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1.
Mol Syst Biol ; 14(8): e8266, 2018 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-30150282

RESUMO

miRNAs are small RNAs that regulate gene expression post-transcriptionally. By repressing the translation and promoting the degradation of target mRNAs, miRNAs may reduce the cell-to-cell variability in protein expression, induce correlations between target expression levels, and provide a layer through which targets can influence each other's expression as "competing RNAs" (ceRNAs). However, experimental evidence for these behaviors is limited. Combining mathematical modeling with RNA sequencing of individual human embryonic kidney cells in which the expression of two distinct miRNAs was induced over a wide range, we have inferred parameters describing the response of hundreds of miRNA targets to miRNA induction. Individual targets have widely different response dynamics, and only a small proportion of predicted targets exhibit high sensitivity to miRNA induction. Our data reveal for the first time the response parameters of the entire network of endogenous miRNA targets to miRNA induction, demonstrating that miRNAs correlate target expression and at the same time increase the variability in expression of individual targets across cells. The approach is generalizable to other miRNAs and post-transcriptional regulators to improve the understanding of gene expression dynamics in individual cell types.


Assuntos
Redes Reguladoras de Genes/genética , MicroRNAs/genética , RNA Mensageiro/genética , Análise de Célula Única , Biologia Computacional , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Modelos Teóricos , Análise de Sequência de RNA
2.
Nat Commun ; 8(1): 457, 2017 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-28878244

RESUMO

In Saccharomyces cerevisiae, deletion of large ribosomal subunit protein-encoding genes increases the replicative lifespan in a Gcn4-dependent manner. However, how Gcn4, a key transcriptional activator of amino acid biosynthesis genes, increases lifespan, is unknown. Here we show that Gcn4 acts as a repressor of protein synthesis. By analyzing the messenger RNA and protein abundance, ribosome occupancy and protein synthesis rate in various yeast strains, we demonstrate that Gcn4 is sufficient to reduce protein synthesis and increase yeast lifespan. Chromatin immunoprecipitation reveals Gcn4 binding not only at genes that are activated, but also at genes, some encoding ribosomal proteins, that are repressed upon Gcn4 overexpression. The promoters of repressed genes contain Rap1 binding motifs. Our data suggest that Gcn4 is a central regulator of protein synthesis under multiple perturbations, including ribosomal protein gene deletions, calorie restriction, and rapamycin treatment, and provide an explanation for its role in longevity and stress response.The transcription factor Gcn4 is known to regulate yeast amino acid synthesis. Here, the authors show that Gcn4 also acts as a repressor of protein biosynthesis in a range of conditions that enhance yeast lifespan, such as ribosomal protein knockout, calorie restriction or mTOR inhibition.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Fases de Leitura Aberta/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Regulação para Cima/genética
3.
Nucleic Acids Res ; 45(5): 2341-2353, 2017 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-28031372

RESUMO

High-throughput sequencing has greatly facilitated the discovery of long and short non-coding RNAs (ncRNAs), which frequently guide ribonucleoprotein complexes to RNA targets, to modulate their metabolism and expression. However, for many ncRNAs, the targets remain to be discovered. In this study, we developed computational methods to map C/D box snoRNA target sites using data from core small nucleolar ribonucleoprotein crosslinking and immunoprecipitation and from transcriptome-wide mapping of 2΄-O-ribose methylation sites. We thereby assigned the snoRNA guide to a known methylation site in the 18S rRNA, we uncovered a novel partially methylated site in the 28S ribosomal RNA, and we captured a site in the 28S rRNA in interaction with multiple snoRNAs. Although we also captured mRNAs in interaction with snoRNAs, we did not detect 2΄-O-methylation of these targets. Our study provides an integrated approach to the comprehensive characterization of 2΄-O-methylation targets of snoRNAs in species beyond those in which these interactions have been traditionally studied and contributes to the rapidly developing field of 'epitranscriptomics'.


