th1 polarization of allogeneic CD4+ T cells was severely compromised. In contrast, both cDC subsets from resistant mice produced significant amounts of Th1-polarizing cytokines and demonstrated greater capability in differentiating allogeneic T cells into Th1 cells compared to cDCs from BALB/c mice. Collectively, our results indicate that in the early stages of mousepox, splenic cDC subpopulations from the resistant mouse strain can better elicit a Th1 cell-mediated response than the susceptible strain can, probably contributing to the induction of the protective immune responses necessary for the control of virus dissemination and for survival from ECTV challenge.


Assuntos
Ectromelia Infecciosa , Infecções por Poxviridae , Humanos , Animais , Camundongos , Camundongos Endogâmicos C57BL , Citocinas , Células Dendríticas
2.
Int J Mol Sci ; 23(19)2022 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-36232439

RESUMO

Kae1 is a subunit of the highly evolutionarily conserved KEOPS/EKC complex, which is involved in universal (t6A37) tRNA modification. Several reports have discussed the participation of this complex in transcription regulation in yeast and human cells, including our previous observations of KaeA, an Aspergillus nidulans homologue of Kae1p. The aim of this project was to confirm the role of KaeA in transcription, employing high-throughput transcriptomic (RNA-Seq and ChIP-Seq) and proteomic (LC-MS) analysis. We confirmed that KaeA is a subunit of the KEOPS complex in A. nidulans. An analysis of kaeA19 and kaeA25 mutants showed that, although the (t6A37) tRNA modification is unaffected in both mutants, they reveal significantly altered transcriptomes compared to the wild type. The finding that KaeA is localized in chromatin and identifying its protein partners allows us to postulate an additional nuclear function for the protein. Our data shed light on the universal bi-functional role of this factor and proves that the activity of this protein is not limited to tRNA modification in cytoplasm, but also affects the transcriptional activity of a number of nuclear genes. Data are available via the NCBI's GEO database under identifiers GSE206830 (RNA-Seq) and GSE206874 (ChIP-Seq), and via ProteomeXchange with identifier PXD034554 (proteomic).


Assuntos
Aspergillus nidulans , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Cromatina/metabolismo , Humanos , Proteômica , RNA de Transferência/genética , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/metabolismo
3.
Nucleic Acids Res ; 50(22): e132, 2022 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-36259646

RESUMO

Analysis of the protein coding transcriptome by the RNA sequencing requires either enrichment of the desired fraction of coding transcripts or depletion of the abundant non-coding fraction consisting mainly of rRNA. We propose an alternative mRNA enrichment strategy based on the RNA-binding properties of the human IFIT1, an antiviral protein recognizing cap 0 RNA. Here, we compare for Saccharomyces cerevisiae an IFIT1-based mRNA pull-down with yeast targeted rRNA depletion by the RiboMinus method. IFIT1-based RNA capture depletes rRNA more effectively, producing high quality RNA-seq data with an excellent coverage of the protein coding transcriptome, while depleting cap-less transcripts such as mitochondrial or some non-coding RNAs. We propose IFIT1 as a cost effective and versatile tool to prepare mRNA libraries for a variety of organisms with cap 0 mRNA ends, including diverse plants, fungi and eukaryotic microbes.


Assuntos
Saccharomyces cerevisiae , Transcriptoma , Humanos , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Capuzes de RNA , RNA Ribossômico/genética , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de RNA/métodos
4.
Pathogens ; 9(10)2020 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-33020446

RESUMO

Dendritic cells (DCs) and macrophages are the first line of antiviral immunity. Viral pathogens exploit these cell populations for their efficient replication and dissemination via the modulation of intracellular signaling pathways. Disruption of the noncanonical nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling has frequently been observed in lymphoid cells upon infection with oncogenic viruses. However, several nononcogenic viruses have been shown to manipulate the noncanonical NF-κB signaling in different cell types. This study demonstrates the modulating effect of ectromelia virus (ECTV) on the components of the noncanonical NF-κB signaling pathway in established murine cell lines: JAWS II DCs and RAW 264.7 macrophages. ECTV affected the activation of TRAF2, cIAP1, RelB, and p100 upon cell treatment with both canonical and noncanonical NF-κB stimuli and thus impeded DNA binding by RelB and p52. ECTV also inhibited the expression of numerous genes related to the noncanonical NF-κB pathway and RelB-dependent gene expression in the cells treated with canonical and noncanonical NF-κB activators. Thus, our data strongly suggest that ECTV influenced the noncanonical NF-κB signaling components in the in vitro models. These findings provide new insights into the noncanonical NF-κB signaling components and their manipulation by poxviruses in vitro.

