RESUMO
DND1 is essential to maintain germ cell identity. Loss of Dnd1 function results in germ cell differentiation to teratomas in some inbred strains of mice or to somatic fates in zebrafish. Using our knock-in mouse line in which a functional fusion protein between DND1 and GFP is expressed from the endogenous locus (Dnd1GFP), we distinguished two male germ cell (MGC) populations during late gestation cell cycle arrest (G0), consistent with recent reports of heterogeneity among MGCs. Most MGCs express lower levels of DND1-GFP (DND1-GFP-lo), but some MGCs express elevated levels of DND1-GFP (DND1-GFP-hi). A RNA-seq time course confirmed high Dnd1 transcript levels in DND1-GFP-hi cells along with 5-10-fold higher levels for multiple epigenetic regulators. Using antibodies against DND1-GFP for RNA immunoprecipitation (RIP)-sequencing, we identified multiple epigenetic and translational regulators that are binding targets of DND1 during G0 including DNA methyltransferases (Dnmts), histone deacetylases (Hdacs), Tudor domain proteins (Tdrds), actin dependent regulators (Smarcs), and a group of ribosomal and Golgi proteins. These data suggest that in DND1-GFP-hi cells, DND1 hosts coordinating mRNA regulons that consist of functionally related and localized groups of epigenetic enzymes and translational components.
Assuntos
Espermatogônias , Peixe-Zebra , Animais , Feminino , Masculino , Camundongos , Gravidez , Cromatina/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Ligação a RNA/genética , Espermatogônias/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
The adult spermatogonial stem cell population arises from pluripotent primordial germ cells (PGCs) that enter the fetal testis around embryonic day (E)10.5. PGCs undergo rapid mitotic proliferation, then enter prolonged cell cycle arrest (G1/G0), during which they transition to pro-spermatogonia. In mice homozygous for the Ter mutation in the RNA-binding protein Dnd1 (Dnd1Ter/Ter ), many male germ cells (MGCs) fail to enter G1/G0 and instead form teratomas: tumors containing many embryonic cell types. To investigate the origin of these tumors, we sequenced the MGC transcriptome in Dnd1Ter/Ter mutants at E12.5, E13.5 and E14.5, immediately prior to teratoma formation, and correlated this information with DO-RIP-Seq-identified DND1 direct targets. Consistent with previous results, we found DND1 controls downregulation of many genes associated with pluripotency and active cell cycle, including mTor, Hippo and Bmp/Nodal signaling pathway elements. However, DND1 targets also include genes associated with male differentiation, including a large group of chromatin regulators activated in wild-type but not mutant MGCs during the E13.5 and E14.5 transition. Results suggest multiple DND1 functions and link DND1 to initiation of epigenetic modifications in MGCs.
Assuntos
Reprogramação Celular/genética , Epigênese Genética , Células Germinativas/citologia , Células Germinativas/metabolismo , Proteínas de Neoplasias/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Apoptose/genética , Sequência de Bases , Ciclo Celular/genética , Cromatina/metabolismo , Elementos de DNA Transponíveis/genética , Regulação para Baixo/genética , Embrião de Mamíferos/citologia , Feminino , Homozigoto , Masculino , Camundongos , Mutação/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/genética , Transcrição Gênica , Regulação para Cima/genéticaRESUMO
In vertebrates, the RNA-binding protein (RBP) dead end 1 (DND1) is essential for primordial germ cell (PGC) survival and maintenance of cell identity. In multiple species, Dnd1 loss or mutation leads to severe PGC loss soon after specification or, in some species, germ cell transformation to somatic lineages. Our investigations into the role of DND1 in PGC specification and differentiation have been limited by the absence of an available antibody. To address this problem, we used CRISPR/Cas9 gene editing to establish a transgenic mouse line carrying a DND1GFP fusion allele. We present imaging analysis of DND1GFP expression showing that DND1GFP expression is heterogeneous among male germ cells (MGCs) and female germ cells (FGCs). DND1GFP was detected in MGCs throughout fetal life but lost from FGCs at meiotic entry. In postnatal and adult testes, DND1GFP expression correlated with classic markers for the premeiotic spermatogonial population. Utilizing the GFP tag for RNA immunoprecipitation (RIP) analysis in MGCs validated this transgenic as a tool for identifying in vivo transcript targets of DND1. The DND1GFP mouse line is a novel tool for isolation and analysis of embryonic and fetal germ cells, and the spermatogonial population of the postnatal and adult testis.