Assuntos
Algoritmos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Guia de Cinetoplastídeos/genética , RNA Nucleolar Pequeno/genética , Ribonucleoproteínas Nucleolares Pequenas/genética , Transcriptoma , Sequência de Bases , Reagentes de Ligações Cruzadas/química , Bases de Dados Genéticas , Imunoprecipitação , Metilação , Ligação Proteica , RNA Guia de Cinetoplastídeos/metabolismo , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , RNA Ribossômico 28S/genética , RNA Ribossômico 28S/metabolismo , RNA Nucleolar Pequeno/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Ribose/metabolismo , Software
4.
J Invest Dermatol ; 136(12): 2345-2355, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27365293

RESUMO

Trex2 is a keratinocyte-specific 3'-deoxyribonuclease that participates in the maintenance of skin homeostasis after DNA damage. Here, we show that this exonuclease is strongly upregulated in human psoriasis, a hyperproliferative and inflammatory skin disease. Similarly, the imiquimod (IMQ)- and Il23-induced mouse psoriasis was associated with a substantial upregulation of Trex2, which was recruited into fragmented chromatin in keratinocytes that were undergoing impaired proliferation, differentiation, and cell death, indicating an important role in DNA processing. Using Trex2 knockout mice, we have found that Trex2 deficiency attenuated IMQ-induced psoriasis-like skin inflammation and enhanced IMQ-induced parakeratosis. Also, Il23-induced ear swelling was diminished in Trex2 knockout mice in comparison with wild-type (wt) mice. Transcriptome analysis identified multiple genes that were deregulated by Trex2 loss after treatment with IMQ. Specifically, immune response genes and pathways normally associated with inflammation were downregulated, whereas those related to skin differentiation and chromatin biology showed increased expression. Interestingly, Trex2 deficiency led to decreased IMQ-induced keratinocyte death via both cell autonomous and noncell autonomous mechanisms. Hence, our data indicate that Trex2 acts as a critical factor in the pathogenesis of psoriasis by promoting keratinocyte apoptosis and enucleation and thereby influencing skin immune responses.


Assuntos
Aminoquinolinas/farmacologia , Exodesoxirribonucleases/genética , Regulação da Expressão Gênica , Psoríase/genética , Animais , Apoptose/genética , Biópsia por Agulha , Estudos de Casos e Controles , Sobrevivência Celular/genética , Células Cultivadas , Modelos Animais de Doenças , Humanos , Imiquimode , Imuno-Histoquímica , Queratinócitos/citologia , Camundongos , Camundongos Knockout , Fenótipo , Prognóstico , Psoríase/tratamento farmacológico , Psoríase/patologia , Índice de Gravidade de Doença , Regulação para Cima
5.
Methods ; 85: 100-107, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25952948

RESUMO

The generation of dynamic models of biological processes critically depends on the determination of precise cellular concentrations of biomolecules. Measurements of system-wide absolute protein levels are particularly valuable information in systems biology. Recently, mass spectrometry based proteomics approaches have been developed to estimate protein concentrations on a proteome-wide scale. However, for very complex proteomes, fractionation steps are required, increasing samples number and instrument analysis time. As a result, the number of full proteomes that can be routinely analyzed is limited. Here we combined absolute quantification strategies with the multiplexing capabilities of isobaric tandem mass tags to determine cellular protein abundances in a high throughput and proteome-wide scale even for highly complex biological systems, such as a whole human cell line. We generated two independent data sets to demonstrate the power of the approach regarding sample throughput, dynamic range, quantitative precision and accuracy as well as proteome coverage in comparison to existing mass spectrometry based strategies.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Marcação por Isótopo/métodos , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Células HEK293 , Humanos , Espectrometria de Massas/métodos
6.
FASEB J ; 28(7): 3023-37, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24719356

