RESUMEN
Temporally and spatially controlled accumulation underlies the functions of microRNAs (miRNAs) in various developmental processes. In Caenorhabditis elegans, this is exemplified by the temporal patterning miRNAs lin-4 and let-7, but for most miRNAs, developmental expression patterns remain poorly resolved. Indeed, experimentally observed long half-lives may constrain possible dynamics. Here, we profile miRNA expression throughout C. elegans postembryonic development at high temporal resolution, which identifies dynamically expressed miRNAs. We use mathematical models to explore the underlying mechanisms. For let-7, we can explain, and experimentally confirm, a striking stepwise accumulation pattern through a combination of rhythmic transcription and stage-specific regulation of precursor processing by the RNA-binding protein LIN-28. By contrast, the dynamics of several other miRNAs cannot be explained by regulation of production rates alone. Specifically, we show that a combination of oscillatory transcription and rhythmic decay drive rhythmic accumulation of miR-235, orthologous to miR-92 in other animals. We demonstrate that decay of miR-235 and additional miRNAs depends on EBAX-1, previously implicated in target-directed miRNA degradation (TDMD). Taken together, our results provide insight into dynamic miRNA decay and establish a resource to studying both the developmental functions of, and the regulatory mechanisms acting on, miRNAs.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Regulación del Desarrollo de la Expresión Génica , MicroARNs , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , MicroARNs/metabolismo , MicroARNs/genética , Proteínas Represoras , Estabilidad del ARN/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genéticaRESUMEN
C. elegans develops through four larval stages that are rhythmically terminated by molts, that is, the synthesis and shedding of a cuticular exoskeleton. Each larval cycle involves rhythmic accumulation of thousands of transcripts, which we show here relies on rhythmic transcription. To uncover the responsible gene regulatory networks (GRNs), we screened for transcription factors that promote progression through the larval stages and identified GRH-1, BLMP-1, NHR-23, NHR-25, MYRF-1, and BED-3. We further characterize GRH-1, a Grainyhead/LSF transcription factor, whose orthologues in other animals are key epithelial cell-fate regulators. We find that GRH-1 depletion extends molt durations, impairs cuticle integrity and shedding, and causes larval death. GRH-1 is required for, and accumulates prior to, each molt, and preferentially binds to the promoters of genes expressed during this time window. Binding to the promoters of additional genes identified in our screen furthermore suggests that we have identified components of a core molting-clock GRN. Since the mammalian orthologues of GRH-1, BLMP-1 and NHR-23, have been implicated in rhythmic homeostatic skin regeneration in mouse, the mechanisms underlying rhythmic C. elegans molting may apply beyond nematodes.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Ratones , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Muda/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación del Desarrollo de la Expresión Génica , Mamíferos/genéticaRESUMEN
Argonaute proteins are at the core of the microRNA-mediated gene silencing pathway essential for animals. In C. elegans, the microRNA-specific Argonautes ALG-1 and ALG-2 regulate multiple processes required for proper animal developmental timing and viability. Here we identified a phosphorylation site on ALG-1 that modulates microRNA association. Mutating ALG-1 serine 642 into a phospho-mimicking residue impairs microRNA binding and causes embryonic lethality and post-embryonic phenotypes that are consistent with alteration of microRNA functions. Monitoring microRNA levels in alg-1 phosphorylation mutant animals shows that microRNA passenger strands increase in abundance but are not preferentially loaded into ALG-1, indicating that the miRNA binding defects could lead to microRNA duplex accumulation. Our genetic and biochemical experiments support protein kinase A (PKA) KIN-1 as the putative kinase that phosphorylates ALG-1 serine 642. Our data indicate that PKA triggers ALG-1 phosphorylation to regulate its microRNA association during C. elegans development.
