RESUMEN
RNA capping is a prominent RNA modification that influences RNA stability, metabolism, and function. While it was long limited to the study of the most abundant eukaryotic canonical m7G cap, the field recently went through a large paradigm shift with the discovery of non-canonical RNA capping in bacteria and ultimately all domains of life. The repertoire of non-canonical caps has expanded to encompass metabolite caps, including NAD, FAD, CoA, UDP-Glucose, and ADP-ribose, alongside alarmone dinucleoside polyphosphate caps, and methylated phosphate cap-like structures. This review offers an introduction into the field, presenting a summary of the current knowledge about non-canonical RNA caps. We highlight the often still enigmatic biological roles of the caps together with their processing enzymes, focusing on the most recent discoveries. Furthermore, we present the methods used for the detection and analysis of these non-canonical RNA caps and thus provide an introduction into this dynamic new field.
Asunto(s)
Caperuzas de ARN , Caperuzas de ARN/metabolismo , Caperuzas de ARN/química , Humanos , Estabilidad del ARN , Animales , ARN/química , ARN/metabolismo , ARN/genética , Bacterias/genética , Bacterias/metabolismoRESUMEN
Nicotinamide adenine dinucleotide (NAD) is a critical component of the cellular metabolism and also serves as an alternative 5' cap on various RNAs. However, the function of the NAD RNA cap is still under investigation. We studied NAD capping of RNAs in HIV-1-infected cells because HIV-1 is responsible for the depletion of the NAD/NADH cellular pool and causing intracellular pellagra. By applying the NAD captureSeq protocol to HIV-1-infected and uninfected cells, we revealed that four snRNAs (e.g., U1) and four snoRNAs lost their NAD cap when infected with HIV-1. Here, we provide evidence that the presence of the NAD cap decreases the stability of the U1/HIV-1 pre-mRNA duplex. Additionally, we demonstrate that reducing the quantity of NAD-capped RNA by overexpressing the NAD RNA decapping enzyme DXO results in an increase in HIV-1 infectivity. This suggests that NAD capping is unfavorable for HIV-1 and plays a role in its infectivity.
Asunto(s)
Infecciones por VIH , VIH-1 , NAD , ARN Nuclear Pequeño , ARN Nucleolar Pequeño , Humanos , NAD/metabolismo , ARN Nucleolar Pequeño/metabolismo , ARN Nucleolar Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Infecciones por VIH/virología , Infecciones por VIH/metabolismo , Caperuzas de ARN/metabolismoRESUMEN
The recent expansion of the field of RNA chemical modifications has changed our understanding of post-transcriptional gene regulation. Apart from internal nucleobase modifications, 7-methylguanosine was long thought to be the only eukaryotic RNA cap. However, the discovery of non-canonical RNA caps in eukaryotes revealed a new niche of previously undetected RNA chemical modifications. We are the first to report the existence of a new non-canonical RNA cap, diadenosine tetraphosphate (Ap4 A), in human and rat cell lines. Ap4 A is the most abundant dinucleoside polyphosphate in eukaryotic cells and can be incorporated into RNA by RNA polymerases as a non-canonical initiating nucleotide (NCIN). Using liquid chromatography-mass spectrometry (LC-MS), we show that the amount of capped Ap4 A-RNA is independent of the cellular concentration of Ap4 A. A decapping enzyme screen identifies two enzymes cleaving Ap4 A-RNA,NUDT2 and DXO, both of which also cleave other substrate RNAs in vitro. We further assess the translatability and immunogenicity of Ap4 A-RNA and show that although it is not translated, Ap4 A-RNA is recognized as self by the cell and does not elicit an immune response, making it a natural component of the transcriptome. Our findings open a previously unexplored area of eukaryotic RNA regulation.
