RESUMEN
The trimethylguanosine (TMG) cap is a motif present inter alia at the 5' end of small nuclear RNAs, which are involved in RNA splicing. The TMG cap plays a crucial role in RNA processing and stability as it protects the RNA molecule from degradation by exonucleases and facilitates its export from the nucleus. Additionally, the TMG cap plays a role in the recognition of snRNA by snurportin, a protein that facilitates nuclear import. TMG cap analogs are used in biochemical experiments as molecular tools to substitute the natural TMG cap. To expand the range of available TMG-based tools, here we conjugated the TMG cap to Fluorescent Molecular Rotors (FMRs) to open the possibility of detecting protein-ligand interactions in vitro and, potentially, in vivo, particularly visualizing interactions with snurportin. Consequently, we report the synthesis of 34 differently modified TMG cap-FMR conjugates and their evaluation as molecular probes for snurportin. As FMRs we selected three GFP-like chromophores (derived from green fluorescent protein) and one julolidine derivative. The evaluation of binding affinities for snurportin showed unexpectedly a strong stabilizing effect for TMGpppG-derived dinucleotides containing the FMR at the 2'-O-position of guanosine. These newly discovered compounds are potent snurportin ligands with nanomolar KD (dissociation constant) values, which are two orders of magnitude lower than that of natural TMGpppG. The effect is diminished by â¼50-fold for the corresponding 3'-regioisomers. To deepen the understanding of the structure-activity relationship, we synthesized and tested FMR conjugates lacking the TMG cap moiety. These studies, supported by molecular docking, suggested that the enhanced affinity arises from additional hydrophobic contacts provided by the FMR moiety. The strongest snurportin ligand, which also gave the greatest fluorescence enhancement (Fm/F0) when saturated with the protein, were tested in living cells to detect interactions and visualize complexes by fluorescence lifetime monitoring. This approach has potential applications in the study of RNA processing and RNA-protein interactions.
Asunto(s)
Colorantes Fluorescentes , Guanosina , Ligandos , Humanos , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Guanosina/análogos & derivados , Guanosina/química , Guanosina/metabolismo , Análogos de Caperuza de ARN/química , Análogos de Caperuza de ARN/síntesis química , Análogos de Caperuza de ARN/metabolismo , Células HeLa , Estructura MolecularRESUMEN
Transposable elements are genomic parasites that expand within and spread between genomes1. PIWI proteins control transposon activity, notably in the germline2,3. These proteins recognize their targets through small RNA co-factors named PIWI-interacting RNAs (piRNAs), making piRNA biogenesis a key specificity-determining step in this crucial genome immunity system. Although the processing of piRNA precursors is an essential step in this process, many of the molecular details remain unclear. Here, we identify an endoribonuclease, precursor of 21U RNA 5'-end cleavage holoenzyme (PUCH), that initiates piRNA processing in the nematode Caenorhabditis elegans. Genetic and biochemical studies show that PUCH, a trimer of Schlafen-like-domain proteins (SLFL proteins), executes 5'-end piRNA precursor cleavage. PUCH-mediated processing strictly requires a 7-methyl-G cap (m7G-cap) and a uracil at position three. We also demonstrate how PUCH interacts with PETISCO, a complex that binds to piRNA precursors4, and that this interaction enhances piRNA production in vivo. The identification of PUCH concludes the search for the 5'-end piRNA biogenesis factor in C. elegans and uncovers a type of RNA endonuclease formed by three SLFL proteins. Mammalian Schlafen (SLFN) genes have been associated with immunity5, exposing a molecular link between immune responses in mammals and deeply conserved RNA-based mechanisms that control transposable elements.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Endorribonucleasas , ARN de Interacción con Piwi , Animales , Proteínas Argonautas/metabolismo , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Elementos Transponibles de ADN/genética , Endorribonucleasas/química , Endorribonucleasas/metabolismo , Holoenzimas/química , Holoenzimas/metabolismo , ARN de Interacción con Piwi/química , ARN de Interacción con Piwi/genética , ARN de Interacción con Piwi/metabolismo , Análogos de Caperuza de ARN/química , Análogos de Caperuza de ARN/metabolismoRESUMEN