RESUMO

The endocytic compartment is emerging as a functional platform for controlling important cellular processes. We have found that ∼10 to 15% of total KRas, a protein that is frequently mutated in cancer, is present on endosomes, independent of its activation state. The dynamics of GFP-KRas wild-type (WT) and constitutively active or inactive mutants on endosomes were analyzed by fluorescence recovery after photobleaching (FRAP) microscopy. The measurements revealed an extraordinarily fast recovery of KRas WT [half-time (HT), ∼1.3 s] compared to HRas, Rab5, and EGFR, with the active KRasG12V mutant being significantly faster and more mobile (HT, ∼1 s, and ∼82% of exchangeable fraction) than the inactive KRasS17N (HT, ∼1.6 s, and ∼60% of exchangeable fraction). KRas rapidly switches from the cytoplasm to the endosomal membranes by an electrostatic interaction between its polybasic region and the endosomal acidic phospholipids, mainly phosphatidylserine.-Gelabert-Baldrich, M., Soriano-Castell, D., Calvo, M., Lu, A., Viña-Vilaseca, A., Rentero, C., Pol, A., Grinstein, S. Enrich, C., Tebar, F. Dynamics of KRas on endosomes: involvement of acidic phospholipids in its association.


Assuntos
Endossomos/metabolismo , Fosfolipídeos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Citoplasma/genética , Citoplasma/metabolismo , Endocitose/genética , Endocitose/fisiologia , Endossomos/genética , Fibroblastos/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde/metabolismo , Membranas Intracelulares/metabolismo , Mutação/genética , Proteínas Proto-Oncogênicas p21(ras)/genética
7.
J Biol Chem ; 286(10): 8697-8706, 2011 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-21212261

RESUMO

Cationic amino acid transporter 1 (CAT-1) is responsible for the bulk of the uptake of cationic amino acids in most mammalian cells. Activation of protein kinase C (PKC) leads to down-regulation of the cell surface CAT-1. To examine the mechanisms of PKC-induced down-regulation of CAT-1, a functional mutant of CAT-1 (CAT-1-HA-GFP) was generated in which a hemagglutinin antigen (HA) epitope tag was introduced into the second extracellular loop and GFP was attached to the carboxyl terminus. CAT-1-HA-GFP was stably expressed in porcine aorthic endothelial and human epithelial kidney (HEK) 293 cells. Using the HA antibody internalization assay we have demonstrated that PKC-dependent endocytosis was strongly inhibited by siRNA depletion of clathrin heavy chain, indicating that CAT-1-HA-GFP internalization requires clathrin-coated pits. Internalized CAT-1-HA-GFP was accumulated in early, recycling, and late endosomes. PKC activation also resulted in ubiquitination of CAT-1. CAT-1 ubiquitination and endocytosis in phorbol ester-stimulated porcine aorthic endothelial and HEK293 cells were inhibited by siRNA knockdown of NEDD4-2 and NEDD4-1 E3 ubiquitin ligases, respectively. In contrast, ubiquitination and endocytosis of the dopamine transporter was dependent on NEDD4-2 in all cell types tested. Altogether, our data suggest that ubiquitination mediated by NEDD4-2 or NEDD4-1 leading to clathrin-mediated endocytosis is the common mode of regulation of various transporter proteins by PKC.


Assuntos
Transportador 1 de Aminoácidos Catiônicos/metabolismo , Clatrina/metabolismo , Endocitose/fisiologia , Proteína Quinase C/metabolismo , Animais , Transportador 1 de Aminoácidos Catiônicos/genética , Clatrina/genética , Vesículas Revestidas por Clatrina/genética , Vesículas Revestidas por Clatrina/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Células HEK293 , Humanos , Ubiquitina-Proteína Ligases Nedd4 , Proteína Quinase C/genética , Estrutura Secundária de Proteína , Suínos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia
8.
J Biol Chem ; 285(10): 7645-56, 2010 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-20051513