Assuntos
Células Germinativas/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Neoplasias/genética , Alelos , Animais , Diferenciação Celular/genética , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Células Germinativas/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas de Neoplasias/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/genéticaRESUMO
RNA-binding proteins (RBPs) and noncoding RNAs orchestrate post-transcriptional processes through the recognition of specific sites on targeted transcripts. Thus, understanding the connection between binding to specific sites and active regulation of the whole transcript is essential. Many immunoprecipitation techniques have been developed that identify either whole transcripts or binding sites of RBPs on each transcript using cell lysates. However, none of these methods simultaneously measures the strength of each binding site and quantifies binding to whole transcripts. In this study, we compare current procedures and present digestion optimized (DO)-RIP-seq, a simple method that locates and quantifies RBP binding sites using a continuous metric. We have used the RBP HuR/ELAVL1 to demonstrate that DO-RIP-seq can quantify HuR binding sites with high coverage across the entire human transcriptome, thereby generating metrics of relative RNA binding strength. We demonstrate that this quantitative enrichment of binding sites is proportional to the relative in vitro binding strength for these sites. In addition, we used DO-RIP-seq to quantify and compare HuR's binding to whole transcripts, thus allowing for seamless integration of binding site data with whole-transcript measurements. Finally, we demonstrate that DO-RIP-seq is useful for identifying functional mRNA target sets and binding sites where combinatorial interactions between HuR and AGO-microRNAs regulate the fate of the transcripts. Our data indicate that DO-RIP-seq will be useful for quantifying RBP binding events that regulate dynamic biological processes.
Assuntos
Proteína Semelhante a ELAV 1/metabolismo , Perfilação da Expressão Gênica/métodos , MicroRNAs/genética , RNA Mensageiro/genética , Análise de Sequência de RNA/métodos , Sítios de Ligação , Bases de Dados Genéticas , Proteína Semelhante a ELAV 1/química , Regulação da Expressão Gênica , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ligação ProteicaRESUMO
RNA-binding proteins coordinate the fates of multiple RNAs, but the principles underlying these global interactions remain poorly understood. We elucidated regulatory mechanisms of the RNA-binding protein HuR, by integrating data from diverse high-throughput targeting technologies, specifically PAR-CLIP, RIP-chip, and whole-transcript expression profiling. The number of binding sites per transcript, degree of HuR association, and degree of HuR-dependent RNA stabilization were positively correlated. Pre-mRNA and mature mRNA containing both intronic and 3' UTR binding sites were more highly stabilized than transcripts with only 3' UTR or only intronic binding sites, suggesting that HuR couples pre-mRNA processing with mature mRNA stability. We also observed HuR-dependent splicing changes and substantial binding of HuR in polypyrimidine tracts of pre-mRNAs. Comparison of the spatial patterns surrounding HuR and miRNA binding sites provided functional evidence for HuR-dependent antagonism of proximal miRNA-mediated repression. We conclude that HuR coordinates gene expression outcomes at multiple interconnected steps of RNA processing.
