RESUMO
Argonaute proteins use small RNAs to guide the silencing of complementary target RNAs in many eukaryotes. Although small RNA biogenesis pathways are well studied, mechanisms for removal of guide RNAs from Argonaute are poorly understood. Here we show that the Argonaute2 (Ago2) guide RNA complex is extremely stable, with a half-life on the order of days. However, highly complementary target RNAs destabilize the complex and significantly accelerate release of the guide RNA from Ago2. This "unloading" activity can be enhanced by mismatches between the target and the guide 5' end and attenuated by mismatches to the guide 3' end. The introduction of 3' mismatches leads to more potent silencing of abundant mRNAs in mammalian cells. These findings help to explain why the 3' ends of mammalian microRNAs (miRNAs) rarely match their targets, suggest a mechanism for sequence-specific small RNA turnover, and offer insights for controlling small RNAs in mammalian cells.
Assuntos
Proteínas Argonautas/genética , RNA Complementar/genética , Pareamento Incorreto de Bases , Linhagem Celular , Inativação Gênica , Células HEK293 , Meia-Vida , Humanos , MicroRNAs/genética , Complexo de Inativação Induzido por RNA/genética , Pequeno RNA não TraduzidoRESUMO
Gene expression in eukaryotes is subject to extensive regulation at posttranscriptional levels. One of the most important sites of control involves mRNA 3' untranslated regions (3'utrs), which are recognized by RNA-binding proteins (RBPs) and microRNAs (miRNAs). These factors greatly influence translational efficiency and stability of target mRNAs and often also determine their cellular localization. HuR, a ubiquitously expressed member of the ElaV family of RBPs, has been implicated in regulation of stability and translation of over one hundred mRNAs in mammalian cells. Recent data indicate that some of the effects of HuR can be explained by its interplay with miRNAs. Binding of HuR may suppress the inhibitory effect of mirNAs interacting with the 3'UTR and redirect the repressed mRNA to polysomes for active translation. However, HuR can also synergize with miRNAs. The finding that HuR is able to disengage miRNAs from the repressed mrNa, or render them inactive, provides evidence that miRNa regulation is much more dynamic then originally anticipated. In this chapter we review properties of HuR and describe examples of the cross-talk between the protein and miRNAs, with emphasis on response of the regulation to cellular stress.
Assuntos
Proteína Semelhante a ELAV 1 , MicroRNAs , Regiões 3' não Traduzidas , Animais , Proteínas ELAV , Humanos , MicroRNAs/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Gene expression in eukaryotes is subject to extensive regulation at posttranscriptional levels. One of the most important sites of control involves mRNA 3' untranslated regions (3'UTRs), which are recognized by RNA-binding proteins (RBPs) and microRNAs (miRNAs). These factors greatly influence translational efficiency and stability of target mRNAs and often also determine their cellular localization. HuR, a ubiquitously expressed member of the ELAV family of RBPs, has been implicated in regulation of stability and translation of over one hundred mRNAs in mammalian cells. Recent data indicate that some of the effects of HuR can be explained by its interplay with miRNAs. Binding of HuR may suppress the inhibitory effect of miRNAs interacting with the 3'UTR and redirect the repressed mRNA to polysomes for active translation. However, HuR can also synergize with miRNAs. The finding that HuR is able to disengage miRNAs from the repressed mRNA, or render them inactive, provides evidence that miRNA regulation is much more dynamic then originally anticipated. In this chapter we review properties of HuR and describe examples of the cross-talk between the protein and miRNAs, with emphasis on response of the regulation to cellular stress.
Assuntos
Antígenos de Superfície/fisiologia , MicroRNAs/fisiologia , Proteínas de Ligação a RNA/fisiologia , Proteínas Repressoras/fisiologia , Animais , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Regulação da Expressão Gênica , Genes myc , HumanosRESUMO
New and improved: The incorporation of a 6-chlorotryptophan (6-Cl-Trp) into a beta-peptide (M)-3(14) helix leads to a high-affinity hDM2 inhibitor, as demonstrated by fluorescence fluctuation analysis at single molecule resolution. When conjugated to penetratin, the newly derived hDM2 binder specifically inhibits tumour cell growth in vitro.