RESUMO

RNA interference screen previously revealed that a HECT-domain E3 ubiquitin ligase, neuronal precursor cell expressed, developmentally down-regulated 4-2 (Nedd4-2), is necessary for ubiquitination and endocytosis of the dopamine transporter (DAT) induced by the activation of protein kinase C (PKC). To further confirm the role of Nedd4-2 in DAT ubiquitination and endocytosis, we demonstrated that the depletion of Nedd4-2 by two different small interfering RNA (siRNA) duplexes suppressed PKC-dependent ubiquitination and endocytosis of DAT in human and porcine cells, whereas knock-down of a highly homologous E3 ligase, Nedd4-1, had no effect on DAT. The abolished DAT ubiquitination in Nedd4-2-depleted cells was rescued by expression of recombinant Nedd4-2. Moreover, overexpression of Nedd4-2 resulted in increased PKC-dependent ubiquitination of DAT. Mutational inactivation of the HECT domain of Nedd4-2 inhibited DAT ubiquitination and endocytosis. Structure-function analysis of Nedd4-2-mediated DAT ubiquitination revealed that the intact WW4 domain and to a lesser extent WW3 domain are necessary for PKC-dependent DAT ubiquitination. Moreover, a fragment of the Nedd4-2 molecule containing WW3, WW4, and HECT domains was sufficient for fully potentiating PKC-dependent ubiquitination of DAT. Analysis of DAT ubiquitination using polyubiquitin chain-specific antibodies showed that DAT is mainly conjugated with Lys(63)-linked ubiquitin chains. siRNA analysis demonstrated that this polyubiquitination is mediated by Nedd4-2 cooperation with UBE2D and UBE2L3 E2 ubiquitin-conjugating enzymes. The model is proposed whereby each ubiquitinated DAT molecule is modified by a single four-ubiquitin Lys(63)-linked chain that can be conjugated to various lysine residues of DAT.


Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Lisina/metabolismo , Poliubiquitina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Endocitose/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Lisina/genética , Ubiquitina-Proteína Ligases Nedd4 , Poliubiquitina/genética , Proteína Quinase C/metabolismo , Estrutura Terciária de Proteína , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Suínos , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
9.
Cancer Res ; 69(16): 6676-84, 2009 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-19654293

RESUMO

TREX2 is a proofreading 3'-5' exonuclease that can be involved in genome maintenance; however, its biological role remains undefined. To better understand the function and physiologic relevance of TREX2, we generated mice deficient in TREX2 by targeted disruption of its unique coding exon. The knockout mice are viable and do not show relevant differences in growth, survival, lymphocyte development, or spontaneous tumor incidence compared with their wild-type counterparts over a period of up to 2 years. Also, we did not observe chromosomal instability or defects in cell proliferation and cell cycle upon loss of TREX2. We have observed that TREX2 expression is not ubiquitous, being expressed preferentially in tissues with stratified squamous epithelia, such as the skin or esophagus, and specifically in keratinocytes. Interestingly, TREX2-null mice are more susceptible to skin carcinogenesis induced by 7,12-dimethylbenz(a)anthracene (DMBA) compared with wild-type mice. This phenotype correlates with a reduction of DMBA-induced apoptosis in both the epidermis and keratinocytes of TREX2-null mice. Altogether, our results suggest a tumor suppressor role for TREX2 in skin carcinogenesis through which it contributes to keratinocyte apoptosis under conditions of genotoxic stress.


Assuntos
Carcinoma/genética , Exodesoxirribonucleases/genética , Predisposição Genética para Doença , Neoplasias Cutâneas/genética , 9,10-Dimetil-1,2-benzantraceno , Envelhecimento/genética , Envelhecimento/fisiologia , Animais , Apoptose/genética , Carcinógenos , Carcinoma/induzido quimicamente , Carcinoma/metabolismo , Células Cultivadas , Embrião de Mamíferos , Exodesoxirribonucleases/metabolismo , Feminino , Queratinócitos/metabolismo , Queratinócitos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos/genética , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/metabolismo , Distribuição Tecidual
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