The mRNA cap structure is a major site of dynamic mRNA methylation. mRNA caps exist in either the Cap1 or Cap2 form, depending on the presence of 2'-O-methylation on the first transcribed nucleotide or both the first and second transcribed nucleotides, respectively1,2. However, the identity of Cap2-containing mRNAs and the function of Cap2 are unclear. Here we describe CLAM-Cap-seq, a method for transcriptome-wide mapping and quantification of Cap2. We find that unlike other epitranscriptomic modifications, Cap2 can occur on all mRNAs. Cap2 is formed through a slow continuous conversion of mRNAs from Cap1 to Cap2 as mRNAs age in the cytosol. As a result, Cap2 is enriched on long-lived mRNAs. Large increases in the abundance of Cap1 leads to activation of RIG-I, especially in conditions in which expression of RIG-I is increased. The methylation of Cap1 to Cap2 markedly reduces the ability of RNAs to bind to and activate RIG-I. The slow methylation rate of Cap2 allows Cap2 to accumulate on host mRNAs, yet ensures that low levels of Cap2 occur on newly expressed viral RNAs. Overall, these results reveal an immunostimulatory role for Cap1, and that Cap2 functions to reduce activation of the innate immune response.
Asunto(s)
Senescencia Celular , Epigenoma , Mamíferos , Metilación , Caperuzas de ARN , ARN Mensajero , Animales , Citosol/metabolismo , Proteína 58 DEAD Box , Perfilación de la Expresión Génica , Inmunidad Innata , Mamíferos/genética , Mamíferos/metabolismo , Nucleótidos/química , Nucleótidos/genética , Nucleótidos/metabolismo , Receptores Inmunológicos , Análogos de Caperuza de ARN/química , Análogos de Caperuza de ARN/genética , Análogos de Caperuza de ARN/metabolismo , Caperuzas de ARN/química , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcriptoma , Factores de TiempoRESUMEN
mRNA-based vaccines are relatively new technologies that have been in the field of interest of research centers and pharmaceutical companies in recent years. Such therapeutics are an attractive alternative for DNA-based vaccines since they provide material that can be used with no risk of genomic integration. Additionally, mRNA can be quite easily engineered to introduce modifications for different applications or to modulate its properties, for example, to increase translational efficiency or stability, which is not available for DNA vectors. Here, we describe the use of N2 modified dinucleotide cap analogs as components of mRNA transcripts. The compounds obtained showed very promising biological properties while incorporated into mRNA. The presented N2-guanine modifications within the cap structure ensure proper attachment of the dinucleotide to the transcripts in the IVT reaction, guarantees their incorporation only in the correct orientation, and enables highly efficient translation of mRNA both in the in vitro translation system and in human HEK293 cells.
Asunto(s)
Biosíntesis de Proteínas , Vacunas , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análogos de Caperuza de ARN/química , Células HEK293 , Fosfatos de DinucleósidosRESUMEN
The recent FDA approval of the mRNA vaccine for severe acute respiratory syndrome coronavirus (SARS-CoV-2) emphasizes the importance of mRNA as a powerful tool for therapeutic applications. The chemically modified mRNA cap analogs contain a unique cap structure, m7 G[5']ppp[5']N (where N=G, A, C or U), present at the 5'-end of many eukaryotic cellular and viral RNAs and several non-coding RNAs. The chemical modifications on cap analog influence orientation's nature, translational efficiency, nuclear stability, and binding affinity. The recent invention of a trinucleotide cap analog provides groundbreaking research in the area of mRNA analogs. Notably, trinucleotide cap analogs outweigh dinucleotide cap analogs in terms of capping efficiency and translational properties. This review focuses on the recent development in the synthesis of various dinucleotide cap analogs such as dinucleotide containing a triazole moiety, phosphorothiolate cap, biotinylated cap, cap analog containing N1 modification, cap analog containing N2 modification, dinucleotide containing fluorescence probe and TAT, bacterial caps, and trinucleotide cap analogs. In addition, the biological applications of these novel cap analogs are delineated.