5.
J Cell Commun Signal ; 13(3): 381-394, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30929166

RESUMO

The risk of renal cell carcinoma development is correlated with obesity and type II diabetes. Since insulin and insulin-like growth factors play a key role during development of both metabolic diseases, these molecules may be important in RCC pathophysiology We investigated the effect of insulin and IGFs on RCC cells using in vitro model with 786-O, 769-P, Caki-1, Caki-2, ACHN cancer cell lines. Cancer cells were compared with normal kidney cells - PCS-400-010 and HEK293. The growth, viability of cells as well as migration rate were assessed upon hormonal stimulation. The insulin receptor and Insulin-like growth factor 1 receptor presence were evaluated and the expression of 84 genes related to insulin signaling pathway. In all RCC cell lines IGF-1R expression was confirmed in contrast to IR, which was expressed only in control HEK293 cell line. Insulin and IGFs stimulated RCC cells growth and migration rate. Insulin, IGF-1 and IGF-2 triggered both IR and IGF-1R phosphorylation. Analyzed RCC did not secret insulin, IGF-1 or IGF-2 and were not activated in autocrine-paracrine signaling loop. Insulin and IGFs stimulations triggered down-regulation of PI3K-Akt-mTOR and Ras-MAPK pathway gens, as well as DOK2-3, INS, FRS3, IRS1-2, IGF1R - genes encoding insulin receptor-associated proteins. In conclusion, we showed that IGFs and insulin may play a stimulatory role for renal cancer cells, thus they can possibly affect renal cancer tumorigenesis and progression on cellular level.

6.
FEMS Microbiol Lett ; 366(6)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30939206

RESUMO

In Aspergillus nidulans, nitrogen and carbon metabolism are under the control of wide-domain regulatory systems, including nitrogen metabolite repression, carbon catabolite repression and the nutrient starvation response. Transcriptomic analysis of the wild type strain grown under different combinations of carbon and nitrogen regimes was performed, to identify differentially regulated genes. Carbon metabolism predominates as the most important regulatory signal but for many genes, both carbon and nitrogen metabolisms coordinate regulation. To identify mechanisms coordinating nitrogen and carbon metabolism, we tested the role of AreB, previously identified as a regulator of genes involved in nitrogen metabolism. Deletion of areB has significant phenotypic effects on the utilization of specific carbon sources, confirming its role in the regulation of carbon metabolism. AreB was shown to regulate the expression of areA, tamA, creA, xprG and cpcA regulatory genes suggesting areB has a range of indirect, regulatory effects. Different isoforms of AreB are produced as a result of differential splicing and use of two promoters which are differentially regulated by carbon and nitrogen conditions. These isoforms are likely to be functionally distinct and thus contributing to the modulation of AreB activity.


Assuntos
Aspergillus nidulans/metabolismo , Carbono/metabolismo , Proteínas Fúngicas/metabolismo , Fatores de Transcrição GATA/metabolismo , Regulação Fúngica da Expressão Gênica , Nitrogênio/metabolismo , Aspergillus nidulans/genética , Proteínas Fúngicas/genética , Fatores de Transcrição GATA/genética , Regiões Promotoras Genéticas
7.
Biotechniques ; 65(3): 163-168, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30227741

RESUMO

Comparative transcriptional analyses require appropriate and precise normalization. Here we describe a modified transcription run-on (TRO) method, named quantitative TRO (qTRO), that allows quantification of nascent transcription activity. The most critical improvement it introduces is a new standardization method for RNA isolation and hybridization steps, enabling transcription activity quantification and comparative biological analysis. We used this technique with chromatin immunoprecipitation to investigate RNA polymerase I (RNAPI) transcription activity and its rDNA gene profiles in Saccharomyces cerevisiae. We designed a set of new oligonucleotide probes complementary to nascent ribosomal RNA (rRNA) transcripts and standardized their hybridization strength. The qTRO method could be successfully implemented to study RNAPI transcription in response to oxidative stress and in two mutant strains with impaired rRNA synthesis.


Assuntos
RNA Polimerase I/fisiologia , Saccharomyces cerevisiae/genética , Biotecnologia/métodos , DNA Ribossômico/química , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , RNA Ribossômico/química , Transcrição Gênica
8.
Sci Rep ; 7(1): 8342, 2017 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-28827675

