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1.
Cell ; 186(1): 80-97.e26, 2023 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-36608661

RESUMO

Glucose is a universal bioenergy source; however, its role in controlling protein interactions is unappreciated, as are its actions during differentiation-associated intracellular glucose elevation. Azido-glucose click chemistry identified glucose binding to a variety of RNA binding proteins (RBPs), including the DDX21 RNA helicase, which was found to be essential for epidermal differentiation. Glucose bound the ATP-binding domain of DDX21, altering protein conformation, inhibiting helicase activity, and dissociating DDX21 dimers. Glucose elevation during differentiation was associated with DDX21 re-localization from the nucleolus to the nucleoplasm where DDX21 assembled into larger protein complexes containing RNA splicing factors. DDX21 localized to specific SCUGSDGC motif in mRNA introns in a glucose-dependent manner and promoted the splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings uncover a biochemical mechanism of action for glucose in modulating the dimerization and function of an RNA helicase essential for tissue differentiation.


Assuntos
RNA Helicases DEAD-box , Glucose , Queratinócitos , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , RNA Helicases DEAD-box/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Glucose/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Humanos
2.
Cell ; 176(1-2): 361-376.e17, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30580963

RESUMO

Here, we present Perturb-ATAC, a method that combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells based on the simultaneous detection of CRISPR guide RNAs and open chromatin sites by assay of transposase-accessible chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in ∼4,300 single cells, encompassing more than 63 genotype-phenotype relationships. Perturb-ATAC in human B lymphocytes uncovered regulators of chromatin accessibility, TF occupancy, and nucleosome positioning and identified a hierarchy of TFs that govern B cell state, variation, and disease-associated cis-regulatory elements. Perturb-ATAC in primary human epidermal cells revealed three sequential modules of cis-elements that specify keratinocyte fate. Combinatorial deletion of all pairs of these TFs uncovered their epistatic relationships and highlighted genomic co-localization as a basis for synergistic interactions. Thus, Perturb-ATAC is a powerful strategy to dissect gene regulatory networks in development and disease.


Assuntos
Epigenômica/métodos , Redes Reguladoras de Genes/genética , Análise de Célula Única/métodos , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Redes Reguladoras de Genes/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Análise de Sequência de DNA/métodos , Fatores de Transcrição/metabolismo
3.
Hum Mol Genet ; 29(4): 541-553, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-31628467

RESUMO

Missense mutations in the RNA exosome component exosome component 2 (EXOSC2), also known as ribosomal RNA-processing protein 4 (RRP4), were recently identified in two unrelated families with a novel syndrome known as Short stature, Hearing loss, Retinitis pigmentosa and distinctive Facies (SHRF, #OMIM 617763). Little is known about the mechanism of the SHRF pathogenesis. Here we have studied the effect of mutations in EXOSC2/RRP4 in patient-derived lymphoblasts, clustered regularly interspaced short palindromic repeats (CRISPR)-generated mutant fetal keratinocytes and Drosophila. We determined that human EXOSC2 is an essential gene and that the pathogenic G198D mutation prevents binding to other RNA exosome components, resulting in protein and complex instability and altered expression and/or activities of critical genes, including those in the autophagy pathway. In parallel, we generated multiple CRISPR knockouts of the fly rrp4 gene. Using these flies, as well as rrp4 mutants with Piggy Bac (PBac) transposon insertion in the 3'UTR and RNAi flies, we determined that fly rrp4 was also essential, that fly rrp4 phenotypes could be rescued by wild-type human EXOSC2 but not the pathogenic form and that fly rrp4 is critical for eye development and maintenance, muscle ultrastructure and wing vein development. We found that overexpression of the transcription factor MITF was sufficient to rescue the small eye and adult lethal phenotypes caused by rrp4 inhibition. The autophagy genes ATG1 and ATG17, which are regulated by MITF, had similar effect. Pharmacological stimulation of autophagy with rapamycin also rescued the lethality caused by rrp4 inactivation. Our results implicate defective autophagy in SHRF pathogenesis and suggest therapeutic strategies.