Assuntos
Peptídeos/metabolismo , Peptídeos/farmacologia , Proteínas de Ligação a RNA/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Animais , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Materiais Biomiméticos/farmacologia , Linhagem Celular Tumoral , Desenho de Fármacos , Humanos , Ligantes , Camundongos , Modelos Moleculares , Peptídeos/síntese química , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Proteína Supressora de Tumor p53/químicaRESUMO
RNA-ligand binding often depends crucially on the local RNA secondary structure at the binding site. We develop here a model that quantitatively predicts the effect of RNA secondary structure on effective RNA-ligand binding activities based on equilibrium thermodynamics and the explicit computations of partition functions for the RNA structures. A statistical test for the impact of a particular structural feature on the binding affinities follows directly from this approach. The formalism is extended to describing the effects of hybridizing small "modifier RNAs" to a target RNA molecule outside its ligand binding site. We illustrate the applicability of our approach by quantitatively describing the interaction of the mRNA stabilizing protein HuR with AU-rich elements. We discuss our model and recent experimental findings demonstrating the effectivity of modifier RNAs in vitro in the context of the current research activities in the field of non-coding RNAs. We speculate that modifier RNAs might also exist in nature; if so, they present an additional regulatory layer for fine-tuning gene expression that could evolve rapidly, leaving no obvious traces in the genomic DNA sequences.
Assuntos
Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Algoritmos , Animais , Sequência de Bases , Sítios de Ligação/genética , Humanos , Ligantes , Modelos Estatísticos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , RNA/química , RNA/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TermodinâmicaRESUMO
Chemical biology has emerged as a new scientific discipline to change the way scientists approach and study the interface between chemistry, biology, and physics. By integrating the knowledge base of the human genome with the power of diverse and flexible chemical technology platforms, the ultimate goal is to define the 'rules of engagement' for small molecules and their use in basic biology and in drug discovery. Herein, we highlight the current counterpoles of the chemical biology philosophy in the framework between conformational diversity and informational complexity. Expanding the growing molecular recognition information matrix into classification of diseases and immediate mechanistic in-vivo proof of concept models represent the next development phase in a field that, unlike any other due to its multidisciplinary nature, unifies basic scientists and drug discoverers.
Assuntos
Técnicas de Química Combinatória/métodos , Desenho de Fármacos , Animais , Sítios de Ligação/efeitos dos fármacos , Técnicas de Química Combinatória/instrumentação , Sistemas de Liberação de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Genômica , Humanos , FenótipoRESUMO
Steroid sulfatase is an enzyme that currently enjoys considerable interest as a potential drug target in the treatment of estrogen- and androgen-dependent diseases, in particular breast cancer. We have purified human steroid sulfatase to apparent homogeneity from recombinant Chinese hamster ovary cells, and we established an assay with a new fluorogenic substrate, 3,4-benzocoumarin-7-O-sulfate (1). Substrate 1 features a K(m) value of 22.5 microM, which is close to the value for the natural substrate dehydroepiandrosterone sulfate (26 microM) and much lower than the K(m) values of other synthetic substrates (276-736 microM). Importantly, the cleavage of substrate 1 can be monitored continuously during the enzymatic cleavage, since a change in fluorescence intensity is detectable at the pH where the enzyme is active; in contrast, all other synthetic substrates described so far require alkalization to reveal a measurable absorbance or fluorescence signal. The adaptation of the assay to the 96-well format allows continuous monitoring of multiple wells in a microplate fluorescence reader. Applications of the assay for the determination of IC(50) and K(i) values of novel steroid sulfatase inhibitors are presented. Most importantly the assay was transferred to the nanoscale format (1-microl assay volume) in 2080-well plates with confocal fluorescence detection. This miniaturization will permit screening with a minimum throughput of 20000 compounds per day. The system presented demonstrates that the confocal detection platform used for nanoscreening can be successfully adapted to assays for which conventional ultraviolet dyes like coumarins are necessary. This strongly broadens the application range of confocal readers in drug screening.
