RESUMO
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.
Assuntos
Senescência Celular , Epigenoma , Mamíferos , Metilação , Capuzes de RNA , RNA Mensageiro , Animais , Citosol/metabolismo , Proteína DEAD-box 58 , Perfilação da Expressão Gênica , Imunidade Inata , Mamíferos/genética , Mamíferos/metabolismo , Nucleotídeos/química , Nucleotídeos/genética , Nucleotídeos/metabolismo , Receptores Imunológicos , Análogos de Capuz de RNA/química , Análogos de Capuz de RNA/genética , Análogos de Capuz de RNA/metabolismo , Capuzes de RNA/química , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma , Fatores de TempoRESUMO
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.
Assuntos
Biossíntese de Proteínas , Vacinas , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análogos de Capuz de RNA/química , Células HEK293 , Fosfatos de DinucleosídeosRESUMO
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.
Assuntos
COVID-19 , Vacinas , COVID-19/prevenção & controle , Humanos , Análogos de Capuz de RNA/química , Análogos de Capuz de RNA/metabolismo , RNA Mensageiro/química , SARS-CoV-2 , Vacinas Sintéticas , Vacinas de mRNARESUMO
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.
Assuntos
Capuzes de RNA/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/química , Cristalografia , Metilação , Metiltransferases/química , Metiltransferases/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Análogos de Capuz de RNA/química , Análogos de Capuz de RNA/metabolismo , Capuzes de RNA/química , RNA Mensageiro/química , RNA Viral/química , S-Adenosil-Homocisteína/química , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Síncrotrons , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Naftalenossulfonato de Anilina/química , Bioensaio , Corantes Fluorescentes/química , Proteínas de Neoplasias/química , Proteínas de Ligação ao Cap de RNA/química , Capuzes de RNA/química , Proteínas de Ligação a RNA/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sítios de Ligação , Ligação Competitiva , Humanos , Ligação de Hidrogênio , Cinética , Simulação de Acoplamento Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Conformação Proteica , Análogos de Capuz de RNA/química , Análogos de Capuz de RNA/metabolismo , Proteínas de Ligação ao Cap de RNA/genética , Proteínas de Ligação ao Cap de RNA/metabolismo , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Espectrometria de Fluorescência , Eletricidade Estática , TermodinâmicaRESUMO
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.
Assuntos
Fator de Iniciação 4E em Eucariotos/genética , Guanina/análogos & derivados , Nucleotídeos/química , Fosfatos/química , Triptaminas/química , Endorribonucleases/metabolismo , Variação Genética , Guanina/química , Guanosina/análogos & derivados , Guanosina/química , Humanos , Modelos Moleculares , Proteínas do Tecido Nervoso/metabolismo , Motivos de Nucleotídeos , Nucleotídeos/genética , Biossíntese de Proteínas , Análogos de Capuz de RNA/química , Análogos de Capuz de RNA/genética , RNA Mensageiro/química , RNA Mensageiro/genéticaRESUMO
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.
Assuntos
Isoxazóis/química , Isoxazóis/farmacologia , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Análogos de Capuz de RNA/química , Análogos de Capuz de RNA/farmacologia , Animais , Desenho de Fármacos , Fator de Iniciação 4E em Eucariotos/metabolismo , Isoxazóis/síntese química , Camundongos , Simulação de Acoplamento Molecular , Análogos de Capuz de RNA/síntese química , CoelhosRESUMO
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.
Assuntos
Endorribonucleases/metabolismo , Pirofosfatases/metabolismo , Análogos de Capuz de RNA/metabolismo , Humanos , Hidrólise , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Análogos de Capuz de RNA/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Especificidade por SubstratoRESUMO
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.
Assuntos
Endorribonucleases/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Análogos de Capuz de RNA/farmacologia , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , Animais , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Fosfatos de Dinucleosídeos/química , Fosfatos de Dinucleosídeos/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Células HeLa , Humanos , Camundongos , Modelos Moleculares , Análogos de Capuz de RNA/química , Análogos de Capuz de RNA/metabolismo , Capuzes de RNA/química , Capuzes de RNA/efeitos dos fármacos , Capuzes de RNA/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismoRESUMO
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.