Asunto(s)
COVID-19 , Vacunas , COVID-19/prevención & control , Humanos , Análogos de Caperuza de ARN/química , Análogos de Caperuza de ARN/metabolismo , ARN Mensajero/química , SARS-CoV-2 , Vacunas Sintéticas , Vacunas de ARNmRESUMEN
The genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus has a capping modification at the 5'-untranslated region (UTR) to prevent its degradation by host nucleases. These modifications are performed by the Nsp10/14 and Nsp10/16 heterodimers using S-adenosylmethionine as the methyl donor. Nsp10/16 heterodimer is responsible for the methylation at the ribose 2'-O position of the first nucleotide. To investigate the conformational changes of the complex during 2'-O methyltransferase activity, we used a fixed-target serial synchrotron crystallography method at room temperature. We determined crystal structures of Nsp10/16 with substrates and products that revealed the states before and after methylation, occurring within the crystals during the experiments. Here we report the crystal structure of Nsp10/16 in complex with Cap-1 analog (m7GpppAm2'-O). Inhibition of Nsp16 activity may reduce viral proliferation, making this protein an attractive drug target.
Asunto(s)
Caperuzas de ARN/metabolismo , ARN Mensajero/metabolismo , ARN Viral/metabolismo , SARS-CoV-2/química , Cristalografía , Metilación , Metiltransferasas/química , Metiltransferasas/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Análogos de Caperuza de ARN/química , Análogos de Caperuza de ARN/metabolismo , Caperuzas de ARN/química , ARN Mensajero/química , ARN Viral/química , S-Adenosilhomocisteína/química , S-Adenosilhomocisteína/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Sincrotrones , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Proteínas Reguladoras y Accesorias Virales/química , Proteínas Reguladoras y Accesorias Virales/metabolismoRESUMEN
The interferon-induced proteins with tetratricopeptide repeats (IFITs) are a family of RNA-binding proteins that are very highly expressed during antiviral response of immune system. IFIT proteins recognize and tightly bind foreign RNA particles. These are primarily viral RNAs ended with triphosphate at the 5' or lacking methylation of the first cap-proximal nucleotide but also in vitro transcribed RNA synthesized in the laboratory. Recognition of RNA by IFIT proteins leads to the formation of stable RNA/IFIT complexes and translational shut off of non-self transcripts. Here, we present a fluorescent-based assay to study the interaction between RNA molecules and IFIT family proteins. We have particularly focused on two representatives of this family: IFIT1 and IFIT5. We found a probe that competitively with RNA binds the positively charged tunnel in these IFIT proteins. The use of this probe for IFIT titration allowed us to evaluate the differences in binding affinities of mRNAs with different variants of 5' ends.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Naftalenosulfonatos de Anilina/química , Bioensayo , Colorantes Fluorescentes/química , Proteínas de Neoplasias/química , Proteínas de Unión a Caperuzas de ARN/química , Caperuzas de ARN/química , Proteínas de Unión al ARN/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sitios de Unión , Unión Competitiva , Humanos , Enlace de Hidrógeno , Cinética , Simulación del Acoplamiento Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Unión Proteica , Conformación Proteica , Análogos de Caperuza de ARN/química , Análogos de Caperuza de ARN/metabolismo , Proteínas de Unión a Caperuzas de ARN/genética , Proteínas de Unión a Caperuzas de ARN/metabolismo , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Espectrometría de Fluorescencia , Electricidad Estática , TermodinámicaRESUMEN
Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed in many cancers deregulating translational control of the cell cycle. mRNA 5' cap analogs targeting eIF4E are small molecules with the potential to counteract elevated levels of eIF4E in cancer cells. However, the practical utility of typical cap analogs is limited because of their reduced cell membrane permeability. Transforming the active analogs into their pronucleotide derivatives is a promising approach to overcome this obstacle. 7-Benzylguanosine monophosphate (bn7GMP) is a cap analog that has been successfully transformed into a cell-penetrating pronucleotide by conjugation of the phosphate moiety with tryptamine. In this work, we explored whether a similar strategy is applicable to other cap analogs, particularly phosphate-modified 7-methylguanine nucleotides. We report the synthesis of six new tryptamine conjugates containing N7-methylguanosine mono- and diphosphate and their analogs modified with thiophosphate moiety. These new potential pronucleotides and the expected products of their activation were characterized by biophysical and biochemical methods to determine their affinity towards eIF4E, their ability to inhibit translation in vitro, their susceptibility to enzymatic degradation and their turnover in cell extract. The results suggest that compounds containing the thiophosphate moiety may act as pronucleotides that release low but sustainable concentrations of 7-methylguanosine 5'-phosphorothioate (m7GMPS), which is a translation inhibitor with in vitro potency higher than bn7GMP.