RESUMO

We investigated possibility of predicting whether blooms, if they occur, would be formed of microcystin-producing cyanobacteria. DGGE analysis of 16S-ITS and mcyA genes revealed that only Planktothrix and Microcystis possessed mcy-genes and Planktothrix was the main microcystin producer. qPCR analysis revealed that the proportion of cells with mcy-genes in Planktothrix populations was almost 100%. Microcystin concentration correlated with the number of potentially toxic and total Planktothrix cells and the proportion of Planktothrix within all cyanobacteria, but not with the proportion of cells with mcy-genes in total Planktothrix. The share of Microcystis cells with mcy-genes was low and variable in time. Neither the number of mcy-possessing cells, nor the proportion of these cells in total Microcystis, correlated with the concentration of microcystins. This suggests that it is possible to predict whether the bloom in the Masurian Lakes will be toxic based on Planktothrix occurrence. Two species of toxin producing Planktothrix, P. agardhii and P. rubescens, were identified by phylogenetic analysis of 16S-ITS. Based on morphological and ecological features, the toxic Planktothrix was identified as P. agardhii. However, the very high proportion of cells with mcy-genes suggests P. rubescens. Our study reveals the need of universal primers for mcyA genes from environment.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Cianobactérias/classificação , Proliferação Nociva de Algas , Lagos/microbiologia , Microcystis/classificação , Cianobactérias/genética , Cianobactérias/patogenicidade , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Microcistinas/genética , Microcistinas/metabolismo , Microcystis/genética , Microcystis/patogenicidade , Filogenia , RNA Ribossômico 16S/análise , Análise de Sequência de DNA
9.
Gene ; 573(2): 310-20, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26210809

RESUMO

The kaeA(KAE1) (suDpro) gene, which was identified in Aspergillus nidulans as a suppressor of proline auxotrophic mutations, encodes the orthologue of Saccharomyces cerevisiae Kae1p, a member of the evolutionarily conserved KEOPS/EKC (Kinase, Endopeptidase and Other Proteins of Small size/Endopeptidase-like and Kinase associated to transcribed Chromatin) complex. In yeast, this complex has been shown to be involved in tRNA modification, transcription, and genome maintenance. In A. nidulans, mutations in kaeA result in several phenotypic effects, the derepression of arginine catabolism genes, and changes in the expression levels of several others, including genes involved in amino acid and siderophore metabolism, sulfate transport, carbon/energy metabolism, translation, and transcription regulation, such as rcoA(TUP1), which encodes the global transcriptional corepressor.


Assuntos
Arginina/metabolismo , Aspergillus nidulans/genética , Proteínas Fúngicas/fisiologia , Sequência de Aminoácidos , Aspergillus nidulans/metabolismo , Sequência de Bases , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Pleiotropia Genética , Redes e Vias Metabólicas , Dados de Sequência Molecular , Complexos Multiproteicos/fisiologia , Mutação
10.
Enzymes ; 31: 131-63, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-27166444

RESUMO

The role of the nucleus of a eukaryotic cell during gene expression is not only limited to transcription and RNA processing but also includes the initial stages of RNA surveillance. All of these processes, and more precisely, transcription elongation and termination, 5'-end RNA maturation, and the removal of processing intermediates and aberrant molecules, require the activity of the nuclear 5'-3' exoribonuclease Rat1/Xrn2. This protein, together with its cytoplasmic counterpart, Xrn1, constitutes a highly conserved eukaryotic family of nucleases, whose roles exceed participation in RNA metabolism alone. Despite many years of extensive research and recent findings related to the structure and function of these enzymes revealed almost every year, several aspects are yet to be discovered.

11.
RNA ; 16(12): 2570-80, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20974745

RESUMO

We describe methods for obtaining a quantitative description of RNA processing at high resolution in budding yeast. As a model gene expression system, we constructed tetON (for induction studies) and tetOFF (for repression, derepression, and RNA degradation studies) yeast strains with a series of reporter genes integrated in the genome under the control of a tetO7 promoter. Reverse transcription and quantitative real-time-PCR (RT-qPCR) methods were adapted to allow the determination of mRNA abundance as the average number of copies per cell in a population. Fluorescence in situ hybridization (FISH) measurements of transcript numbers in individual cells validated the RT-qPCR approach for the average copy-number determination despite the broad distribution of transcript levels within a population of cells. In addition, RT-qPCR was used to distinguish the products of the different steps in splicing of the reporter transcripts, and methods were developed to map and quantify 3'-end cleavage and polyadenylation. This system permits pre-mRNA production, splicing, 3'-end maturation and degradation to be quantitatively monitored with unprecedented kinetic detail, suitable for mathematical modeling. Using this approach, we demonstrate that reporter transcripts are spliced prior to their 3'-end cleavage and polyadenylation, that is, cotranscriptionally.