Assuntos
Fosfatos de Dinucleosídeos/química , Fosfatos de Dinucleosídeos/farmacologia , Desenho de Fármacos , Fator de Iniciação 4E em Eucariotos/química , Guanosina/análogos & derivados , Análogos de Capuz de RNA/química , Análogos de Capuz de RNA/farmacologia , RNA Polimerases Dirigidas por DNA/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Guanosina/química , Células HeLa , Humanos , Modelos Moleculares , Análogos de Capuz de RNA/síntese química , Transcrição Gênica/efeitos dos fármacos , Proteínas Virais/metabolismoRESUMO
A trimethylguanosine (TMG) cap is present at the 5' end of several small nuclear and nucleolar RNAs. Recently, it has been reported that the TMG cap is a potential nuclear import signal for nucleus-targeting therapeutic nucleic acids and proteins. The import is mediated by recognition of the TMG cap by the snRNA transporting protein, snurportin1. This work describes the synthesis and properties of a series of dinucleotide TMG cap (m3(2,2,7)GpppG) analogs modified in the 5',5'-triphosphate bridge as tools to study TMG cap-dependent biological processes. The bridge was altered at different positions by introducing either bridging (imidodiphosphate, O to NH and methylenebisphosphonate, O to CH2) or non-bridging (phosphorothioate, O to S and boranophosphate, O to BH3) modifications, or by elongation to tetraphosphate. The stability of novel analogs in blood serum was studied to reveal that the α,ß-bridging O to NH substitution (m3(2,2,7)GppNHpG) confers the highest resistance. Short RNAs capped with analogs containing α,ß-bridging (m3(2,2,7)GppNHpG) or ß-non-bridging (m3(2,2,7)GppSpG D2) modifications were resistant to decapping pyrophosphatase, hNudt16. Preliminary studies on binding by human snurportin1 revealed that both O to NH and O to S substitutions support this binding. Due to favorable properties in all three assays, m3(2,2,7)GppNHpG was selected as a promising candidate for further studies on the efficiency of the TMG cap as a nuclear import signal.
Assuntos
Transporte Ativo do Núcleo Celular , Guanosina/química , Polifosfatos/química , Análogos de Capuz de RNA/síntese química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Pirofosfatases/metabolismo , Análogos de Capuz de RNA/química , Proteínas de Ligação ao Cap de RNA/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de SinaisRESUMO
Modified mRNA cap analogs aid in the study of mRNA-related processes and may enable creation of novel therapeutic interventions. We report the synthesis and properties of 11 dinucleotide cap analogs bearing a single boranophosphate modification at either the α-, ß- or γ-position of the 5',5'-triphosphate chain. The compounds can potentially serve either as inhibitors of translation in cancer cells or reagents for increasing expression of therapeutic proteins in vivo from exogenous mRNAs. The BH3-analogs were tested as substrates and binding partners for two major cytoplasmic cap-binding proteins, DcpS, a decapping pyrophosphatase, and eIF4E, a translation initiation factor. The susceptibility to DcpS was different between BH3-analogs and the corresponding analogs containing S instead of BH3 (S-analogs). Depending on its placement, the boranophosphate group weakened the interaction with DcpS but stabilized the interaction with eIF4E. The first of the properties makes the BH3-analogs more stable and the second, more potent as inhibitors of protein biosynthesis. Protein expression in dendritic cells was 2.2- and 1.7-fold higher for mRNAs capped with m2 (7,2'-O)GppBH3pG D1 and m2 (7,2'-O)GppBH3pG D2, respectively, than for in vitro transcribed mRNA capped with m2 (7,3'-O)GpppG. Higher expression of cancer antigens would make mRNAs containing m2 (7,2'-O)GppBH3pG D1 and m2 (7,2'-O)GppBH3pG D2 favorable for anticancer immunization.
Assuntos
Boranos/química , Fosfatos/química , Inibidores da Síntese de Proteínas/química , Análogos de Capuz de RNA/química , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Células Dendríticas/metabolismo , Endorribonucleases/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Pirofosfatases/metabolismo , Análogos de Capuz de RNA/síntese química , Análogos de Capuz de RNA/metabolismo , Análogos de Capuz de RNA/farmacologia , EstereoisomerismoRESUMO
The translational efficiency and stability of synthetic mRNA in both cultured cells and whole animals can be improved by incorporation of modified cap structures at the 5'-end. mRNAs are synthesized in vitro by a phage RNA polymerase transcribing a plasmid containing the mRNA sequence in the presence of all four NTPs plus a cap dinucleotide. Modifications in the cap dinucleotide at the 2'- or 3'-positions of m(7)Guo, or modifications in the polyphosphate chain, can improve both translational efficiency and stability of the mRNA, thereby increasing the amount and duration of protein expression. In the context of RNA-based immunotherapy, the latter is especially important for antigen production and presentation by dendritic cells. Protocols are presented for synthesis of modified mRNAs, their introduction into cells and whole animals, and measurement of their translational efficiency and stability.