Asunto(s)
Proteínas de Caenorhabditis elegans , MicroARNs , Animales , Caenorhabditis elegans/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Fosforilación , Proteínas de Unión al ARN/metabolismo , Serina/metabolismoRESUMEN
Epithelial to mesenchymal transition (EMT) is a process in which epithelial cells lose cell polarity and cell-cell adhesion and gain migratory and invasive properties to become mesenchymal cells that are very vital for development, wound healing and stem cell behavior and contribute pathologically to fibrosis and cancer progression. miR21, a potent regulator of the tumor suppressor gene PTEN, can be silenced to reverse EMT, thereby providing an attractive target for abrogating the malignant behavior of breast cancer. Here, we report the design, synthesis and binding of a peptidic-aminoglycoside (PA) based chemical library against pre-miR21 that led to the identification of a group of small molecules that bind to pre-miR21 with high affinities and antagonize miR-21 maturation and function, thereby reversing EMT. The approach described here offers a promising miRNA targeting platform where such aminosugar conjugates can be similarly used to target other oncogenic miRNAs. Minor changes in the amino acid sequence allow us to tailor the binding effectiveness and downstream biological effects, thus making this approach a potentially tunable method of regulation of miRNA function.
RESUMEN
Originating as a component of prokaryotic adaptive immunity, the type II CRISPR/Cas9 system has been repurposed for targeted genome editing in various organisms. Although Cas9 can bind and cleave DNA efficiently under in vitro conditions, its activity inside a cell can vary dramatically between targets owing to the differences between genomic loci and the availability of enough Cas9/sgRNA (single guide RNA) complex molecules for cleavage. Most methods have so far relied on Cas9 protein engineering or base modifications in the sgRNA sequence to improve CRISPR/Cas9 activity. Here we demonstrate that a structure based rational design of sgRNAs can enhance the efficiency of Cas9 cleavage in vivo. By appending a naturally forming RNA G-quadruplex motif to the 3' end of sgRNAs we can improve its stability and target cleavage efficiency in zebrafish embryos without inducing off-target activity, thereby underscoring its value in the design of better and optimized genome editing triggers.
Asunto(s)
Proteínas Bacterianas/química , Sistemas CRISPR-Cas/genética , Endonucleasas/química , G-Cuádruplex , Edición Génica , ARN Guía de Kinetoplastida/genética , ARN/genética , Animales , Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR , Línea Celular , ADN/química , Embrión no Mamífero/metabolismo , Endonucleasas/metabolismo , Mutación INDEL , Secuencias Invertidas Repetidas , Ratones , ARN/química , Estabilidad del ARN , ARN Guía de Kinetoplastida/química , Pez CebraRESUMEN
miRNAs constitute an important layer of gene regulation mediated by sequence-specific targeting of mRNAs. Aberrant expression of miRNAs contributes to a host of pathological states. Promoting cancer, miR-21 is upregulated in variety of cancers and promotes tumor progresion by suppressing a network of tumor suppressor genes. Here we describe a novel class of bicyclic RNA analogues, selenomethylene-locked nucleic acid (SeLNA), that display high affinity, improved metabolic stability, and increased potency for miR-21 inhibition. The thermal stability (Tm) for duplexes was increased significantly with incorporation of SeLNA monomers as compared to that of the unmodified DNA-RNA hybrid. A comprehensive thermodynamic profile obtained by isothermal titration calorimetry revealed a favorable increase in the enthalpy of hybridization for SeLNA containing DNA and target RNA heteroduplexes. SeLNA modifications displayed remarkable binding affinity for miR-21 target RNA with a Ka of ≤1.05 × 108 M-1. We also observed enhanced serum stability for SeLNA-RNA duplexes with a half-life of ≤36 h. These in vitro results were well correlated with the antisense activity in cancer cells imparting up to â¼91% inhibition of miR-21. The functional impact of SeLNA modifications on miR-21 inhibition was further gauged by investigating the migration and invasion characterisitics of cancer cells, which were drastically reduced to â¼49 and â¼55%, respectively, with SeLNA having four such modifications. Our findings demonstrate SeLNA as a promising candidate for therapeutics for disease-associated miRNAs.