Asunto(s)
Fosfatos de Dinucleósidos , Caperuzas de ARN , Ratas , Animales , Humanos , Fosfatos de Dinucleósidos/metabolismo , Mamíferos/metabolismo , Hidrolasas Nudix , Monoéster Fosfórico HidrolasasRESUMEN
Recent discoveries of various noncanonical RNA caps, such as dinucleoside polyphosphates (Np n N), coenzyme A (CoA), and nicotinamide adenine dinucleotide (NAD) in all domains of life have led to a revision of views on RNA cap function and metabolism. Enzymes from the NudiX family capable of hydrolyzing a polyphosphate backbone attached to a nucleoside are the strongest candidates for degradation of noncanonically capped RNA. The model plant organism Arabidopsis thaliana encodes as many as 28 NudiX enzymes. For most of them, only in vitro substrates in the form of small molecules are known. In our study, we focused on four A. thaliana NudiX enzymes (AtNUDT6, AtNUDT7, AtNUDT19 and AtNUDT27), and we studied whether these enzymes can cleave RNA capped with Np n Ns (Ap2-5A, Gp3-4G, Ap3-5G, m7Gp3G, m7Gp3A), CoA, ADP-ribose, or NAD(H). While AtNUDT19 preferred NADH-RNA over other types of capped RNA, AtNUDT6 and AtNUDT7 preferentially cleaved Ap4A-RNA. The most powerful decapping enzyme was AtNUDT27, which cleaved almost all types of capped RNA at a tenfold lower concentration than the other enzymes. We also compared cleavage efficiency of each enzyme on free small molecules with RNA capped with corresponding molecules. We found that AtNUDT6 prefers free Ap4A, while AtNUDT7 preferentially cleaved Ap4A-RNA. These findings show that NudiX enzymes may act as RNA-decapping enzymes in A. thaliana and that other noncanonical RNA caps such as Ap4A and NADH should be searched for in plant RNA.
RESUMEN
The Picornavirales include viruses that infect vertebrates, insects, and plants. It was believed that they pack only their genomic mRNA in the particles; thus, we envisaged these viruses as excellent model systems for studies of mRNA modifications. We used LC-MS to analyze digested RNA isolated from particles of the sacbrood and deformed wing iflaviruses as well as of the echovirus 18 and rhinovirus 2 picornaviruses. Whereas in the picornavirus RNAs we detected only N6 -methyladenosine and 2'-O-methylated nucleosides, the iflavirus RNAs contained a wide range of methylated nucleosides, such as 1-methyladenosine (m1 A) and 5-methylcytidine (m5 C). Mapping of m1 A and m5 C through RNA sequencing of the SBV and DWV RNAs revealed the presence of tRNA molecules. Both modifications were detected only in tRNA. Further analysis revealed that tRNAs are present in form of 3' and 5' fragments and they are packed selectively. Moreover, these tRNAs are typically packed by other viruses.
Asunto(s)
Nucleósidos , ARN de Transferencia , Animales , Abejas/genética , ARN , ARN Mensajero , ARN de Transferencia/genética , Virión/genéticaRESUMEN
The ongoing COVID-19 pandemic exemplifies the general need to better understand viral infections. The positive single-strand RNA genome of its causative agent, the SARS coronavirus 2 (SARS-CoV-2), encodes all viral enzymes. In this work, we focused on one particular methyltransferase (MTase), nsp16, which, in complex with nsp10, is capable of methylating the first nucleotide of a capped RNA strand at the 2'-O position. This process is part of a viral capping system and is crucial for viral evasion of the innate immune reaction. In light of recently discovered non-canonical RNA caps, we tested various dinucleoside polyphosphate-capped RNAs as substrates for nsp10-nsp16 MTase. We developed an LC-MS-based method and discovered four types of capped RNA (m7Gp3A(G)- and Gp3A(G)-RNA) that are substrates of the nsp10-nsp16 MTase. Our technique is an alternative to the classical isotope labelling approach for the measurement of 2'-O-MTase activity. Further, we determined the IC50 value of sinefungin to illustrate the use of our approach for inhibitor screening. In the future, this approach may be an alternative technique to the radioactive labelling method for screening inhibitors of any type of 2'-O-MTase.