Assuntos
Biossíntese de Proteínas , Análogos de Capuz de RNA/química , Estabilidade de RNA , RNA Mensageiro , Transfecção/métodos , Animais , Humanos , RNA Mensageiro/síntese química , RNA Mensageiro/química , RNA Mensageiro/genéticaRESUMO
The eukaryotic initiation factor 4E (eIF4E) is the key component of the translational initiation complex that recruits mRNA by binding to a unique "cap" structure located at the 5' end of the mRNA. Overexpression of eIF4E has been implicated in the development of cancer, potentially as a result of increasing the cellular levels of proteins involved in processes that include proliferation and regulation of apoptosis. As a result, the cap-binding site of eIF4E has become a target for the development of anti-cancer therapeutics. The structure of eIF4E bound to the cap mimic 7-methyl-GDP revealed that two tryptophans from different loops in eIF4E sandwiched the 7-methylguanine group between them. This interaction gives rise to a strong exciton coupling signal between the two tryptophans that can be visualized by CD spectroscopy. eIF4E is a challenging protein to work with because of a propensity to aggregate under conditions used in biophysical techniques. CD spectroscopy provides a gentle, solution-based approach to study binding to the cap-binding site of eIF4E. Evidence is provided that the exciton coupling signal can be used to both qualitatively and quantitatively analyze the binding of cap analogs to eIF4E.
Assuntos
Dicroísmo Circular , Fator de Iniciação 4E em Eucariotos/metabolismo , Capuzes de RNA/metabolismo , Sítios de Ligação , Fator de Iniciação 4E em Eucariotos/química , Fator de Iniciação 4E em Eucariotos/genética , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Nucleotídeos/química , Nucleotídeos/metabolismo , Ligação Proteica , Redobramento de Proteína , Estrutura Terciária de Proteína , Análogos de Capuz de RNA/química , Análogos de Capuz de RNA/metabolismo , Capuzes de RNA/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Ribavirina/química , Ribavirina/metabolismo , Soluções/químicaRESUMO
Herein we describe the first simple and short method for specific labeling of mono- and trimethylated dinucleotide mRNA cap analogues with (13)C and (14)C isotopes. The labels were introduced within the cap structures either at the N7 for monomethylguanosine cap or N7 and N2 position for trimethylguanosine cap. The compounds designed for structural and biochemical studies will be useful tools for better understanding the role of the mRNA cap structures in pre-mRNA splicing, nucleocytoplasmic transport, translation initiation and mRNA degradation.
Assuntos
Fosfatos de Dinucleosídeos/química , Análogos de Capuz de RNA/síntese química , RNA Mensageiro/metabolismo , Isótopos de Carbono/química , Radioisótopos de Carbono/química , Marcação por Isótopo , Análogos de Capuz de RNA/química , Splicing de RNA , RNA Mensageiro/químicaRESUMO
Metazoan spliced leader (SL) trans-splicing generates mRNAs with an m(2,2,7)G-cap and a common downstream SL RNA sequence. The mechanism for eIF4E binding an m²²7G-cap is unknown. Here, we describe the first structure of an eIF4E with an m(2,2,7)G-cap and compare it to the cognate m7G-eIF4E complex. These structures and Nuclear Magnetic Resonance (NMR) data indicate that the nematode Ascaris suum eIF4E binds the two different caps in a similar manner except for the loss of a single hydrogen bond on binding the m(2,2,7)G-cap. Nematode and mammalian eIF4E both have a low affinity for m(2,2,7)G-cap compared with the m7G-cap. Nematode eIF4E binding to the m7G-cap, m(2,2,7)G-cap and the m(2,2,7)G-SL 22-nt RNA leads to distinct eIF4E conformational changes. Additional interactions occur between Ascaris eIF4E and the SL on binding the m(2,2,7)G-SL. We propose interactions between Ascaris eIF4E and the SL impact eIF4G and contribute to translation initiation, whereas these interactions do not occur when only the m(2,2,7)G-cap is present. These data have implications for the contribution of 5'-UTRs in mRNA translation and the function of different eIF4E isoforms.