Assuntos
Antígenos de Superfície/metabolismo , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Motivos de Aminoácidos , Antígenos de Superfície/química , Antígenos de Superfície/fisiologia , Sítios de Ligação , Biologia Computacional , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Regulação da Expressão Gênica , Células HEK293 , Humanos , MicroRNAs/metabolismo , MicroRNAs/fisiologia , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/fisiologia , SoftwareRESUMO
The 18-kb Xist long noncoding RNA (lncRNA) is essential for X-chromosome inactivation during female eutherian mammalian development. Global structural architecture, cell-induced conformational changes, and protein-RNA interactions within Xist are poorly understood. We used selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) to examine these features of Xist at single-nucleotide resolution both in living cells and ex vivo. The Xist RNA forms complex well-defined secondary structure domains and the cellular environment strongly modulates the RNA structure, via motifs spanning one-half of all Xist nucleotides. The Xist RNA structure modulates protein interactions in cells via multiple mechanisms. For example, repeat-containing elements adopt accessible and dynamic structures that function as landing pads for protein cofactors. Structured RNA motifs create interaction domains for specific proteins and also sequester other motifs, such that only a subset of potential binding sites forms stable interactions. This work creates a broad quantitative framework for understanding structure-function interrelationships for Xist and other lncRNAs in cells.
Assuntos
Conformação de Ácido Nucleico , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/genética , Acilação/genética , Animais , Feminino , Camundongos , Mutação , RNA Longo não Codificante/química , Proteínas de Ligação a RNA/química , Cromossomo X/genética , Inativação do Cromossomo X/genéticaRESUMO
BACKGROUND/AIMS: Myofibroblasts (MF) derived from quiescent nonfibrogenic hepatic stellate cells (HSC) are the major sources of fibrous matrix in cirrhosis. Because many factors interact to regulate expansion and regression of MF-HSC populations, efforts to prevent cirrhosis by targeting any one factor have had limited success, motivating research to identify mechanisms that integrate these diverse inputs. As key components of RNA regulons, RNA binding proteins (RBPs) may fulfill this function by orchestrating changes in the expression of multiple genes that must be coordinately regulated to affect the complex phenotypic modifications required for HSC transdifferentiation. METHODS: We profiled the transcriptomes of quiescent and MF-HSC to identify RBPs that were differentially-expressed during HSC transdifferentiation, manipulated the expression of the most significantly induced RBP, insulin like growth factor 2 binding protein 3 (Igf2bp3), and evaluated transcriptomic and phenotypic effects. RESULTS: Depleting Igf2bp3 changed the expression of thousands of HSC genes, including multiple targets of TGF-ß signaling, and caused HSCs to reacquire a less proliferative, less myofibroblastic phenotype. RNA immunoprecipitation assays demonstrated that some of these effects were mediated by direct physical interactions between Igf2bp3 and mRNAs that control proliferative activity and mesenchymal traits. Inhibiting TGF-ß receptor-1 signaling revealed a microRNA-dependent mechanism that induces Igf2bp3. CONCLUSIONS: The aggregate results indicate that HSC transdifferentiation is ultimately dictated by Igf2bp3-dependent RNA regulons and thus, can be controlled simply by manipulating Igf2bp3.
Assuntos
Transdiferenciação Celular , Regulação da Expressão Gênica , Células Estreladas do Fígado/citologia , Miofibroblastos/citologia , Proteínas de Ligação a RNA/genética , Transcriptoma , Animais , Células Cultivadas , Células Estreladas do Fígado/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Miofibroblastos/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Post-transcriptional regulation of gene expression by RNA binding proteins (RBPs) and non-coding RNAs plays an important role in global gene expression. Many post-transcriptional regulators are misexpressed and misregulated in cancers, resulting in altered programs of protein biosynthesis that can drive tumor progression. While comparative studies of several RBPs and microRNAs expressed in various cancer types have been reported, a model system that can be used to quantify RBP regulation and functional outcomes during the initiation and early stages of tumorigenesis is lacking. It was previously demonstrated that oncogenic transformation of normal human cells can be induced by expressing hTERT, p53DD, cyclin D1, CDK4R24C, C-MYCT58A and H-RASG12V. Here we describe a user-friendly method for generating this genetically defined model of step-wise tumorigenesis beginning with normal donor-derived human cells. This method immortalizes a donor's normal cells in about a week, reducing the chances of senescence. The entire stable system can be established in less than 12weeks. We then demonstrate the utility of such a system in elucidating the expression of multiple RBPs at an early step of tumor formation. We identify significant changes in the expression levels of transcripts encoding RBPs prior to transformation, suggesting that our described donor-derived isogenic system can provide insight about post-transcriptional regulation during the earliest stages of tumorigenesis in the context of diverse genetic backgrounds.