Asunto(s)
Movimiento Celular , Proliferación Celular , MicroARNs/antagonistas & inhibidores , Oligonucleótidos/química , Selenio/química , Apoptosis , Western Blotting , Dicroismo Circular , Humanos , Luciferasas/metabolismo , Células MCF-7 , MicroARNs/genética , Oligonucleótidos/administración & dosificación , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
miRNAs are highly conserved class of small ncRNAs whose involvement in human pathophysiologies is extensively investigated. MiR-21 is a well established oncogenic miRNA whose deregulation plays a significant role in onset and progression of cancer. The need of novel approaches to downregulate miR-21 is rapidly expanding. Potent inhibition of miR-21 is achieved by chemically modified 2'-O-methyl RNA oligonucleotide. The serinol capping at 3' and 5'ends and the interspersed 2'-O-(R-2-amino-3-methoxypropyl) uridine units enhance the nuclease resistance and efficacy of 2'-O-methyl RNA for the inhibition of miR-21. This represents a simple and novel modification for developing oligonucleotide-based therapeutics.
Asunto(s)
Antagomirs/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/antagonistas & inhibidores , Serina/química , Uridina/análogos & derivados , Antagomirs/síntesis química , Antagomirs/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Secuencia de Bases , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Genes Reporteros , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Células MCF-7 , Metilación , MicroARNs/genética , MicroARNs/metabolismo , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
BLUF domains are flavin-based photoreceptors which receive the blue light signal and are involved in the sensory transduction. We report a short BLUF photoreceptor (SnfB) in Stenotrophomonas sp. We have investigated photodynamic properties of C terminus truncated and several mutated SnfB proteins. Deletion of the extended C-terminal residues alters the thermal recovery kinetics and also affects the integrity of the SnfB protein. Mutagenesis studies demonstrated that the conserved residues within and outside the flavin-binding pocket also regulates the photocycle properties of the protein. These studies suggest that the C-terminal residues outside the BLUF domain can tune the photodynamic properties of the BLUF protein.
Asunto(s)
Proteínas Bacterianas/metabolismo , Stenotrophomonas/metabolismo , Proteínas Bacterianas/genética , Mutagénesis , Dominios Proteicos , Stenotrophomonas/genéticaRESUMEN
Aptamers, the chemical-antibody substitute to conventional antibodies, are primarily discovered through SELEX technology involving multi-round selections and enrichment. Circumventing conventional methodology, here we report an in silico selection of aptamers to estrogen receptor alpha (ERα) using RNA analogs of human estrogen response elements (EREs). The inverted repeat nature of ERE and the ability to form stable hairpins were used as criteria to obtain aptamer-alike sequences. Near-native RNA analogs of selected single stranded EREs were modelled and their likelihood to emerge as ERα aptamer was examined using AutoDock Vina, HADDOCK and PatchDock docking. These in silico predictions were validated by measuring the thermodynamic parameters of ERα -RNA interactions using isothermal titration calorimetry. Based on the in silico and in vitro results, we selected a candidate RNA (ERaptR4; 5'-GGGGUCAAGGUGACCCC-3') having a binding constant (Ka) of 1.02 ± 0.1 × 10(8) M(-1) as an ERα-aptamer. Target-specificity of the selected ERaptR4 aptamer was confirmed through cytochemistry and solid-phase immunoassays. Furthermore, stability analyses identified ERaptR4 resistant to serum and RNase A degradation in presence of ERα. Taken together, an efficient ERα-RNA aptamer is identified using a non-SELEX procedure of aptamer selection. The high-affinity and specificity can be utilized in detection of ERα in breast cancer and related diseases.