Assuntos
Fator de Iniciação 4E em Eucariotos/química , Proteínas de Helminto/química , Iniciação Traducional da Cadeia Peptídica , Análogos de Capuz de RNA/química , Animais , Ascaris suum , Fosfatos de Dinucleosídeos/química , Fator de Iniciação 4E em Eucariotos/metabolismo , Proteínas de Helminto/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , RNA Líder para Processamento/químicaRESUMO
A method is described for the detection of certain nucleotide modifications adjacent to the 5' 7-methyl guanosine cap of mRNAs from individual genes. The method quantitatively measures the relative abundance of 2'-O-methyl and N(6),2'-O-dimethyladenosine, two of the most common modifications. In order to identify and quantitatify the amounts of N(6),2'-O-dimethyladenosine, a novel method for the synthesis of modified adenosine phosphoramidites was developed. This method is a one step synthesis and the product can directly be used for the production of N(6),2'-O-dimethyladenosine containing RNA oligonucleotides. The nature of the cap-adjacent nucleotides were shown to be characteristic for mRNAs from individual genes transcribed in liver and testis.
Assuntos
Análogos de Capuz de RNA/síntese química , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Adenosina/química , Animais , Sequência de Bases , Cromatografia em Camada Fina , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Análogos de Capuz de RNA/química , Análogos de Capuz de RNA/genética , RNA Mensageiro/genética , Testículo/metabolismoRESUMO
The first example of the synthesis and biological evaluation of a new analog containing 2'-OH modification on m(7)G moiety, that is, m(7,2'-)(O-Allyl)GpppG is reported. The effect of the 2'-O-allyl substitution on cap analog has been evaluated with respect to its in vitro transcription by using T7 RNA polymerase, capping efficiency, and translational activity. The gel shift assay indicates that the new cap analog has 59% capping efficiency whereas the standard cap analog, m(7)GpppG has a capping efficiency of 70%. The capping efficiency experiment clearly demonstrates that the new analog was a substrate for T7 RNA polymerase. The nature of the orientation has been determined by HPLC that reveals that the new analog incorporates exclusively in the forward orientation. It is noteworthy that the mRNA poly(A) capped with 2'-O-allyl substituted cap analog was translated â¼1.7-fold more efficiently than the mRNA capped with standard cap analog. Based on the higher translational data compared to the standard cap analog, it is likely that the new analog may find application that utilize mRNA transfection such as protein production, anti-cancer immunization, and gene therapy.
Assuntos
Fosfatos de Dinucleosídeos/química , Biossíntese de Proteínas/efeitos dos fármacos , Análogos de Capuz de RNA/síntese química , RNA Mensageiro/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Fosfatos de Dinucleosídeos/síntese química , Fosfatos de Dinucleosídeos/farmacologia , Desenho de Fármacos , Células HeLa , Humanos , Análogos de Capuz de RNA/química , Análogos de Capuz de RNA/farmacologia , Proteínas Virais/metabolismoRESUMO
Vaccination with in vitro transcribed RNA coding for tumor antigens is considered a promising approach for cancer immunotherapy and has already entered human clinical testing. One of the basic objectives for development of RNA as a drug is the optimization of immunobioavailability of the encoded antigen in vivo. By analyzing the effect of different synthetic 5' mRNA cap analogs on the kinetics of the encoded protein, we found that m(2)(7,2'-O)Gpp(S)pG (beta-S-ARCA) phosphorothioate caps, in particular the D1 diastereoisomer, profoundly enhance RNA stability and translational efficiency in immature but not mature dendritic cells. Moreover, in vivo delivery of the antigen as beta-S-ARCA(D1)-capped RNA species is superior for protein expression and for efficient priming and expansion of naïve antigen-specific T cells in mice. Our findings establish 5' mRNA cap analogs as yet another module for tuning immunopharmacological properties of recombinant antigen-encoding RNA for vaccination purposes.
Assuntos
Vacinas Anticâncer/genética , Células Dendríticas/imunologia , Análogos de Capuz de RNA/química , Estabilidade de RNA , Apresentação de Antígeno , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/metabolismo , Células Dendríticas/metabolismo , Genes Reporter , Meia-Vida , Humanos , Luciferases/análise , Oligonucleotídeos Fosforotioatos/química , Biossíntese de Proteínas , RNA/química , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/metabolismoRESUMO
Cap analogues having differently methylated adenosine at 2' and N6 position, m(7)G(5')pppApG which is existed in plant mRNA (plant type), m(7)G(5')pppAmpG (animal type), m(7)G(5')pppm(6)AmpG (mammalian type) and m(7)G(5')pppm(6)ApG (unnnatural type), were synthesized. In order to clarify the function of these methyl groups, luciferase mRNAs having differently methylated adenosine at the 5'-terminus, were successfully prepared by in vitro transcription using the synthesized cap anologues. As the preliminary results of in vitro translation with rabbit reticulocyte lysate and luciferase assay, luciferase mRNA having the mammalian type of cap structure, m(7)G(5')pppm(6)AmpG, was most efficiently translated. In the case of m(7)G(5')pppApG (plant type) efficiency of translation was lowest.