Assuntos
Complexo Multienzimático de Ribonucleases do Exossomo/genética , Proteínas de Ligação a RNA/genética , Animais , Autofagia/genética , Modelos Animais de Doenças , Drosophila/genética , Nanismo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Exossomos/metabolismo , Feminino , Genômica/métodos , Células HEK293 , Perda Auditiva/genética , Humanos , Masculino , Mutação de Sentido Incorreto/genética , Fenótipo , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Retinose Pigmentar/genética , Síndrome
4.
Nat Methods ; 15(3): 207-212, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29400715

RESUMO

RNA-protein interactions play numerous roles in cellular function and disease. Here we describe RNA-protein interaction detection (RaPID), which uses proximity-dependent protein labeling, based on the BirA* biotin ligase, to rapidly identify the proteins that bind RNA sequences of interest in living cells. RaPID displays utility in multiple applications, including in evaluating protein binding to mutant RNA motifs in human genetic disorders, in uncovering potential post-transcriptional networks in breast cancer, and in discovering essential host proteins that interact with Zika virus RNA. To improve the BirA*-labeling component of RaPID, moreover, a new mutant BirA* was engineered from Bacillus subtilis, termed BASU, that enables >1,000-fold faster kinetics and >30-fold increased signal-to-noise ratio over the prior standard Escherichia coli BirA*, thereby enabling direct study of RNA-protein interactions in living cells on a timescale as short as 1 min.


Assuntos
Biotina/química , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Proteínas Virais/metabolismo , Zika virus/metabolismo , Bacillus subtilis/metabolismo , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Células HEK293 , Humanos , Neurônios/citologia , Neurônios/metabolismo , RNA/química , RNA/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Zika virus/genética
5.
Nat Immunol ; 9(12): 1371-8, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18997794

RESUMO

Recent studies suggest that nuclear factor kappaB-inducing kinase (NIK) is suppressed through constitutive proteasome-mediated degradation regulated by TRAF2, TRAF3 and cIAP1 or cIAP2. Here we demonstrated that the degradation of NIK occurs upon assembly of a regulatory complex through TRAF3 recruitment of NIK and TRAF2 recruitment of cIAP1 and cIAP2. In contrast to TRAF2 and TRAF3, cIAP1 and cIAP2 seem to play redundant roles in the degradation of NIK, as inhibition of both cIAPs was required for noncanonical NF-kappaB activation and increased survival and proliferation of primary B lymphocytes. Furthermore, the lethality of TRAF3 deficiency in mice could be rescued by a single NIK gene, highlighting the importance of tightly regulated NIK.


Assuntos
Linfócitos B/imunologia , Diferenciação Celular/imunologia , Proteínas Inibidoras de Apoptose/imunologia , NF-kappa B/imunologia , Fator 2 Associado a Receptor de TNF/imunologia , Fator 3 Associado a Receptor de TNF/imunologia , Animais , Linfócitos B/citologia , Sobrevivência Celular , Células Cultivadas , Ativação Enzimática/imunologia , Immunoblotting , Imunoprecipitação , Proteínas Inibidoras de Apoptose/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , NF-kappa B/genética , NF-kappa B/metabolismo , RNA Interferente Pequeno , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/metabolismo , Transfecção
6.
Nat Methods ; 13(6): 489-92, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27111506

RESUMO

The complexity of transcriptome-wide protein-RNA interaction networks is incompletely understood. While emerging studies are greatly expanding the known universe of RNA-binding proteins, methods for the discovery and characterization of protein-RNA interactions remain resource intensive and technically challenging. Here we introduce a UV-C crosslinking and immunoprecipitation platform, irCLIP, which provides an ultraefficient, fast, and nonisotopic method for the detection of protein-RNA interactions using far less material than standard protocols.