Asunto(s)
Factor 4E Eucariótico de Iniciación/genética , Guanina/análogos & derivados , Nucleótidos/química , Fosfatos/química , Triptaminas/química , Endorribonucleasas/metabolismo , Variación Genética , Guanina/química , Guanosina/análogos & derivados , Guanosina/química , Humanos , Modelos Moleculares , Proteínas del Tejido Nervioso/metabolismo , Motivos de Nucleótidos , Nucleótidos/genética , Biosíntesis de Proteínas , Análogos de Caperuza de ARN/química , Análogos de Caperuza de ARN/genética , ARN Mensajero/química , ARN Mensajero/genéticaRESUMEN
Herein we describe a synthesis of new isoxazole-containing 5' mRNA cap analogues via a cycloaddition reaction. The obtained analogues show a capability to inhibit cap-dependent translation in vitro and are characterized by a new binding mode in which an isoxazolic ring, instead of guanine, is involved in the stacking effect. Our study provides valuable information toward designing new compounds that can be potentially used as anticancer therapeutics.
Asunto(s)
Isoxazoles/química , Isoxazoles/farmacología , Iniciación de la Cadena Peptídica Traduccional/efectos de los fármacos , Análogos de Caperuza de ARN/química , Análogos de Caperuza de ARN/farmacología , Animales , Diseño de Fármacos , Factor 4E Eucariótico de Iniciación/metabolismo , Isoxazoles/síntesis química , Ratones , Simulación del Acoplamiento Molecular , Análogos de Caperuza de ARN/síntesis química , ConejosRESUMEN
This article describes a simple, reliable, efficient, and general method for the synthesis of 7-methylguanosine nucleotides such as 7-methylguanosine 5'-O-monophosphate (m7 GMP), 7-methylguanosine 5'-O-diphosphate (m7 GDP), 7-methyl-2'-deoxyguanosine 5'-O-triphosphate (m7 2'dGTP), and 7-methylguanosine 5'-O-triphosphate (m7 GTP) starting from the corresponding guanosine nucleotide is described. The present protocol involves methylation reaction of guanosine nucleotide using dimethyl sulfate as a methylating agent and water as a solvent at room temperature to provide the corresponding 7-methylguanosine nucleotide in good yields with high purity (>99.5%). It is noteworthy that the present methylation reaction proceeds smoothly under aqueous conditions that is highly regioselective to afford exclusive 7-methylguanosine nucleotide. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Synthesis of 7-methylguanosine nucleotides.
Asunto(s)
Guanosina Difosfato/análogos & derivados , Análogos de Caperuza de ARN/síntesis química , Guanosina Difosfato/síntesis química , Guanosina Difosfato/química , Indicadores y Reactivos/química , Metilación , Análogos de Caperuza de ARN/química , Solventes/química , Ésteres del Ácido Sulfúrico/químicaRESUMEN
The mRNA 5' cap consists of N7-methylguanosine bound by a 5',5'-triphosphate bridge to the first nucleotide of the transcript. The cap interacts with various specific proteins and participates in all key mRNA-related processes, which may be of therapeutic relevance. There is a growing demand for new biophysical and biochemical methods to study cap-protein interactions and identify the factors which inhibit them. The development of such methods can be aided by the use of properly designed fluorescent molecular probes. Herein, we synthesized a new class of m7Gp3G cap derivatives modified with an alkyne handle at the N1-position of guanosine and, using alkyne-azide cycloaddition, we functionalized them with fluorescent tags to obtain potential probes. The cap derivatives and probes were evaluated in the context of two cap-binding proteins, eukaryotic translation initiation factor (eIF4E) and decapping scavenger (DcpS). Biochemical and biophysical studies revealed that N1-propargyl moiety did not significantly disturb cap-protein interaction. The fluorescent properties of the probes turned out to be in line with microscale thermophoresis (MST)-based binding assays.