Assuntos
Carcinogênese/genética , Progressão da Doença , Neoplasias/genética , Processamento Pós-Transcricional do RNA/fisiologia , Proteínas de Ligação a RNA/genética , Carcinogênese/metabolismo , Linhagem Celular Transformada , Células HEK293 , Humanos , Neoplasias/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Post-transcriptional processes orchestrate gene expression through dynamic protein-RNA interactions. These interactions occur at specific sites determined by RNA sequence, secondary structure, or nucleotide modifications. Methods have been developed either to quantify binding of whole transcripts or to identify the binding sites, but there is none proven to quantify binding at both the whole transcript and binding site levels. Here we describe digestion optimized RNA immunoprecipitation with deep sequencing (DO-RIP-seq) as a method that quantitates at the whole transcript target (RIP-Seq-Like or RSL) level and at the binding site level (BSL) using continuous metrics. DO-RIP-seq methodology was developed using the RBP HuR/ELAVL1 as a test case (Nicholson et al., 2016). DO-RIP-seq employs treatment of cell lysates with a nuclease under optimized conditions to yield partially digested RNA fragments bound by RNA binding proteins, followed by immunoprecipitations that capture the digested RNA-protein complexes and assess non-specific or background interactions. Analyses of sequenced cDNA libraries made from the bound RNA fragments yielded two types of enrichment scores; one for RSL binding events and the other for BSL events (Nicholson et al., 2016). These analyses plus the extensive read coverage of DO-RIP-seq allows seamless integration of binding site and whole transcript information. Therefore, DO-RIP-seq is useful for quantifying RBP binding events that are regulated during dynamic biological processes.
Assuntos
Proteína Semelhante a ELAV 1/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Imunoprecipitação/métodos , Proteínas de Ligação a RNA/genética , RNA/química , Transcriptoma , Anticorpos/química , Sequência de Bases , Sítios de Ligação , Proteína Semelhante a ELAV 1/metabolismo , Biblioteca Gênica , Células HEK293 , Humanos , Conformação de Ácido Nucleico , Ligação Proteica , RNA/genética , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleases/química , Análise de Sequência de RNARESUMO
Global mRNA abundance depends on the balance of synthesis and decay of a population of mRNAs. To account for this balance during activation of T cells, we used metabolic labeling to quantify the contributions of RNA transcription and decay over a 4 h time course during activation of leukemia-derived Jurkat T cells. While prior studies suggested more than half of the changes in mRNA abundance were due to RNA stability, we found a smaller but more interesting population of mRNAs changed stability. These mRNAs clustered into functionally related subpopulations that included replicative histones, ribosomal biogenesis and cell motility functions. We then applied a novel analysis based on integrating global protein-RNA binding with concurrent changes in RNA stability at specific time points following activation. This analysis demonstrated robust stabilization of mRNAs by the HuR RNA-binding protein 4 h after activation. Our unexpected findings demonstrate that the temporal regulation of mRNA stability coordinates vital cellular pathways and is in part controlled by the HuR RNA binding protein in Jurkat T cells following activation.
