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1.
J Invest Dermatol ; 135(2): 569-578, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25118157

RESUMO

UV damage to the skin leads to the release of noncoding RNA (ncRNA) from necrotic keratinocytes that activates Toll-like receptor 3 (TLR3). This release of ncRNA triggers inflammation in the skin following UV damage. Recently, TLR3 activation was also shown to aid wound repair and increase the expression of genes associated with permeability barrier repair. Here, we sought to test whether skin barrier repair after UVB damage is dependent on the activation of TLR3. We observed that multiple ncRNAs induced expression of skin barrier repair genes, that the TLR3 ligand Poly (I:C) also induced expression and function of tight junctions, and that the ncRNA U1 acts in a TLR3-dependent manner to induce expression of skin barrier repair genes. These observations were shown to have functional relevance as Tlr3-/- mice displayed a delay in skin barrier repair following UVB damage. Combined, these data further validate the conclusion that recognition of endogenous RNA by TLR3 is an important step in the program of skin barrier repair.


Assuntos
Pele/efeitos da radiação , Receptor 3 Toll-Like/fisiologia , Raios Ultravioleta/efeitos adversos , Animais , Citocinas/genética , Feminino , Queratinócitos/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Permeabilidade , Poli I-C/farmacologia , Pele/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/fisiologia
2.
Dev Biol ; 373(2): 373-82, 2013 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-23123965

RESUMO

Activating mutations in the KRAS oncogene are associated with three related human syndromes, which vary in hair and skin phenotypes depending on the involved allele. How variations in RAS signals are interpreted during hair and skin development is unknown. In this study, we investigated the developmental and transcriptional response of skin and hair to changes in RAS activity, using mouse genetic models and microarray analysis. While activation of Kras (Kras(G12D)) in the skin had strong effects on hair growth and hair shape, steady state changes in downstream RAS/MAPK effectors were subtle and detected only by transcriptional responses. To model the transcriptional response of multiple developmental pathways to active RAS, the effects of growth factor stimulation were studied in skin explants. Here FGF acutely suppressed Shh transcription within 90 min but had significantly less effect on Eda, WNT, Notch or BMP pathways. Furthermore, in vivo Fgfr2 loss-of-function in the ectoderm caused derepression of Shh, revealing a role for FGF in Shh regulation in the hair follicle. These studies define both dosage sensitive effects of RAS signaling on hair morphogenesis and reveal acute mechanisms for fine-tuning Shh levels in the hair follicle.


Assuntos
Regulação para Baixo/genética , Folículo Piloso/crescimento & desenvolvimento , Folículo Piloso/metabolismo , Proteínas Hedgehog/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Regulação da Expressão Gênica no Desenvolvimento , Folículo Piloso/citologia , Folículo Piloso/enzimologia , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Tamanho do Órgão , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/genética , Pele/crescimento & desenvolvimento , Pele/metabolismo , Transcrição Gênica
3.
PLoS Biol ; 7(10): e1000213, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19806183

RESUMO

Biogenesis of ribosomes is an essential cellular process conserved across all eukaryotes and is known to require >170 genes for the assembly, modification, and trafficking of ribosome components through multiple cellular compartments. Despite intensive study, this pathway likely involves many additional genes. Here, we employ network-guided genetics-an approach for associating candidate genes with biological processes that capitalizes on recent advances in functional genomic and proteomic studies-to computationally identify additional ribosomal biogenesis genes. We experimentally evaluated >100 candidate yeast genes in a battery of assays, confirming involvement of at least 15 new genes, including previously uncharacterized genes (YDL063C, YIL091C, YOR287C, YOR006C/TSR3, YOL022C/TSR4). We associate the new genes with specific aspects of ribosomal subunit maturation, ribosomal particle association, and ribosomal subunit nuclear export, and we identify genes specifically required for the processing of 5S, 7S, 20S, 27S, and 35S rRNAs. These results reveal new connections between ribosome biogenesis and mRNA splicing and add >10% new genes-most with human orthologs-to the biogenesis pathway, significantly extending our understanding of a universally conserved eukaryotic process.