Assuntos
Imunoprecipitação/métodos , Proteínas de Ligação a RNA/análise , Raios Ultravioleta , Sítios de Ligação , Reagentes de Ligações Cruzadas/química , DNA Complementar/genética , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Processos Fotoquímicos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/efeitos da radiação , Sensibilidade e Especificidade , Transcriptoma
7.
RNA ; 21(1): 135-43, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25411354

RESUMO

RNA-protein interactions are central to biological regulation. Cross-linking immunoprecipitation (CLIP)-seq is a powerful tool for genome-wide interrogation of RNA-protein interactomes, but current CLIP methods are limited by challenging biochemical steps and fail to detect many classes of noncoding and nonhuman RNAs. Here we present FAST-iCLIP, an integrated pipeline with improved CLIP biochemistry and an automated informatic pipeline for comprehensive analysis across protein coding, noncoding, repetitive, retroviral, and nonhuman transcriptomes. FAST-iCLIP of Poly-C binding protein 2 (PCBP2) showed that PCBP2-bound CU-rich motifs in different topologies to recognize mRNAs and noncoding RNAs with distinct biological functions. FAST-iCLIP of PCBP2 in hepatitis C virus-infected cells enabled a joint analysis of the PCBP2 interactome with host and viral RNAs and their interplay. These results show that FAST-iCLIP can be used to rapidly discover and decipher mechanisms of RNA-protein recognition across the diversity of human and pathogen RNAs.


Assuntos
Perfilação da Expressão Gênica , RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Sequência Consenso , Hepacivirus/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Imunoprecipitação , Ligação Proteica , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Transcriptoma
8.
Trends Genet ; 29(1): 31-40, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23141808

RESUMO

More than 100 human genetic skin diseases, impacting over 20% of the population, are characterized by disrupted epidermal differentiation. A significant proportion of the 90 genes identified in these disorders to date are concentrated within several functional pathways, suggesting the emergence of organizing themes in epidermal differentiation. Among these are the Notch, transforming growth factor ß (TGFß), IκB kinase (IKK), Ras/mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), p63, and Wnt signaling pathways, as well as core biological processes mediating calcium homeostasis, tissue integrity, cornification, and lipid biogenesis. Here, we review recent results supporting the central role of these pathways in epidermal differentiation, highlighting the integration of genetic information with functional studies to illuminate the biological actions of these pathways in humans as well as to guide development of future therapeutics to correct their dysfunction.


Assuntos
Diferenciação Celular/genética , Epiderme/fisiologia , Transdução de Sinais/genética , Dermatopatias/genética , Dermatopatias/fisiopatologia , Animais , Epiderme/metabolismo , Redes Reguladoras de Genes/fisiologia , Humanos , Modelos Biológicos , Dermatopatias/etiologia
9.
Am J Hum Genet ; 91(3): 435-43, 2012 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-22922031

RESUMO

The basis for impaired differentiation in TP63 mutant ankyloblepharon-ectodermal dysplasia-clefting (AEC) syndrome is unknown. Human epidermis harboring AEC TP63 mutants recapitulated this impairment, along with downregulation of differentiation activators, including HOPX, GRHL3, KLF4, PRDM1, and ZNF750. Gene-set enrichment analysis indicated that disrupted expression of epidermal differentiation programs under the control of ZNF750 and KLF4 accounted for the majority of disrupted epidermal differentiation resulting from AEC mutant TP63. Chromatin immunoprecipitation (ChIP) analysis and ChIP-sequencing of TP63 binding in differentiated keratinocytes revealed ZNF750 as a direct target of wild-type and AEC mutant TP63. Restoring ZNF750 to AEC model tissue rescued activator expression and differentiation, indicating that AEC TP63-mediated ZNF750 inhibition contributes to differentiation defects in AEC. Incorporating disease-causing mutants into regenerated human tissue can thus dissect pathomechanisms and identify targets that reverse disease features.


Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Displasia Ectodérmica/genética , Anormalidades do Olho/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Diferenciação Celular/genética , Epiderme/metabolismo , Pálpebras/anormalidades , Humanos , Fator 4 Semelhante a Kruppel , Mutação , Técnicas de Cultura de Órgãos/métodos , Transcriptoma
10.
bioRxiv ; 2024 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-39386644

RESUMO

RNA binding proteins ( RBPs ) control varied processes, including RNA splicing, stability, transport, and translation 1-3 . Dysfunctional RNA-RBP interactions contribute to the pathogenesis of human disease 1,4,5 , however, characterizing the nature and dynamics of multiprotein assemblies on RNA has been challenging. To address this, non-isotopic ligation-based ultraviolet crosslinking immunoprecipitation 6 was combined with mass spectrometry ( irCLIP-RNP ) to identify RNA-dependent associated proteins ( RDAPs ) co-bound to RNA with any RBP of interest. irCLIP-RNP defined landscapes of multimeric protein assemblies on RNA, uncovering previously unknown patterns of RBP-RNA associations, including cell-type-selective combinatorial relationships between RDAPs and primary RBPs. irCLIP-RNP also defined dynamic RDAP remodeling in response to epidermal growth factor ( EGF ), uncovering EGF-induced recruitment of UPF1 adjacent to HNRNPC to effect splicing surveillance of cell proliferation mRNAs. To identify the RNAs simultaneously co-bound by multiple studied RBPs, a sequential immunoprecipitation irCLIP ( Re-CLIP ) method was also developed. Re-CLIP confirmed binding relationships seen in irCLIP-RNP and detected simultaneous HNRNPC and UPF1 co-binding on RND3 and DDX3X mRNAs. irCLIP-RNP and Re-CLIP provide a framework to identify and characterize dynamic RNA-protein assemblies in living cells.

11.
Curr Protoc ; 3(1): e659, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36705610

RESUMO

UV cross-linking-based methods are the most common tool to explore in vivo RNA-protein interactions. UV cross-linking enables the freezing of direct interactions in the cell, which can then be mapped by high-throughput sequencing through a family of methods termed CLIP-seq. CLIP-seq measures the distribution of cross-link events by purifying a protein of interest and sequencing the covalently bound RNA fragments. However, there are disagreements and ambiguities as to which proteins are RNA-binding proteins and what interactions are significant as all proteins contact all RNAs at some frequency. Here we describe a protocol for both determining RNA-protein interactions through a combination of RNA library preparation and the measurement of absolute cross-link rates, which helps determine what proteins are RNA-binding proteins and what interactions are significant. This protocol, comprising an updated form of the easyCLIP protocol, describes guidelines for RNA library preparation, oligo and protein standard construction, and the measurement of cross-link rates. These methods are easily visualizable through their fluorescent labels and can be adapted to study RNA-binding properties of both functional, high affinity RNA-binding proteins, and the accidental RNA interactions of non-RNA-binding proteins. © 2023 Wiley Periodicals LLC. Basic Protocol 1: RNA library construction Basic Protocol 2: Determining UV cross-link rates Support Protocol 1: Cross-linking and lysing cells Support Protocol 2: Adapter preparation Support Protocol 3: Preparation of cross-linked RBP standard.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação , Proteínas de Ligação a RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA/genética , RNA/química , RNA/metabolismo , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos
12.
J Exp Med ; 203(11): 2413-8, 2006 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-17015635