Asunto(s)
Aptámeros de Nucleótidos/genética , Receptor alfa de Estrógeno/genética , Elementos de Respuesta/genética , Técnica SELEX de Producción de Aptámeros , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Secuencia de Bases/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/terapia , Biología Computacional , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor alfa de Estrógeno/química , Receptor alfa de Estrógeno/metabolismo , Estrógenos/genética , Estrógenos/metabolismo , Femenino , Humanos , Secuencias Invertidas Repetidas/genética , Simulación del Acoplamiento Molecular , Unión ProteicaRESUMEN
MicroRNAs (miRNA) are small RNAs that have a regulatory role in gene expression. Because of this regulatory role, miRNAs have become a new target for therapeutic compounds. Here, we outline an approach to target specific miRNAs using a high throughput capable assay and a 215 compound peptidic-aminosugar (PA) library. Aminosugars have been shown in a number of recent reports as important lead compounds that bind miRNA. In order to screen for compounds that bind miRNA, we have developed a high throughput displacement assay using a fluorescein-neomycin conjugated molecule (F-neo) as a probe for competitive miRNA binding compounds. We have applied the F-neo assay to four different miRNA constructs and the assay is applicable to most miRNAs, at various stages of processing. The results of the screen were validated by the determination of the IC50 for a select group of compounds from the library. For example, we identified eight compounds that bind to hsa-miR 504 with higher affinity than the parent neomycin. From the F-neo displacement assay we found that the number of binding sites differs for each miRNA, and the binding sites appear to differ both physically and chemically, with different affinity of the compounds resulting from the size of the molecule as well as the chemical structure. Additionally, the affinity of the compounds was dependent on the identity and position of the amino acid position of conjugation and the affinity of the compounds relative to other compounds in the library was miRNA dependent with the introduction of a second amino acid.
Asunto(s)
Aminoglicósidos/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , MicroARNs/metabolismo , Biblioteca de Péptidos , Péptidos/metabolismo , Aminoácidos/metabolismo , Secuencia de Bases , Sitios de Unión , Fluoresceína/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Concentración 50 Inhibidora , Cinética , Células MCF-7 , MicroARNs/química , MicroARNs/genética , Datos de Secuencia Molecular , Neomicina/química , Neomicina/farmacología , Conformación de Ácido Nucleico , Unión Proteica/efectos de los fármacos , Procesamiento Postranscripcional del ARN/efectos de los fármacosRESUMEN
The discovery of microRNAs (miRNAs) has added a new dimension to the gene regulatory networks, making aberrantly expressed miRNAs as therapeutically important targets. Small molecules that can selectively target and modulate miRNA levels can thus serve as lead structures. Cationic cyclic peptides containing sugar amino acids represent a new class of small molecules that can target miRNA selectively. Upon treatment of these small molecules in breast cancer cell line, we profiled 96 therapeutically important miRNAs associated with cancer and observed that these peptides can selectively target paralogous miRNAs of the same seed family. This selective inhibition is of prime significance in cases when miRNAs of the same family have tissue-specific expression and perform different functions. During these conditions, targeting an entire miRNA family could lead to undesired adverse effects. The selective targeting is attributable to the difference in the three-dimensional structures of precursor miRNAs. Hence, the core structure of these peptides can be used as a scaffold for designing more potent inhibitors of miRNA maturation and hence function.
Asunto(s)
Aminoácidos/análisis , Carbohidratos/química , MicroARNs/química , Péptidos Cíclicos/farmacología , Precursores del ARN/química , Cationes , Humanos , Células MCF-7 , Péptidos Cíclicos/químicaRESUMEN
(S,S)- and (R,R)-ß,γ-Bis-substituted PNAs were synthesized from the C-2 symmetric vicinal diamine system embedded in 1,4 dihydroxybutane and 1,4-dimethoxybutane scaffolds. (R,R)-ß,γ-Bis-methoxymethyl-PNA derived from d-tartaric acid was found to be in the right configuration and conformation to be an excellent mimic of PNA, endowed with superior ability to enter into cells.
Asunto(s)
Células/metabolismo , ADN Complementario/química , Ácidos Nucleicos de Péptidos/metabolismo , ARN/química , Sitios de Unión , Células HCT116 , Humanos , Conformación de Ácido Nucleico , Ácidos Nucleicos de Péptidos/síntesis química , Ácidos Nucleicos de Péptidos/químicaRESUMEN
miRNAs are nodal regulators of gene expression and deregulation of miRNAs is causally associated with different diseases, including cancer. Modulation of miRNA expression is thus of therapeutic importance. Small molecules are currently being explored for their potential to downregulate miRNAs. Peptides have shown to have better potency and selectivity toward their targets but their potential in targeting and modulating miRNAs remain unexplored. Herein, using phage display we found a very selective peptide against pre-miR-21. Interestingly, the peptide has the potential to downregulate miR-21, by binding to pre-miR-21 and hindering Dicer processing. It is selective towards miR-21 inside the cell. By antagonising miR-21 function, the peptide is able to increase the expression of its target proteins and thereby increase apoptosis and suppress cell proliferation, invasion and migration. This peptide can further be explored for its anti-cancer activity in vivo and may be even extended to clinical studies.