Assuntos
Biologia Computacional/métodos , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Centrifugação com Gradiente de Concentração , Análise por Conglomerados , Mineração de Dados , Espectrometria de Massas , Precursores de RNA/metabolismo , Splicing de RNA , Subunidades Ribossômicas/genética , Subunidades Ribossômicas/metabolismo , Ribossomos/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia
4.
Mol Biol Cell ; 19(2): 735-44, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18077551

RESUMO

We previously showed that nuclear export of the large (60S) ribosomal subunit relies on Nmd3 in a Crm1-dependent manner. Recently the general mRNA export factor, the Mtr2/Mex67 heterodimer, was shown to act as an export receptor in parallel with Crm1. These observations raise the possibility that nuclear export of the 60S subunit in Saccharomyces cerevisiae requires multiple export receptors. Here, we show that the previously characterized 60S subunit biogenesis factor, Arx1, also acts as an export receptor for the 60S subunit. We found that deletion of ARX1 was synthetic lethal with nmd3 and mtr2 mutants and was synthetic sick with several nucleoporin mutants. Deletion of ARX1 led to accumulation of pre-60S particles in the nucleus that were enriched for Nmd3, Crm1, Mex67, and Mtr2, suggesting that in the absence of Arx1, 60S export is impaired even though the subunit is loaded with export receptors. Finally, Arx1 interacted with several nucleoporins in yeast two-hybrid as well as in vitro assays. These results show that Arx1 can directly bridge the interaction between the pre-60S particle and the NPC and thus is a third export receptor for the 60S subunit in yeast.


Assuntos
Núcleo Celular/metabolismo , Receptores de Superfície Celular/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Deleção de Genes , Dados de Sequência Molecular , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
5.
Proc Natl Acad Sci U S A ; 104(5): 1558-63, 2007 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-17242366

RESUMO

J-proteins and Hsp70 chaperones function together in diverse cellular processes. We identified a cytosolic J-protein, Jjj1, of Saccharomyces cerevisiae that is associated with 60S ribosomal particles. Unlike Zuo1, a 60S subunit-associated J-protein that is a component of the chaperone machinery that binds nascent polypeptide chains upon their exit from the ribosome, Jjj1 plays a role in ribosome biogenesis. Cells lacking Jjj1 have phenotypes very similar to those lacking Rei1, a ribosome biogenesis factor associated with pre-60S ribosomal particles in the cytosol. Jjj1 stimulated the ATPase activity of the general cytosolic Hsp70 Ssa, but not Ssb, Zuo1's ribosome-associated Hsp70 partner. Overexpression of Jjj1, which is normally approximately 40-fold less abundant than Zuo1, can partially rescue the phenotypes of cells lacking Zuo1 as well as cells lacking Ssb. Together, these results are consistent with the idea that Jjj1 normally functions with Ssa in a late, cytosolic step of the biogenesis of 60S ribosomal subunits. In addition, because of its ability to bind 60S subunits, we hypothesize that Jjj1, when overexpressed, is able to partially substitute for the Zuo1:Ssb chaperone machinery by recruiting Ssa to the ribosome, facilitating its interaction with nascent polypeptide chains.


Assuntos
Citosol/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP40/fisiologia , Ribossomos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/metabolismo , Dimerização , Regulação Fúngica da Expressão Gênica , Proteínas de Choque Térmico HSP40/química , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares , Fenótipo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Temperatura
6.
Mol Cell Biol ; 26(10): 3718-27, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16648468

RESUMO

Arx1 and Rei1 are found on late pre-60S ribosomal particles containing the export adaptor Nmd3. Arx1 is related to methionine aminopeptidases (MetAPs), and Rei1 is a C2H2 zinc finger protein whose function in ribosome biogenesis has not been previously characterized. Arx1 and Rei1 localized predominately to the nucleus and cytoplasm, respectively, but could be coimmunoprecipitated, suggesting that they are transiently in the same 60S complex. arx1delta mutants showed a modest accumulation of 60S subunits in the nucleus, suggesting that Arx1 enhances 60S export. Deletion of REI1 led to cold sensitivity and redistribution of Arx1 to the cytoplasm, where it remained bound to free 60S subunits. However, deletion of ARX1 or the fusion of enhanced GFP (eGFP) to Rpl25 suppressed the cold sensitivity of an rei1delta mutant. The presence of eGFP on Rpl25 or its neighboring protein Rpl35 reduced the binding of Arx1 to 60S subunits, suggesting that Arx1 binds to 60S subunits in the vicinity of the exit tunnel. Mutations in Arx1 that disrupted its binding to 60S also suppressed an rei1delta mutant and restored the normal nuclear localization of Arx1. These results indicate that the cold sensitivity of rei1delta cells is due to the persistence of Arx1 on 60S subunits in the cytoplasm. Furthermore, these results suggest that Rei1 is needed for release of Arx1 from nascent 60S subunits after export to the cytoplasm but not for the subsequent nuclear import of Arx1.


Assuntos
Núcleo Celular/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Western Blotting , Fracionamento Celular , Centrifugação com Gradiente de Concentração , Cicloeximida/farmacologia , Citoplasma/metabolismo , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Deleção de Genes , Genes Fúngicos , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência , Modelos Moleculares , Dados de Sequência Molecular , Testes de Precipitina , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Ribossomos/química , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos
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