RESUMO

Proper activation of nuclear factor (NF)-kappaB transcription factors is critical in regulating fundamental biological processes such as cell survival and proliferation, as well as in inflammatory and immune responses. Recently, the NF-kappaB signaling pathways have been categorized into the canonical pathway, which results in the nuclear translocation of NF-kappaB complexes containing p50, and the noncanonical pathway, which involves the induced processing of p100 to p52 and the formation of NF-kappaB complexes containing p52 (Bonizzi, G., and M. Karin. 2004. Trends Immunol. 25:280-288). We demonstrate that loss of tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) results in constitutive noncanonical NF-kappaB activity. Importantly, TRAF3-/- B cells show ligand-independent up-regulation of intracellular adhesion molecule 1 and protection from spontaneous apoptosis during in vitro culture. In addition, we demonstrate that loss of TRAF3 results in profound accumulation of NF-kappaB-inducing kinase in TRAF3-/- cells. Finally, we show that the early postnatal lethality observed in TRAF3-deficient mice is rescued by compound loss of the noncanonical NF-kappaB p100 gene. Thus, these genetic data clearly demonstrate that TRAF3 is a critical negative modulator of the noncanonical NF-kappaB pathway and that constitutive activation of the noncanonical NF-kappaB pathway causes the lethal phenotype of TRAF3-deficient mice.


Assuntos
Subunidade p52 de NF-kappa B/deficiência , Subunidade p52 de NF-kappa B/genética , Fator 3 Associado a Receptor de TNF/deficiência , Fator 3 Associado a Receptor de TNF/genética , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Cultivadas , Regulação para Baixo , Genes Letais , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Subunidade p52 de NF-kappa B/antagonistas & inibidores , Subunidade p52 de NF-kappa B/fisiologia , Processamento de Proteína Pós-Traducional
13.
Nature ; 439(7073): 208-11, 2006 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-16306936

RESUMO

Type I interferon (IFN) production is a critical component of the innate defence against viral infections. Viral products induce strong type I IFN responses through the activation of Toll-like receptors (TLRs) and intracellular cytoplasmic receptors such as protein kinase R (PKR). Here we demonstrate that cells lacking TRAF3, a member of the TNF receptor-associated factor family, are defective in type I IFN responses activated by several different TLRs. Furthermore, we show that TRAF3 associates with the TLR adaptors TRIF and IRAK1, as well as downstream IRF3/7 kinases TBK1 and IKK-epsilon, suggesting that TRAF3 serves as a critical link between TLR adaptors and downstream regulatory kinases important for IRF activation. In addition to TLR stimulation, we also show that TRAF3-deficient fibroblasts are defective in their type I IFN response to direct infection with vesicular stomatitis virus, indicating that TRAF3 is also an important component of TLR-independent viral recognition pathways. Our data demonstrate that TRAF3 is a major regulator of type I IFN production and the innate antiviral response.


Assuntos
Imunidade Inata/imunologia , Interferon Tipo I/imunologia , Fator 3 Associado a Receptor de TNF/metabolismo , Receptores Toll-Like/metabolismo , Viroses/imunologia , Viroses/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Quinase I-kappa B/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 7 de Interferon/metabolismo , Interferon Tipo I/biossíntese , Quinases Associadas a Receptores de Interleucina-1 , Camundongos , Camundongos Endogâmicos C57BL , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptor 3 Toll-Like/imunologia , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , Receptor 7 Toll-Like/imunologia , Receptor 7 Toll-Like/metabolismo , Receptor Toll-Like 9/imunologia , Receptor Toll-Like 9/metabolismo , Receptores Toll-Like/imunologia
14.
Proc Natl Acad Sci U S A ; 105(9): 3503-8, 2008 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-18292232

RESUMO

Articles in recent years have described two separate and distinct NF-kappaB activation pathways that result in the differential activation of p50- or p52-containing NF-kappaB complexes. Studies examining tumor-necrosis factor receptor-associated factors (TRAFs) have identified positive roles for TRAF2, TRAF5, and TRAF6, but not TRAF3, in canonical (p50-dependent) NF-kappaB activation. Conversely, it recently was reported that TRAF3 functions as an essential negative regulator of the noncanonical (p52-dependent) NF-kappaB pathway. In this article, we provide evidence that TRAF3 potently suppresses canonical NF-kappaB activation and gene expression in vitro and in vivo. We also demonstrate that deregulation of the canonical NF-kappaB pathway in TRAF3-deficient cells results from accumulation of NF-kappaB-inducing kinase (NIK), the essential kinase mediating noncanonical NF-kappaB activation. Thus, our data demonstrate that inhibition of TRAF3 results in coordinated activation of both NF-kappaB activation pathways.