Assuntos
Proteína Semelhante a ELAV 1/metabolismo , Ativação Linfocitária/genética , Estabilidade de RNA , RNA Mensageiro/genética , Linfócitos T/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Células Jurkat , Ativação Linfocitária/imunologia , RNA Mensageiro/metabolismo , Linfócitos T/imunologia , Transcrição GênicaRESUMO
We demonstrate that both Hepatitis C virus (HCV) and Bovine Viral Diarrhea virus (BVDV) contain regions in their 5' UTRs that stall and repress the enzymatic activity of the cellular 5'-3' exoribonuclease XRN1, resulting in dramatic changes in the stability of cellular mRNAs. We used biochemical assays, virus infections, and transfection of the HCV and BVDV 5' untranslated regions in the absence of other viral gene products to directly demonstrate the existence and mechanism of this novel host-virus interaction. In the context of HCV infection, we observed globally increased stability of mRNAs resulting in significant increases in abundance of normally short-lived mRNAs encoding a variety of relevant oncogenes and angiogenesis factors. These findings suggest that non-coding regions from multiple genera of the Flaviviridae interfere with XRN1 and impact post-transcriptional processes, causing global dysregulation of cellular gene expression which may promote cell growth and pathogenesis.
Assuntos
Regiões 5' não Traduzidas , Vírus da Diarreia Viral Bovina/patogenicidade , Exorribonucleases/metabolismo , Hepacivirus/patogenicidade , Interações Hospedeiro-Parasita/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Estabilidade de RNA/genética , Replicação Viral/genética , Regiões 5' não Traduzidas/genética , Animais , Western Blotting , Bovinos , Linhagem Celular , Vírus da Diarreia Viral Bovina/genética , Hepacivirus/genética , Humanos , Reação em Cadeia da Polimerase , RNA Mensageiro , TransfecçãoRESUMO
The cellular growth cycle is initiated and maintained by punctual, yet agile, regulatory events involving modifications of cell cycle proteins as well as coordinated gene expression to support cyclic checkpoint decisions. Recent evidence indicates that post-transcriptional partitioning of messenger RNA subsets by RNA-binding proteins help physically localize, temporally coordinate, and efficiently translate cell cycle proteins. This dynamic organization of mRNAs encoding cell cycle components contributes to the overall economy of the cell cycle consistent with the post-transcriptional RNA regulon model of gene expression. This review examines several recent studies demonstrating the coordination of mRNA subsets encoding cell cycle proteins during nuclear export and subsequent coupling to protein synthesis, and discusses evidence for mRNA coordination of p53 targets and the DNA damage response pathway. We consider how these observations may connect to upstream and downstream post-transcriptional coordination and coupling of splicing, export, localization, and translation. Published examples from yeast, nematode, insect, and mammalian systems are discussed, and we consider genetic evidence supporting the conclusion that dysregulation of RNA regulons may promote pathogenic states of growth such as carcinogenesis.
Assuntos
Ciclo Celular , Proliferação de Células , RNA Mensageiro/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Dano ao DNA , Expressão Gênica , Humanos , Interferência de RNA , Transporte de RNA , RNA Mensageiro/genética , RegulonRESUMO
UNLABELLED: The ability of CD8+ T cells to effectively limit HIV-1 replication and block HIV-1 acquisition is determined by the capacity to rapidly respond to HIV-1 antigens. Understanding both the functional properties and regulation of an effective CD8+ response would enable better evaluation of T cell-directed vaccine strategies and may inform the design of new therapies. We assessed the antigen specificity, cytokine signature, and mechanisms that regulate antiviral gene expression in CD8+ T cells from a cohort of HIV-1-infected virus controllers (VCs) (<5,000 HIV-1 RNA copies/ml and CD4+ lymphocyte counts of >400 cells/µl) capable of soluble inhibition of HIV-1. Gag p24 and Nef CD8+ T cell-specific soluble virus inhibition was common among the VCs and correlated with substantial increases in the abundance of mRNAs encoding the antiviral cytokines macrophage inflammatory proteins MIP-1α, MIP-1αP (CCL3L1), and MIP-1ß; granulocyte-macrophage colony-stimulating factor (GM-CSF); lymphotactin (XCL1); tumor necrosis factor receptor superfamily member 9 (TNFRSF9); and gamma interferon (IFN-γ). The induction of several of these mRNAs was driven through a coordinated response of both increased transcription and stabilization of mRNA, which together accounted for the observed increase in mRNA abundance. This coordinated response allows rapid and robust induction of mRNA messages that can enhance the CD8+ T cells' ability to inhibit virus upon antigen encounter. IMPORTANCE: We show that mRNA stability, in addition to transcription, is key in regulating the direct anti-HIV-1 function of antigen-specific memory CD8+ T cells. Regulation at the level of RNA helps enable rapid recall of memory CD8+ T cell effector functions for HIV-1 inhibition. By uncovering and understanding the mechanisms employed by CD8+ T cell subsets with antigen-specific anti-HIV-1 activity, we can identify new strategies for comprehensive identification of other important antiviral genes. This will, in turn, enhance our ability to inhibit virus replication by informing both cure strategies and HIV-1 vaccine designs that aim to reduce transmission and can aid in blocking HIV-1 acquisition.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Citocinas/biossíntese , Regulação da Expressão Gênica , Antígenos HIV/imunologia , HIV-1/imunologia , Biossíntese de Proteínas , Transcrição Gênica , Estudos de Coortes , Citocinas/genética , Perfilação da Expressão Gênica , Sobreviventes de Longo Prazo ao HIV , Humanos , RNA Mensageiro/análise , RNA Mensageiro/genéticaRESUMO
The ubiquitously expressed RNA-binding protein HuR increases the stability and translation of mRNAs encoding growth regulatory proteins that promote proliferation in a variety of cell types. However, the three neuron-specific ELAV/Hu proteins, HuB, HuC and HuD, while binding to the same types of mRNAs, are required instead for neuronal differentiation, and it becomes difficult to reconcile these contrary functions when all four Hu proteins are expressed in the same neuron. HuR mRNA exists as three alternatively polyadenylated variants, a 1.5-kb testes-specific mRNA isoform, a ubiquitous 2.4-kb isoform and a 6.0-kb isoform that we now show is induced during neuronal differentiation and appears to be neuron-specific. This 6.0-kb neuron-specific mRNA isoform is inherently less stable and produces less HuR protein than the ubiquitous 2.4-kb mRNA. Furthermore, we show that neuronal HuB, HuC and HuD, as well as HuR itself, can bind at the 2.4-kb mRNA polyadenylation site, and when overexpressed can affect alternative polyadenylation to generate an extended HuR 3'-UTR that is translationally suppressed. We propose that the regulation of HuR protein expression by alternative polyadenylation allows neurons to post-transcriptionally regulate mRNAs-encoding factors required for proliferation versus differentiation to facilitate neuronal differentiation.
Assuntos
Proteínas ELAV/genética , Neurogênese/genética , Neurônios/metabolismo , Poliadenilação , Regiões 3' não Traduzidas , Linhagem Celular , Proteínas ELAV/biossíntese , Proteínas ELAV/metabolismo , Regulação da Expressão Gênica , Humanos , Neurônios/citologia , Biossíntese de Proteínas , Isoformas de RNA/metabolismo , Estabilidade de RNARESUMO
Maintenance of genomic stability depends on the DNA damage response, a biologic barrier in early stages of cancer development. Failure of this response results in genomic instability and high predisposition toward lymphoma, as seen in patients with ataxia-telangiectasia mutated (ATM) dysfunction. ATM activates multiple cell-cycle checkpoints and DNA repair after DNA damage, but its influence on posttranscriptional gene expression has not been examined on a global level. We show that ionizing radiation modulates the dynamic association of the RNA-binding protein HuR with target mRNAs in an ATM-dependent manner, potentially coordinating the genotoxic response as an RNA operon. Pharmacologic ATM inhibition and use of ATM-null cells revealed a critical role for ATM in this process. Numerous mRNAs encoding cancer-related proteins were differentially associated with HuR depending on the functional state of ATM, in turn affecting expression of encoded proteins. The findings presented here reveal a previously unidentified role of ATM in controlling gene expression posttranscriptionally. Dysregulation of this DNA damage response RNA operon is probably relevant to lymphoma development in ataxia-telangiectasia persons. These novel RNA regulatory modules and genetic networks provide critical insight into the function of ATM in oncogenesis.