Asunto(s)
COVID-19/virología , Metiltransferasas/metabolismo , SARS-CoV-2/enzimología , Proteínas no Estructurales Virales/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Cromatografía Liquida , Regulación Viral de la Expresión Génica , Humanos , Espectrometría de Masas , Metilación , Metiltransferasas/genética , Caperuzas de ARN , ARN Viral/genética , SARS-CoV-2/genética , Especificidad por Sustrato , Proteínas no Estructurales Virales/genética , Proteínas Reguladoras y Accesorias Virales/genéticaRESUMEN
Chemical modifications of viral RNA are an integral part of the viral life cycle and are present in most classes of viruses. To date, more than 170 RNA modifications have been discovered in all types of cellular RNA. Only a few, however, have been found in viral RNA, and the function of most of these has yet to be elucidated. Those few we have discovered and whose functions we understand have a varied effect on each virus. They facilitate RNA export from the nucleus, aid in viral protein synthesis, recruit host enzymes, and even interact with the host immune machinery. The most common methods for their study are mass spectrometry and antibody assays linked to next-generation sequencing. However, given that the actual amount of modified RNA can be very small, it is important to pair meticulous scientific methodology with the appropriate detection methods and to interpret the results with a grain of salt. Once discovered, RNA modifications enhance our understanding of viruses and present a potential target in combating them. This review provides a summary of the currently known chemical modifications of viral RNA, the effects they have on viral machinery, and the methods used to detect them.
Asunto(s)
Procesamiento Postranscripcional del ARN , ARN Viral/genética , ARN Viral/metabolismo , Replicación Viral , Núcleo Celular/metabolismo , Humanos , ARN MensajeroRESUMEN
Dinucleoside polyphosphates (NpnNs) were discovered 50 years ago in all cells. They are often called alarmones, even though the molecular target of the alarm has not yet been identified. Recently, we showed that they serve as noncanonical initiating nucleotides (NCINs) and fulfill the role of 5' RNA caps in Escherichia coli. Here, we present molecular insight into their ability to be used as NCINs by T7 RNA polymerase in the initiation phase of transcription. In general, we observed NpnNs to be equally good substrates as canonical nucleotides for T7 RNA polymerase. Surprisingly, the incorporation of ApnGs boosts the production of RNA 10-fold. This behavior is due to the pairing ability of both purine moieties with the -1 and +1 positions of the antisense DNA strand. Molecular dynamic simulations revealed noncanonical pairing of adenosine with the thymine of the DNA.
Asunto(s)
Fosfatos de Dinucleósidos/genética , ARN/genética , Iniciación de la Transcripción Genética , Bacteriófago T7/enzimología , Emparejamiento Base , ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Fosfatos de Dinucleósidos/metabolismo , Simulación de Dinámica Molecular , Unión Proteica , ARN/metabolismo , Caperuzas de ARN/genética , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
It has been more than 50 years since the discovery of dinucleoside polyphosphates (NpnNs) and yet their roles and mechanisms of action remain unclear. Here, we show that both methylated and non-methylated NpnNs serve as RNA caps in Escherichia coli. NpnNs are excellent substrates for T7 and E. coli RNA polymerases (RNAPs) and efficiently initiate transcription. We demonstrate, that the E. coli enzymes RNA 5'-pyrophosphohydrolase (RppH) and bis(5'-nucleosyl)-tetraphosphatase (ApaH) are able to remove the NpnN-caps from RNA. ApaH is able to cleave all NpnN-caps, while RppH is unable to cleave the methylated forms suggesting that the methylation adds an additional layer to RNA stability regulation. Our work introduces a different perspective on the chemical structure of RNA in prokaryotes and on the role of RNA caps. We bring evidence that small molecules, such as NpnNs are incorporated into RNA and may thus influence the cellular metabolism and RNA turnover.
Asunto(s)
Ácido Anhídrido Hidrolasas/metabolismo , Fosfatos de Dinucleósidos/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Caperuzas de ARN/genética , ARN Polimerasas Dirigidas por ADN/genética , Metilación , Conformación de Ácido Nucleico , Estabilidad del ARN , ARN Bacteriano/genéticaRESUMEN
The mechanism of action of various viruses has been the primary focus of many studies. Yet, the data on RNA modifications in any type of virus are scarce. Methods for the sensitive analysis of RNA modifications have been developed only recently and they have not been applied to viruses. In particular, the RNA composition of HIV-1 virions has never been determined with sufficiently exact methods. Here, we reveal that the RNA of HIV-1 virions contains surprisingly high amount of the 1-methyladenosine. We are the first to use a liquid chromatography-mass spectrometry analysis (LC/MS) of virion RNA, which we combined with m1A profiling and deep sequencing. We found that m1A was present in the tRNA, but not in the genomic HIV-1 RNA and the abundant 7SL RNA. We were able to calculate that an HIV-1 virion contains per 2 copies of genomic RNA and 14 copies of 7SL RNA also 770 copies of tRNA, which is approximately 10 times more than thus far expected. These new insights into the composition of the HIV-1 virion can help in future studies to identify the role of nonprimer tRNAs in retroviruses. Moreover, we present a promising new tool for studying the compositions of virions.