Assuntos
NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fator 3 Associado a Receptor de TNF/fisiologia , Animais , Linhagem Celular , Quinase I-kappa B/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Subunidade p50 de NF-kappa B/metabolismo , Subunidade p52 de NF-kappa B/metabolismo , Quinase Induzida por NF-kappaB
15.
J Exp Med ; 200(4): 437-45, 2004 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-15302901

RESUMO

Numerous bacterial products such as lipopolysaccharide potently induce type I interferons (IFNs); however, the contribution of this innate response to host defense against bacterial infection remains unclear. Although mice deficient in either IFN regulatory factor (IRF)3 or the type I IFN receptor (IFNAR)1 are highly susceptible to viral infection, we show that these mice exhibit a profound resistance to infection caused by the Gram-positive intracellular bacterium Listeria monocytogenes compared with wild-type controls. Furthermore, this enhanced bacterial clearance is accompanied by a block in L. monocytogenes-induced splenic apoptosis in IRF3- and IFNAR1-deficient mice. Thus, our results highlight the disparate roles of type I IFNs during bacterial versus viral infections and stress the importance of proper IFN modulation in host defense.


Assuntos
Apoptose/imunologia , Proteínas de Ligação a DNA/deficiência , Interferon Tipo I/imunologia , Listeriose/imunologia , Receptores de Interferon/deficiência , Fatores de Transcrição/deficiência , Animais , Primers do DNA , Suscetibilidade a Doenças , Ensaio de Imunoadsorção Enzimática , Immunoblotting , Marcação In Situ das Extremidades Cortadas , Fator Regulador 3 de Interferon , Fígado/patologia , Macrófagos/imunologia , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase/métodos , Receptor de Interferon alfa e beta , Baço/imunologia
16.
Nat Commun ; 11(1): 6163, 2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-33268787

RESUMO

Long noncoding RNAs are thought to regulate gene expression by organizing protein complexes through unclear mechanisms. XIST controls the inactivation of an entire X chromosome in female placental mammals. Here we develop and integrate several orthogonal structure-interaction methods to demonstrate that XIST RNA-protein complex folds into an evolutionarily conserved modular architecture. Chimeric RNAs and clustered protein binding in fRIP and eCLIP experiments align with long-range RNA secondary structure, revealing discrete XIST domains that interact with distinct sets of effector proteins. CRISPR-Cas9-mediated permutation of the Xist A-repeat location shows that A-repeat serves as a nucleation center for multiple Xist-associated proteins and m6A modification. Thus modular architecture plays an essential role, in addition to sequence motifs, in determining the specificity of RBP binding and m6A modification. Together, this work builds a comprehensive structure-function model for the XIST RNA-protein complex, and suggests a general strategy for mechanistic studies of large ribonucleoprotein assemblies.


Assuntos
Adenina/análogos & derivados , Células-Tronco Embrionárias Murinas/metabolismo , RNA Longo não Codificante/química , Ribonucleoproteínas/química , Adenina/metabolismo , Animais , Sequência de Bases , Sistemas CRISPR-Cas , Linhagem Celular , Sequência Conservada , Reagentes de Ligações Cruzadas , Feminino , Ficusina/química , Formaldeído/química , Técnicas de Introdução de Genes , Humanos , Células K562 , Masculino , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Conformação de Ácido Nucleico , Gravidez , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Análise de Sequência de RNA
17.
Elife ; 92020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32379046