Assuntos
Proteínas de Ciclo Celular/genética , Dano ao DNA , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Linfócitos/metabolismo , Óperon/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Supressoras de Tumor/genética , Antígenos de Superfície/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Reparo do DNA , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Redes Reguladoras de Genes , Humanos , Linfoma/etiologia , Proteínas Mutantes , Ligação Proteica/efeitos da radiação , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Radiação IonizanteRESUMO
HuR emerged as a posttranscriptional regulator of mRNAs involved in cellular control, stress, and immunity but its role in governing such responses remains elusive. In this study, we assessed HuR's role in the staged progression of thymic T cell differentiation by means of its genetic ablation. Mice with an early deletion of HuR in thymocytes possess enlarged thymi but display a substantial loss of peripheral T cells. We show that this discordant phenotype related to specific defects in thymic cellular processes, which demonstrated HuR's involvement in: 1) intrinsic checkpoint signals suppressing the cell cycle of immature thymocyte progenitors, 2) TCR and antigenic signals promoting the activation and positive selection of mature thymocytes, 3) antigenic and death-receptor signals promoting thymocyte deletion, and 4) chemokine signals driving the egress of postselection thymocytes to the periphery. The cellular consequences of HuR's dysfunction were underlined by the aberrant expression of selective cell cycle regulators, TCR, and death-receptor signaling components. Our studies reveal the signal-dependent context of HuR's cellular activities in thymocytes and its importance in the generation of a physiological T cell pool.
Assuntos
Antígenos de Superfície/fisiologia , Proteínas de Ligação a RNA/fisiologia , Linfócitos T/citologia , Timo/imunologia , Animais , Proteínas de Ciclo Celular , Diferenciação Celular , Quimiocinas , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Camundongos , Receptores de Morte Celular , Células-Tronco/citologia , Timo/citologiaRESUMO
Transcriptomics is used to quantify changes in accumulated levels of mRNAs following cellular activation. These changes arise from the opposing fluxes of transcription and mRNA decay, both of which affect the functional dynamics of global gene expression. A study published recently in BMC Genomics focuses on the contribution made by mRNA stability in shaping the kinetics of gene responses in mammalian cells.
Assuntos
Estabilidade de RNA , RNA Mensageiro/genética , Transcrição Gênica , Animais , Humanos , RNA Mensageiro/metabolismoRESUMO
The process of mRNA localization typically utilizes cis-targeting elements and trans-recognition factors to direct the compartmental organization of translationally suppressed mRNAs. mRNA localization to the endoplasmic reticulum (ER), in contrast, occurs via a co-translational, signal sequence/signal recognition particle (SRP)-dependent mechanism. We have utilized cell fractionation/cDNA microarray analysis, shRNA-mediated suppression of SRP expression, and mRNA reporter construct studies to define the role of the SRP pathway in ER-directed mRNA localization. Cell fractionation studies of mRNA partitioning between the cytosol and ER demonstrated the expected enrichment of cytosolic/nucleoplasmic protein-encoding mRNAs and secretory/integral membrane protein-encoding mRNAs in the cytosol and ER fractions, respectively, and identified a subpopulation of cytosolic/nucleoplasmic protein-encoding mRNAs in the membrane-bound mRNA pool. The latter finding suggests a signal sequence-independent pathway of ER-directed mRNA localization. Extending from these findings, mRNA partitioning was examined in stable SRP54 shRNA knockdown HeLa cell lines. shRNA-directed reductions in SRP did not globally alter mRNA partitioning patterns, although defects in membrane protein processing were observed, further suggesting the existence of multiple pathways for mRNA localization to the ER. ER localization of GRP94-encoding mRNA was observed when translation was disabled by mutation of the start codon/insertion of a 5'UTR stem-loop structure or upon deletion of the encoded signal sequence. Combined, these data indicate that the mRNA localization to the ER can be conferred independent of the signal sequence/SRP pathway and suggest that mRNA localization to the ER may utilize cis-encoded targeting information.