Asunto(s)
Análogos de Caperuza de ARN/síntesis química , Proteínas de Unión a Caperuzas de ARN/metabolismo , Química Clic , Reacción de Cicloadición , Guanosina/química , Humanos , Análogos de Caperuza de ARN/química , Análogos de Caperuza de ARN/metabolismo , Proteínas de Unión a Caperuzas de ARN/químicaRESUMEN
Augmenting the mRNA translation efficiency and stability by replacing the standard 7-methylguanosine 5'-cap with properly designed analogues is a viable strategy for increasing the in vivo expression of proteins from exogenously delivered mRNA. However, the development of novel cap analogues with superior biological properties is hampered by the challenges associated with the synthesis of such highly modified nucleotides. To provide a simpler alternative to traditional methods for cap analogue preparation, we have recently proposed a click-chemistry-based strategy for the synthesis of dinucleotide cap analogues and identified several triazole-containing compounds with promising biochemical properties. Here, we further explored the concept of CuAAC-mediated cap synthesis by designing and studying 'second generation' triazole-modified caps, which were derived from the most promising 'first generation' compounds by modifying the oligophosphate chain length, altering the position of the triazole moiety, or replacing chemically labile P-N bonds with P-O bonds. The biochemical properties of the new analogues were evaluated by determining their affinity for eIF4E, susceptibility to hDcp2-catalysed decapping, and translation efficiencies in vitro and in cultured cells. The results led to identification of cap analogues that have superior translational properties compared to standard caps and the parent triazole-modified compounds as well as provided directions for future improvements.
Asunto(s)
Biosíntesis de Proteínas/efectos de los fármacos , Análogos de Caperuza de ARN/química , Análogos de Caperuza de ARN/farmacología , Triazoles/química , Diseño de Fármacos , Estabilidad de Medicamentos , Endorribonucleasas/metabolismo , Factor 4E Eucariótico de Iniciación/metabolismo , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Análogos de Caperuza de ARN/metabolismo , Estabilidad del ARN , ARN Mensajero/genéticaRESUMEN
Human Nudt16 (hNudt16) is a member of the Nudix family of hydrolases, comprising enzymes catabolizing various substrates including canonical (d)NTPs, oxidized (d)NTPs, nonnucleoside polyphosphates, and capped mRNAs. Decapping activity of the Xenopus laevis (X29) Nudt16 homolog was observed in the nucleolus, with a high specificity toward U8 snoRNA. Subsequent studies have reported cytoplasmic localization of mammalian Nudt16 with cap hydrolysis activity initiating RNA turnover, similar to Dcp2. The present study focuses on hNudt16 and its hydrolytic activity toward dinucleotide cap analogs and short capped oligonucleotides. We performed a screening assay for potential dinucleotide and oligonucleotide substrates for hNudt16. Our data indicate that dinucleotide cap analogs and capped oligonucleotides containing guanine base in the first transcribed nucleotide are more susceptible to enzymatic digestion by hNudt16 than their counterparts containing adenine. Furthermore, unmethylated dinucleotides (GpppG and ApppG) and respective oligonucleotides (GpppG-16nt and GpppA-16nt) were hydrolyzed by hNudt16 with greater efficiency than were m7GpppG and m7GpppG-16nt. In conclusion, we found that hNudt16 hydrolysis of dinucleotide cap analogs and short capped oligonucleotides displayed a broader spectrum specificity than is currently known.