RESUMO

The Xist lncRNA mediates X chromosome inactivation (XCI). Here we show that Spen, an Xist-binding repressor protein essential for XCI , binds to ancient retroviral RNA, performing a surveillance role to recruit chromatin silencing machinery to these parasitic loci. Spen loss activates a subset of endogenous retroviral (ERV) elements in mouse embryonic stem cells, with gain of chromatin accessibility, active histone modifications, and ERV RNA transcription. Spen binds directly to ERV RNAs that show structural similarity to the A-repeat of Xist, a region critical for Xist-mediated gene silencing. ERV RNA and Xist A-repeat bind the RRM domains of Spen in a competitive manner. Insertion of an ERV into an A-repeat deficient Xist rescues binding of Xist RNA to Spen and results in strictly local gene silencing in cis. These results suggest that Xist may coopt transposable element RNA-protein interactions to repurpose powerful antiviral chromatin silencing machinery for sex chromosome dosage compensation.


The genetic material inside cells is often packaged into thread-like structures called chromosomes. In humans, mice and other mammals, a pair of sex chromosomes determines the genetic or chromosomal sex of each individual. Those who inherit two "X" chromosomes are said to be chromosomally female, while chromosomal males have one "X" and one "Y" chromosome. This means females have twice as many copies of genes on the X chromosome as a male does, which turns out to be double the number that the body needs. To solve this problem, mammals have developed a strategy known as dosage compensation. The second X chromosome in females becomes "silent": its DNA remains unchanged, but none of the genes are active. A long noncoding RNA molecule called Xist is responsible for switching off the extra X genes in female cells. It does this by coating the entirety of the second X chromosome. Normally, RNA molecules transmit the coded instructions in genes to the cellular machinery that manufactures proteins. "Noncoding" RNAs like Xist, however, are RNAs that have taken on different jobs inside the cell. Researchers believe that the ancestral Xist gene may have once encoded a protein but changed over time to produce only a noncoding RNA. Carter, Xu et al. therefore set out to find out how exactly this might have happened, and also how Xist might have acquired its ability to switch genes off. Initial experiments used mouse cells grown in the laboratory, in which a protein called Spen was deleted. Spen is known to help Xist silence the X chromosome. In female cells lacking Spen, the second X chromosome remained active. Other chromosomes in male and female cells also had stretches of DNA that became active upon Spen's removal. These DNA sequences, termed endogenous retroviruses, were remnants of ancestral viral infections. In other words, Spen normally acted as an antiviral defense. Analysis of genetic sequences showed that Spen recognized endogenous retrovirus sequences resembling a key region in Xist, a region which was needed for Xist to work properly. Inserting fragments of endogenous retroviruses into a defective version of Xist lacking this region also partially restored its ability to inactivate genes, suggesting that X chromosome silencing might work by hijacking cellular defenses against viruses. That is, female cells essentially 'pretend' there is a viral infection on the second X chromosome by coating it with Xist (which mimics endogenous retroviruses), thus directing Spen to shut it down. This research is an important step towards understanding how female cells carry out dosage compensation in mammals. More broadly, it sheds new light on how ancient viruses may have shaped the evolution of noncoding RNAs in the human genome.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Retrovirus Endógenos/genética , Células-Tronco Embrionárias Murinas/virologia , RNA Longo não Codificante/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Inativação do Cromossomo X , Cromossomo X , Animais , Sítios de Ligação , Linhagem Celular , Proteínas de Ligação a DNA/genética , Mecanismo Genético de Compensação de Dose , Retrovirus Endógenos/metabolismo , Feminino , Interações Hospedeiro-Patógeno , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Ligação Proteica , RNA Longo não Codificante/genética , RNA Viral/genética , Proteínas de Ligação a RNA/genética
18.
Adv Exp Med Biol ; 597: 48-59, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17633016

RESUMO

Tumor necrosis factor receptor associated factor 3 (TRAF3) is one of the most enigmatic members in the TRAF family that consists of six members, TRAF1 to 6. Despite its similarities with other TRAFs in terms of structure and protein-protein association, overexpression of TRAF3 does not induce activation of the commonly known TRAF-inducible signaling pathways, namely NF-kappaB and JNK. This lack of a simple functional assay in combination with the mysterious early lethality of the TRAF3-deficient mice has made the study of the biological function of TRAF3 challenging for almost ten years. Excitingly, TRAF3 has been identified recently to perform two seemingly distinct roles. Namely, TRAF3 functions as a negative regulator of the NF-kappaB pathway and separately, as a positive regulator of type I IFN production, placing itself as a critical regulator of both innate and adaptive immune responses.