Assuntos
Genes Reporter , Processamento de Terminações 3' de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Saccharomyces cerevisiae , Algoritmos , Estudos de Avaliação como Assunto , Processamento de Imagem Assistida por Computador , Hibridização in Situ Fluorescente/métodos , Cinética , Modelos Biológicos , Modelos Genéticos , Processamento de Terminações 3' de RNA/fisiologia , Precursores de RNA/análise , Precursores de RNA/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
12.
Genes Dev ; 22(8): 1069-81, 2008 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-18413717

RESUMO

During transcription termination by RNA polymerase II on protein-coding genes, the nuclear 5' exonuclease Rat1/Xrn2 degrades the nascent transcript downstream from the polyadenylation site and "torpedoes" the polymerase. We report that the activity of Rat1 is also required for efficient termination by RNA polymerase I (Pol I) on the rDNA. In strains lacking catalytically active Rat1 or its cofactor Rai1, Pol I reads through the major, "Reb1-dependent" terminator (T1) but stops downstream at the "fail-safe" terminator (T2) and replication fork barrier (RFB). The absence of both Rat1 and the RFB-binding protein Fob1 increased Pol I read-through of T2 and the RFB. We propose that cotranscriptional cleavage of the pre-rRNA by the endonuclease Rnt1 generates a loading site for the Rat1/Rai1 complex, which then degrades the nascent transcript. When Rat1 catches Pol I, which is predicted to be paused at T1, transcription is terminated.


Assuntos
Exorribonucleases/metabolismo , RNA Polimerase I/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Regiões Terminadoras Genéticas/genética , Transcrição Gênica , DNA Ribossômico/metabolismo , Modelos Biológicos , Modelos Genéticos , Proteínas Nucleares/metabolismo , RNA Ribossômico 5,8S/metabolismo , Proteínas de Ligação a RNA , Saccharomyces cerevisiae/metabolismo
13.
Biol Chem ; 388(2): 135-44, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17261076

RESUMO

Expression of the arginase structural gene (agaA) in Aspergillus nidulans is subject to complex transcriptional and post-transcriptional regulation. Arginase mRNA has a long 5'-UTR sequence. Analysis of this sequence in silico revealed its putative complex secondary structure, the presence of arginine-binding motifs (arginine aptamers) and a short intron with two potential 3' splicing sites. In this report we present evidence that L-arginine (i) binds directly to the arginase 5'-UTR; (ii) invokes drastic changes in the secondary structure of the 5'-UTR, unlike several other L-amino acids and D-arginine; and (iii) forces the selection of one of two 3' splice sites of an intron present in the 5'-UTR. We postulate that expression of the eukaryotic structural gene coding for arginase in A. nidulans is regulated at the level of mRNA stability, depending on riboswitch-mediated alternative splicing of the 5'-UTR intron.


Assuntos
Arginase , Arginina/farmacologia , Aspergillus nidulans/efeitos dos fármacos , Aspergillus nidulans/enzimologia , RNA Mensageiro , Arginase/química , Arginase/efeitos dos fármacos , Arginase/fisiologia , Sequência de Bases , Sítios de Ligação , Ativação Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Íntrons , Lisina/farmacologia , Dados de Sequência Molecular , RNA Mensageiro/análise , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Soluções/química , Relação Estrutura-Atividade
14.
Fungal Genet Biol ; 38(2): 175-86, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12620254

RESUMO

The arginine catabolism gene otaA encoding ornithine transaminase (OTAse) is specifically induced by arginine and is under the control of the broad-domain carbon and nitrogen repression systems. Arginine induction is mediated by a product of arcA gene coding for Zn(2)C(6) activator. We have identified a region responsible for arginine induction in the otaA promoter (AnUAS(arg)). Deletions within this region result in non-inducibility of OTAse by arginine, whether in an arcA(+) strain or in the presence of the arcA(d)47 gain of function allele. AnUAS(arg) is very similar to the Saccharomyces cerevisiae UAS(arg), a sequence bound by the Zn(2)C(6) activator (ArgRIIp), acting in a complex with two MADS-box proteins (McmIp and ArgRIp). We demonstrate here that two CREA in vitro binding sites in the otaA promoter are functional in vivo. CREA is directly involved in carbon repression of the otaA gene and it also reduces its basal level of expression. Although AREA binds to the otaA promoter in vitro, it probably does not participate in nitrogen metabolite repression of the gene in vivo. We show here that another putative negatively acting GATA factor AREB participates directly or indirectly in otaA nitrogen repression. We also demonstrate that the high levels of OTAse activity are an important factor in the suppression of proline auxotrophic mutations. This suppression can be achieved neither by growing of the proline auxotroph under carbon/nitrogen derepressing conditions nor by introducing of a creA(d) mutation.


Assuntos
Arginina/metabolismo , Aspergillus nidulans/genética , Ornitina-Oxo-Ácido Transaminase/genética , Regiões Promotoras Genéticas , Aspergillus nidulans/metabolismo , Sítios de Ligação , Carbono/metabolismo , Indução Enzimática , Proteínas Fúngicas/metabolismo , Fatores de Transcrição GATA , Regulação da Expressão Gênica , Glucose/metabolismo , Nitrogênio/metabolismo , Ornitina-Oxo-Ácido Transaminase/biossíntese , Prolina , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Ureo-Hidrolases/metabolismo
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