Asunto(s)
Antineoplásicos/farmacología , MicroARNs/antagonistas & inhibidores , Péptidos/farmacología , Antineoplásicos/química , Antineoplásicos/metabolismo , Apoptosis , Sitios de Unión , Línea Celular , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Técnicas de Visualización de Superficie Celular , Células MCF-7 , MicroARNs/química , MicroARNs/metabolismo , Invasividad Neoplásica , Neoplasias/patología , Nucleótidos/química , Péptidos/química , Péptidos/metabolismo , Precursores del ARN/metabolismoRESUMEN
MicroRNAs (miRNAs) are a class of genomically encoded small RNA molecules (â¼22nts in length), which regulate gene expression post transcriptionally. The term microRNA or miRNA was coined in 2001, and research in the past decade has shed light on their widespread occurrence, evolutionary conservation and tissue specific functions. It is estimated that they modulate the gene expression of approximately 60% of the mammalian genes by regulating the levels of target mRNAs to which they can bind on the basis of sequence complementarities. miRNAs are produced in a well coordinated series of steps from being transcribed in the nucleus to exerting their function in the cytoplasm. miRNAs are now implicated in diverse biological phenomena ranging from development to stress response which makes miRNAs one of the central regulatory molecules which modulate information flow along the central dogma of gene expression. More importantly, like any regulatory molecule, deregulation of miRNAs is causally associated with several diseases (mainly cancer) and is directly involved in a variety of pathophysiologies owing to their aberrant expression. Thus, modulation of miRNA levels is of prime therapeutic importance. Conventional methods of miRNA knockdown using chemically modified antisense-oligonucleotides have been explored extensively but face the challenges of modes of delivery, biostability and biodistribution. This calls for the development of more alternative and non-conventional methods to target miRNA. Small molecules targeting RNA chemical and structural space provide one such timely opportunity. In this article we first provide a brief overview of miRNA biogenesis and its disease associations. We then summarize the major developments in conventional oligonucleotide based approaches to miRNA knockdown and its status. We then focus on the more non-conventional methods like oligonucleotide enzymes and small molecules and provide an outlook on the future of such methods.
Asunto(s)
Descubrimiento de Drogas/métodos , MicroARNs/antagonistas & inhibidores , Animales , Enfermedad/etiología , Enfermedad/genética , Humanos , MicroARNs/biosíntesis , MicroARNs/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/uso terapéuticoRESUMEN
miRNAs are important components of regulatory networks that control gene expression and have implications in various diseases including cancer. Targeting oncogenic miRNAs with small molecules is currently being explored to develop cancer therapeutics. Here, we report the development of dual binding neomycin-bisbenzimidazole conjugates that target oncogenic miR-27a with high affinity (Ka = 1.2 to 7.4 × 108 M-1). These conjugates bring significant reduction (â¼65% at 5 µM) in mature miRNA levels and penetrate easily in the cells where they localise both in the cytoplasm and the nucleus. Cell cycle analysis showed significant increase in the G0/G1 phase (â¼15%) and decrease in the S phase (â¼7%) upon treatment with neomycin-bisbenzimidazole conjugates, suggesting inhibition of cell proliferation. Using the conjugation approach, we show that moderately binding ligands can be covalently combined into high affinity binders. This study also highlights the role of linker optimization in designing high affinity ligands for miR-27a targeting.
RESUMEN
Three quinazoline based small molecules showed global upregulation of miRNA expression with a selective enrichment of tumor suppressor miRNAs. The target genes of the upregulated miRNAs were predicted to be enriched for apoptotic pathways. Apoptotic induction following treatment with quinazoline compounds was confirmed by in cellulo studies. Thus, these small molecules having the core structural moiety (2,4-diphenyl-quinazoline) can be used as scaffolds to design activators of miRNA expression paving the way for novel anti-cancer drugs.