Assuntos
Retículo Endoplasmático/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/metabolismo , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , Primers do DNA/genética , Células HeLa , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Biossíntese de Proteínas , Frações Subcelulares/metabolismoRESUMO
Although RNA-binding proteins (RBPs) coordinate many key decisions during cell growth and differentiation, the dynamics of RNA-RBP interactions have not been extensively studied on a global basis. We immunoprecipitated endogenous ribonucleoprotein complexes containing HuR and PABP throughout a T-cell activation time course and identified the associated mRNA populations using microarrays. We used Gaussian mixture modeling as a discriminative model, treating RBP association as a discrete variable (target or not target), and as a generative model, treating RBP-association as a continuous variable (probability of association). We report that HuR interacts with different populations of mRNAs during T-cell activation. These populations encode functionally related proteins that are members of the Wnt pathway and proteins mediating T-cell receptor signaling pathways. Moreover, the mRNA targets of HuR were found to overlap with the targets of other posttranscriptional regulatory factors, indicating combinatorial interdependence of posttranscriptional regulatory networks and modules after activation. Applying HuR mRNA dynamics as a quantitative phenotype in the drug-gene-phenotype Connectivity Map, we identified candidate small molecule effectors of HuR and T-cell activation. We show that one of these candidates, resveratrol, exerts T-cell activation-dependent posttranscriptional effects that are rescued by HuR. Thus, we describe a strategy to systematically link an RBP and condition-specific posttranscriptional effects to small molecule drugs.
Assuntos
Ativação Linfocitária/genética , RNA Mensageiro/análise , Linfócitos T/imunologia , Antígenos de Superfície/metabolismo , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Regulação da Expressão Gênica , Humanos , Células Jurkat , Cinética , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Ligação a Poli(A)/metabolismo , Proteínas de Ligação a RNA/metabolismo , Resveratrol , Ribonucleoproteínas/metabolismo , Estilbenos/farmacologiaRESUMO
Brain tumors are horrific diseases with almost universally fatal outcomes; new therapeutics are desperately needed and will come from improved understandings of glioma biology. Exosomes are endosomally derived 30-100 nm membranous vesicles released from many cell types into the extracellular milieu; surprisingly, exosomes are virtually unstudied in neuro-oncology. These microvesicles were used as vaccines in other tumor settings, but their immunological significance is unevaluated in brain tumors. Our purpose here is to report the initial biochemical, proteomic, and immunological studies on murine brain tumor exosomes, following known procedures to isolate exosomes. Our findings show that these vesicles have biophysical characteristics and proteomic profiles similar to exosomes from other cell types but that brain tumor exosomes have unique features (e.g., very basic isoelectric points, expressing the mutated tumor antigen EGFRvIII and the putatively immunosuppressive cytokine TGF-beta). Administration of such exosomes into syngeneic animals produced both humoral and cellular immune responses in immunized hosts capable of rejecting subsequent tumor challenges but failed to prolong survival in established orthotopic models. Control animals received saline or cell lysate vaccines and showed no antitumor responses. Exosomes and microvesicles isolated from sera of patients with brain tumors also possess EGFR, EGFRvIII, and TGF-beta. We conclude that exosomes released from brain tumor cells are biochemically/biophysically like other exosomes and have immune-modulating properties. They can escape the blood-brain barrier, with potential systemic and distal signaling and immune consequences.