Asunto(s)
Adenosina/análogos & derivados , VIH-1/genética , ARN Citoplasmático Pequeño/genética , ARN Viral/genética , Partícula de Reconocimiento de Señal/genética , Virión/genética , Adenosina/metabolismo , Secuencia de Bases , Línea Celular Tumoral , Cromatografía Liquida/métodos , Genoma Viral/genética , VIH-1/fisiología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Espectrometría de Masas/métodos , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , ARN Viral/metabolismo , Virión/metabolismo , Ensamble de Virus/genéticaRESUMEN
Synthesis of base-modified dNTPs through the Suzuki or Sonogashira cross-coupling reactions of halogenated dNTPs with boronic acids or alkynes is reported, as well as the use of these modified dNTPs in polymerase incorporations to oligonucleotides or DNA by primer extension or PCR.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , ADN/biosíntesis , Nucleótidos/biosíntesis , ADN/química , Halogenación , Nucleótidos/químicaRESUMEN
Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction in the major groove of DNA containing 5-ethynyluracil (UE ) with azides was used for turning off sequence-specific protein-DNA interactions. The concept was first demonstrated on switching off cleavage of short modified DNA by restriction endonuclease BamHI-HF. Finally, DNA template containing UE was used for in vitro transcription with E.â coli RNA polymerase and the transcription was turned off by CuAAC with 3-azidopropane-1,2-diol or 3-azido-7-hydroxycoumarin.
Asunto(s)
Alquinos/química , Azidas/química , Cumarinas/química , ARN Polimerasas Dirigidas por ADN/química , ADN/química , Escherichia coli/química , ARN Bacteriano/química , Catálisis , Química Clic , Cobre , Reacción de Cicloadición , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Bacteriano/metabolismo , Uracilo/análogos & derivadosRESUMEN
Here we describe a protocol for NAD captureSeq that allows for the identification of nicotinamide-adenine dinucleotide (NAD)-capped RNA sequences in total RNA samples from different organisms. NAD-capped RNA is first chemo-enzymatically biotinylated with high efficiency, permitting selective capture on streptavidin beads. Then, a highly efficient library preparation protocol tailored to immobilized, 5'-modified RNA is applied, with adaptor ligation to the RNA's 3' terminus and reverse transcription (RT) performed on-bead. Then, cDNA is released into solution, tailed, ligated to a second adaptor and PCR-amplified. After next-generation sequencing (NGS) of the DNA library, enriched sequences are identified by comparison with a control sample in which the first step of chemo-enzymatic biotinylation is omitted. Because the downstream protocol does not necessarily rely on NAD-modified but on 'clickable' or biotin-modified RNA, it can be applied to other RNA modifications or RNA-biomolecule interactions. The central part of this protocol can be completed in â¼7 d, excluding preparatory steps, sequencing and bioinformatic analysis.
Asunto(s)
NAD/metabolismo , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , Análisis de Secuencia de ARN/métodos , Biotinilación , Cartilla de ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Reacción en Cadena de la PolimerasaRESUMEN
A complete series of 5-substituted uracil or cytosine, as well as 7-substituted 7-deazaadenine and 7-deazaguanine 2'-deoxyribonucleoside triphosphates (dNTPs) bearing substituents of increasing bulkiness (H, Me, vinyl, ethynyl, and phenyl) were systematically studied in competitive primer extension in the presence of their natural counterparts (nonmodified dNTPs), and their kinetic data were determined. The results show that modified dNTPs bearing π-electron-containing substituents (vinyl, ethynyl, Ph) are typically excellent substrates for DNA polymerases comparable to or better than natural dNTPs. The kinetic studies revealed that these modified dNTPs have higher affinity to the active site of the enzyme-primer-template complex, and the calculations (semiempirical quantum mechanical scoring function) suggest that it is due to the cation-π interaction of the modified dNTP with Arg629 in the active site of Bst DNA polymerase.