Asunto(s)
Endorribonucleasas/metabolismo , Pirofosfatasas/metabolismo , Análogos de Caperuza de ARN/metabolismo , Humanos , Hidrólisis , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Análogos de Caperuza de ARN/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Especificidad por SustratoRESUMEN
The hydrolysis of nucleoside 5'-monophosphates to the corresponding nucleosides and inorganic phosphate is catalysed by 5'-nucleotidases, thereby contributing to the control of endogenous nucleotide turnover and affecting the fate of exogenously delivered nucleotide- and nucleoside-derived therapeutics in cells. A recently identified nucleotidase cNIIIB shows preference towards 7-methylguanosine monophosphate (m7GMP) as a substrate, which suggests its potential involvement in mRNA degradation. However, the extent of biological functions and the significance of cNIIIB remains to be elucidated. Here, we synthesised a series of m7GMP analogues carrying a 1,2,3-triazole moiety at the 5' position as the potential inhibitors of human cNIIIB. The compounds were synthesised by using the copper-catalysed azide-alkyne cycloaddition (CuAAC) between 5'-azido-5'-deoxy-7-methylguanosine and different phosphate or phosphonate derivatives carrying terminal alkyne. The analogues were evaluated as cNIIIB inhibitors using HPLC and malachite green assays, demonstrating that compound 1a, carrying a 1,2,3-triazoylphosphonate moiety, inhibits cNIIIB activity at micromolar concentrations (IC50 87.8⯱â¯7.5⯵M), while other analogues showed no activity. In addition, compound 1d was identified as an artifical substrate for HscNIIIB. Further characterization of inhibitor 1a revealed that it is poorly recognised by other m7G-binding proteins, eIF4E and DcpS, indicating its selectivity towards cNIIIB. The first inhibitor (1a) and unnatural substrate (1d) of cNIIIB, identified here, can be used as molecular probes for the elucidation of biological roles of cNIIIB, including the verification of its proposed function in mRNA metabolism.
Asunto(s)
5'-Nucleotidasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Análogos de Caperuza de ARN/farmacología , Triazoles/farmacología , 5'-Nucleotidasa/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Estructura Molecular , Análogos de Caperuza de ARN/síntesis química , Análogos de Caperuza de ARN/química , Relación Estructura-Actividad , Triazoles/químicaRESUMEN
Analogues of the mRNA 5'-cap are useful tools for studying mRNA translation and degradation, with emerging potential applications in novel therapeutic interventions including gene therapy. We report the synthesis of novel mono- and dinucleotide cap analogues containing dihalogenmethylenebisphosphonate moiety (i.e. one of the bridging O atom substituted with CCl2 or CF2) and their properties in the context of cellular translational and decapping machineries, compared to phosphate-unmodified and previously reported CH2-substituted caps. The analogues were bound tightly to eukaryotic translation initiation factor 4E (eIF4E), with CCl2-substituted analogues having the highest affinity. When incorporated into mRNA, the CCl2-substituted dinucleotide most efficiently promoted cap-dependent translation. Moreover, the CCl2-analogues were potent inhibitors of translation in rabbit reticulocyte lysate. The crystal structure of eIF4E in complex with the CCl2-analogue revealed a significantly different ligand conformation compared to that of the unmodified cap analogue, which likely contributes to the improved binding. Both CCl2- and CF2- analogues showed lower susceptibility to hydrolysis by the decapping scavenger enzyme (DcpS) and, when incorporated into RNA, conferred stability against major cellular decapping enzyme (Dcp2) to transcripts. Furthermore, the use of difluoromethylene cap analogues was exemplified by the development of 19F NMR assays for DcpS activity and eIF4E binding.