Assuntos
Cumarínicos/síntese química , Cumarínicos/farmacologia , Esteril-Sulfatase/análise , Algoritmos , Animais , Células CHO , Fenômenos Químicos , Físico-Química , Cricetinae , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Feminino , Fluorometria , Humanos , Indicadores e Reagentes , Cinética , Microscopia Confocal , Nanotecnologia , Fotoquímica , Espectrofotometria Ultravioleta , Esteril-Sulfatase/antagonistas & inibidores , Raios UltravioletaRESUMO
Hu-antigen R (HuR) is a ubiquitous RNA-binding protein that comprises three RNA recognition motifs (RRMs). The first two tandem RRMs are known to bind to AU-rich elements (AREs) in the 3'-untranslated region of many mRNAs. The third RRM is connected to the second RRM through a basic hinge region that contains a localization signal termed HuR nucleocytoplasmic shuttling. Binding of HuR to the ARE in the 3'-untranslated region of mRNA leads to nuclear export, stabilization, and/or translational de-repression of the mRNA, resulting in upregulation of the encoded protein. Among the various ARE binding proteins known to date, HuR is still the only known ubiquitous antagonist of posttranscriptional gene silencing by AREs. Given the wide repertoire of known and suspected targets of HuR, it is considered to be a central node in the ARE pathway. Here, the x-ray crystal structure of the first RRM of HuR (amino acids 18-99) at 2.0 A resolution is presented. The overall fold consists of two alpha-helices and a four-stranded beta-sheet, with a beta1-alpha1-beta2-beta3-alpha2-beta4 topology and a beta-hairpin between alpha2 and beta4. The asymmetric unit consists of four chains. The large crystal contact interfaces observed between chains A/B and C/D contain hydrophobic residues located at the alpha-helix side of the fold, opposite to the RNA-binding interface. This hydrophobic region structurally resembles the protein-protein interaction site of RRM domains of other proteins. Because the nature of the assumed HuR homodimerization is mechanistically not well understood to date, we used site-directed mutagenesis, analytical size-exclusion chromatography and multiangle light scattering to investigate HuR interactions via the RRM hydrophobic region. Our data indicate that in vitro, HuR RRM1 and RRM1,2 homodimerization involves a disulfide bond at cysteine 13. This homodimerization mode may have a functional significance in redox modulation of HuR activity in response to oxidative stress. Because HuR is involved in many diseases (e.g., cancer, cachexia, and inflammatory bowel disease), the presented structure may provide a basis for rational drug design.
Assuntos
Antígenos de Superfície/química , Antígenos de Superfície/fisiologia , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/fisiologia , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Regulação da Expressão Gênica , Humanos , Mutagênese Sítio-Dirigida , Oxirredução , Conformação Proteica , Multimerização Proteica , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
In eukaryotic cells, proteins and RNAs are transported between the nucleus and the cytoplasm by nuclear import and export receptors. Over the past decade, small molecules that inhibit the nuclear export receptor CRM1 have been identified, most notably leptomycin B. However, up to now no small molecule inhibitors of nuclear import have been described. Here we have used our automated confocal nanoscanning and bead picking method (CONA) for on-bead screening of a one-bead one-compound library to identify the first such import inhibitor, karyostatin 1A. Karyostatin 1A binds importin ß with high nanomolar affinity and specifically inhibits importin α/ß mediated nuclear import at low micromolar concentrations in vitro and in living cells, without perturbing transportin mediated nuclear import or CRM1 mediated nuclear export. Surface plasmon resonance binding experiments suggest that karyostatin 1A acts by disrupting the interaction between importin ß and the GTPase Ran. As a selective inhibitor of the importin α/ß import pathway, karyostatin 1A will provide a valuable tool for future studies of nucleocytoplasmic trafficking.
Assuntos
Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , beta Carioferinas/antagonistas & inibidores , beta Carioferinas/metabolismo , Células HeLa , Humanos , Ligação Proteica/efeitos dos fármacos , beta Carioferinas/química , Proteína ran de Ligação ao GTP/metabolismoRESUMO
Screening of one-bead one-compound libraries by incubating beads with fluorescently labeled target protein requires isolation and structure elucidation of a large number of primary hit beads. However, the potency of the identified ligands is only revealed after time consuming and expensive larger scale resynthesis and testing in solution. Often, many of the resynthesized compounds turn out to be weak target binders in solution due to large differences between surface and solution binding affinities. For an industry style high-throughput screening (HTS) process a high false positive rate is detrimental. We have therefore combined single bead and single molecule/single cell techniques into an integrated HTS process in which the picomole amount of substance contained on one isolated hit bead is sufficient for quality control, structure determination, and precise affinity determination to the target protein in solution.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Descoberta de Drogas/métodos , Fluorescência , Ligantes , Métodos , Microesferas , Técnicas de Sonda Molecular , Bibliotecas de Moléculas PequenasRESUMO
Posttranscriptional regulation and RNA metabolism have become central topics in the understanding of mammalian gene expression and cell signalling, with the 3' untranslated region emerging as the coordinating unit. The 3' untranslated region trans-acting factor Hu protein R (HuR) forms a central posttranscriptional pathway node bridging between AU-rich element-mediated processes and microRNA regulation. While (m)RNA control by HuR has been extensively characterized, the molecular mode of action still remains elusive. Here we describe the identification of the first RRM3 (RNA recognition motif 3) targeted low molecular weight HuR inhibitors from a one-bead-one-compound library screen using confocal nanoscanning. A further compound characterization revealed the presence of an ATP-binding pocket within HuR RRM3, associated with enzymatic activity. Centered around a metal-ion-coordinating DxD motif, the catalytic site mediates 3'-terminal adenosyl modification of non-polyadenylated RNA substrates by HuR. These findings suggest that HuR actively contributes to RNA modification and maturation and thereby shed an entirely new light on the role of HuR in RNA metabolism.