Assuntos
Fator 3 Associado a Receptor de TNF/fisiologia , Animais , Humanos , Fator 3 Associado a Receptor de TNF/química , Fator 3 Associado a Receptor de TNF/deficiência , Fator 3 Associado a Receptor de TNF/metabolismo
19.
Dev Cell ; 43(2): 227-239.e5, 2017 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-28943242

RESUMO

Somatic progenitors sustain tissue self-renewal while suppressing premature differentiation. Protein arginine methyltransferases (PRMTs) affect many processes; however, their role in progenitor function is incompletely understood. PRMT1 was found to be the most highly expressed PRMT in epidermal progenitors and the most downregulated PRMT during differentiation. In targeted mouse knockouts and in long-term regenerated human mosaic epidermis in vivo, epidermal PRMT1 loss abolished progenitor self-renewal and led to premature differentiation. Mass spectrometry of the PRMT1 protein interactome identified the CSNK1a1 kinase, which also proved essential for progenitor maintenance. CSNK1a1 directly bound and phosphorylated PRMT1 to control its genomic targeting to PRMT1-sustained proliferation genes as well as PRMT1-suppressed differentiation genes. Among the latter were GRHL3, whose derepression was required for the premature differentiation seen with PRMT1 and CSNK1a1 loss. Maintenance of the progenitors thus requires cooperation by PRMT1 and CSNK1a1 to sustain proliferation gene expression and suppress premature differentiation driven by GRHL3.


Assuntos
Caseína Quinase Ialfa/metabolismo , Autorrenovação Celular/fisiologia , Células Epidérmicas , Queratinócitos/citologia , Proteína-Arginina N-Metiltransferases/fisiologia , Células-Tronco/citologia , Animais , Diferenciação Celular , Células Cultivadas , Epiderme/metabolismo , Humanos , Recém-Nascido , Queratinócitos/metabolismo , Camundongos , Camundongos Knockout , Fosforilação , Células-Tronco/metabolismo
20.
Nat Genet ; 48(1): 53-8, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26595770

RESUMO

Small nucleolar RNAs (snoRNAs) are conserved noncoding RNAs best studied as ribonucleoprotein (RNP) guides in RNA modification. To explore their role in cancer, we compared 5,473 tumor-normal genome pairs to identify snoRNAs with frequent copy number loss. The SNORD50A-SNORD50B snoRNA locus was deleted in 10-40% of 12 common cancers, where its loss was associated with reduced survival. A human protein microarray screen identified direct SNORD50A and SNORD50B RNA binding to K-Ras. Loss of SNORD50A and SNORD50B increased the amount of GTP-bound, active K-Ras and hyperactivated Ras-ERK1/ERK2 signaling. Loss of these snoRNAs also increased binding by farnesyltransferase to K-Ras and increased K-Ras prenylation, suggesting that KRAS mutation might synergize with SNORD50A and SNORD50B loss in cancer. In agreement with this hypothesis, CRISPR-mediated deletion of SNORD50A and SNORD50B in KRAS-mutant tumor cells enhanced tumorigenesis, and SNORD50A and SNORD50B deletion and oncogenic KRAS mutation co-occurred significantly in multiple human tumor types. SNORD50A and SNORD50B snoRNAs thus directly bind and inhibit K-Ras and are recurrently deleted in human cancer.


Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Proteínas ras/metabolismo , Animais , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Feminino , Deleção de Genes , Guanosina Trifosfato/metabolismo , Humanos , Camundongos Endogâmicos NOD , Mutação , Neoplasias/mortalidade , Prenilação , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/genética
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