Asunto(s)
Endorribonucleasas/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Análogos de Caperuza de ARN/farmacología , Procesamiento Postranscripcional del ARN/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , Animales , Sitios de Unión/efectos de los fármacos , Cristalografía por Rayos X , Fosfatos de Dinucleósidos/química , Fosfatos de Dinucleósidos/metabolismo , Factor 4E Eucariótico de Iniciación/metabolismo , Células HeLa , Humanos , Ratones , Modelos Moleculares , Análogos de Caperuza de ARN/química , Análogos de Caperuza de ARN/metabolismo , Caperuzas de ARN/química , Caperuzas de ARN/efectos de los fármacos , Caperuzas de ARN/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismoAsunto(s)
Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Precursores del ARN/química , Precursores del ARN/metabolismo , ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/metabolismo , Secuencia de Bases , Cristalografía por Rayos X , Modelos Moleculares , Análogos de Caperuza de ARN/química , Análogos de Caperuza de ARN/metabolismo , Proteínas de Unión al ARN , Proteínas del Complejo SMN , Especificidad por SustratoRESUMEN
The 7-methylguanosine triphosphate cap present at the 5' ends of eukaryotic mRNAs plays numerous roles in mRNA expression and metabolism. The identification and studies on cap-binding partners can be significantly advanced using tailored chemical tools such as synthetic cap analogues or RNAs carrying modified cap structures. Here we provide protocols for the production of mRNAs specifically labeled within the 5' cap with a nucleoside capable of being photo-activated, either 6-thioguanosine or 7-methyl-6-thioguanosine, which can be used in photo-cross-linking experiments to identify or characterize cap-binding biomolecules. We also describe a protocol for the cross-linking experiments with capped RNAs to map histone H4 cap-binding pocket.
Asunto(s)
Guanosina/análogos & derivados , Análogos de Caperuza de ARN/metabolismo , ARN Mensajero/química , Tionucleósidos/metabolismo , Animales , Reactivos de Enlaces Cruzados , Guanosina/metabolismo , Histonas/metabolismo , Humanos , Estructura Molecular , Biosíntesis de Proteínas , Análogos de Caperuza de ARN/química , ARN Mensajero/metabolismo , Rayos UltravioletaRESUMEN
Fluorescent mRNA molecules offer a wide range of applications for studying capping/decapping reactions, translation, and other biophysical studies. Furthermore, fluorescent tags prove invaluable for tracking RNA molecules in cells. Here, we describe an efficient synthesis of a fluorescent cap analog, anthranioyl-GTP, its purification, and in vitro cap labeling of transcribed mRNA catalyzed by the recombinant vaccinia capping enzyme to produce anthranioyl-m(7)GpppG-capped RNA.
Asunto(s)
Análogos de Caperuza de ARN/síntesis química , ARN Mensajero/química , Guanosina/análogos & derivados , Guanosina/química , Estructura Molecular , Biosíntesis de Proteínas , Análogos de Caperuza de ARN/química , ARN Mensajero/genética , Espectrometría de Fluorescencia , Transcripción GenéticaRESUMEN
The RNA polymerase of influenza virus consists of three subunits: PA, PB1 and PB2. It uses a unique `cap-snatching' mechanism for the transcription of viral mRNAs. The cap-binding domain of the PB2 subunit (PB2cap) in the viral polymerase binds the cap of a host pre-mRNA molecule, while the endonuclease of the PA subunit cleaves the RNA 10-13 nucleotides downstream from the cap. The capped RNA fragment is then used as the primer for viral mRNA transcription. The structure of PB2cap from influenza virus H1N1 A/California/07/2009 and of its complex with the cap analog m(7)GTP were solved at high resolution. Structural changes are observed in the cap-binding site of this new pandemic influenza virus strain, especially the hydrophobic interactions between the ligand and the target protein. m(7)GTP binds deeper in the pocket than some other virus strains, much deeper than the host cap-binding proteins. Analysis of the new H1N1 structures and comparisons with other structures provide new insights into the design of small-molecule inhibitors that will be effective against multiple strains of both type A and type B influenza viruses.
Asunto(s)
Proteínas Virales/química , Antivirales/química , Dominio Catalítico , Cristalografía por Rayos X , Guanosina Trifosfato/química , Enlace de Hidrógeno , Subtipo H1N1 del Virus de la Influenza A/enzimología , Modelos Moleculares , Análogos de Caperuza de ARN/químicaRESUMEN
The first example of the synthesis of new dinucleotide cap analog containing propargyl group such as m(7,3'-O-propargyl)G[5']ppp[5']G is reported. The effect of propargyl cap analog with standard cap was evaluated with respect to their capping efficiency, in vitro T7 RNA polymerase transcription efficiency, and translation activity using cultured HeLa cells. It is noteworthy that propargyl cap analog outperforms standard cap by 3.1 fold in terms of translational properties. The propargyl cap analog forms a more stable complex with translation initiation factor eIF4E based on the molecular modeling studies.