Assuntos
Antígenos de Superfície/metabolismo , RNA Nucleotidiltransferases/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Humanos , Metais/metabolismo , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
Careful regulation of mRNA half-lives is a fundamental mechanism allowing cells to quickly respond to changing environmental conditions. The mRNA-binding Hu proteins are important for stabilization of short-lived mRNAs. Here we describe the identification and mechanistic characterization of the first low-molecular-weight inhibitors for Hu protein R (HuR) from microbial broths (Actinomyces sp.): dehydromutactin (1), MS-444 (2) and okicenone (3). These compounds interfere with HuR RNA binding, HuR trafficking, cytokine expression and T-cell activation. A mathematical and experimental analysis of the compounds' mode of action suggests that HuR homodimerizes before RNA binding and that the compounds interfere with the formation of HuR dimers. Our results demonstrate the chemical drugability of HuR; to our knowledge HuR is the first example of a drugable protein within the Hu family. MS-444, dehydromutactin and okicenone may become valuable tools for studying HuR function. An assessment of HuR inhibition as a central node in malignant processes might open up new conceptual routes toward combatting cancer.
Assuntos
Antígenos de Superfície/química , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas de Ligação a RNA/química , Actinomyces/metabolismo , Antracenos/química , Anti-Infecciosos/química , Ligação Competitiva , Relação Dose-Resposta a Droga , Desenho de Fármacos , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Polarização de Fluorescência , Furanos/química , Humanos , Cinética , Modelos Teóricos , Dados de Sequência Molecular , Naftóis/química , Pironas/químicaRESUMO
Approximately 3 000 genes are regulated in a time-, tissue-, and stimulus-dependent manner by degradation or stabilization of their mRNAs. The process is mediated by interaction of AU-rich elements (AREs) in the mRNA's 3'-untranslated regions with trans-acting factors. AU-rich element-controlled genes of fundamentally different functional relevance depend for their activation on one positive regulator, HuR. Here we present a methodology to exploit this central regulatory process for specific manipulation of AU-rich element-controlled gene expression at the mRNA level. With a combination of single-molecule spectroscopy, computational biology, and molecular and cellular biochemistry, we show that mRNA recognition by HuR is dependent on the presentation of the sequence motif NNUUNNUUU in single-stranded conformation. The presentation of the HuR binding site in the mRNA secondary structure appears to act analogously to a regulatory on/off switch that specifically controls HuR access to mRNAs in cis. Based on this finding we present a methodology for manipulating ARE mRNA levels by actuating this conformational switch specifically in a target mRNA. Computationally designed oligonucleotides (openers) enhance the NNUUNNUUU accessibility by rearranging the mRNA conformation. Thereby they increase in vitro and endogenous HuR-mRNA complex formation which leads to specific mRNA stabilization (as demonstrated for TNFalpha and IL-2, respectively). Induced HuR binding both inside and outside the AU-rich element promotes functional IL-2 mRNA stabilization. This opener-induced mRNA stabilization mimics the endogenous IL-2 response to CD28 stimulation in human primary T-cells. We therefore propose that controlled modulation of the AU-rich element conformation by mRNA openers or closers allows message stabilization or destabilization in cis to be specifically triggered. The described methodology might provide a means for studying distinct pathways in a complex cellular network at the node of mRNA stability control. It allows ARE gene expression to be potentially silenced or boosted. This will be of particular value for drug-target validation, allowing the diseased phenotype to ameliorate or deteriorate. Finally, the mRNA openers provide a rational starting point for target-specific mRNA stability assays to screen for low-molecular-weight compounds acting as inhibitors or activators of an mRNA structure rearrangement.