Asunto(s)
ADN Polimerasa Dirigida por ADN/química , Nucleótidos/química , Purinas/química , Pirimidinas/química , Cinética , Teoría Cuántica , Especificidad por SustratoRESUMEN
New types of double-headed 2'-deoxycytidine 5'-O-triphosphates (dC(XC)TPs) bearing another cytosine or 5-fluorocytosine linked through a flexible propargyl, homopropargyl or pent-1-ynyl linker to position 5 were prepared by the aqueous Sonogashira cross-coupling reactions of 5-iodo-dCTP with the corresponding (fluoro)cytosine-alkynes. The modified dC(XC)TPs were good substrates for DNA polymerases and were used for enzymatic synthesis of cytosine-functionalized DNA by primer extension or PCR. The cytosine- or fluorocytosine-linked DNA probes did not significantly inhibit DNA methyltransferases and did not cross-link to these proteins.
Asunto(s)
Citosina/química , Citosina/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/química , ADN/metabolismo , Nucleótidos de Desoxicitosina/síntesis química , Nucleótidos de Desoxicitosina/metabolismo , Citosina/farmacología , ADN/biosíntesis , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasas/química , Nucleótidos de Desoxicitosina/química , Nucleótidos de Desoxicitosina/farmacología , Modelos Moleculares , Estructura MolecularRESUMEN
A distinctive feature of prokaryotic gene expression is the absence of 5'-capped RNA. In eukaryotes, 5',5'-triphosphate-linked 7-methylguanosine protects messenger RNA from degradation and modulates maturation, localization and translation. Recently, the cofactor nicotinamide adenine dinucleotide (NAD) was reported as a covalent modification of bacterial RNA. Given the central role of NAD in redox biochemistry, posttranslational protein modification and signalling, its attachment to RNA indicates that there are unknown functions of RNA in these processes and undiscovered pathways in RNA metabolism and regulation. The unknown identity of NAD-modified RNAs has so far precluded functional analyses. Here we identify NAD-linked RNAs from bacteria by chemo-enzymatic capture and next-generation sequencing (NAD captureSeq). Among those identified, specific regulatory small RNAs (sRNAs) and sRNA-like 5'-terminal fragments of certain mRNAs are particularly abundant. Analogous to a eukaryotic cap, 5'-NAD modification is shown in vitro to stabilize RNA against 5'-processing by the RNA-pyrophosphohydrolase RppH and against endonucleolytic cleavage by ribonuclease (RNase) E. The nudix phosphohydrolase NudC decaps NAD-RNA and thereby triggers RNase-E-mediated RNA decay, while being inactive against triphosphate-RNA. In vivo, â¼13% of the abundant sRNA RNAI is NAD-capped in the presence, and â¼26% in the absence, of functional NudC. To our knowledge, this is the first description of a cap-like structure and a decapping machinery in bacteria.
Asunto(s)
Escherichia coli/genética , NAD/metabolismo , Caperuzas de ARN/química , Caperuzas de ARN/metabolismo , ARN Bacteriano/química , ARN Bacteriano/aislamiento & purificación , Ácido Anhídrido Hidrolasas/metabolismo , Endorribonucleasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Pirofosfatasas/metabolismo , Caperuzas de ARN/aislamiento & purificación , Estabilidad del ARN , ARN Bacteriano/metabolismo , Análisis de SecuenciaRESUMEN
Enzymatic construction of single-nucleobase redox-labelled oligonucleotides was developed either based on polymerase incorporation of a single modified nucleoside triphosphate (dNTP) followed by primer extension (PEX) with natural dNTPs or based on PEX with a biotinylated one-nucleotide overhang template, magnetoseparation and the second PEX with a full-length template.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Nucleótidos/química , Oligonucleótidos/biosíntesis , Cartilla de ADN/metabolismo , Compuestos Ferrosos/química , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Metalocenos , Nitrobencenos/química , Oligonucleótidos/química , Oxidación-ReducciónAsunto(s)
División del ADN/efectos de los fármacos , Desoxirribonucleasas de Localización Especificada Tipo II/antagonistas & inhibidores , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Inhibidores Enzimáticos/farmacología , Compuestos de Organosilicio/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Estructura Molecular , Compuestos de Organosilicio/síntesis química , Compuestos de Organosilicio/química , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Aqueous Suzuki-Miyaura cross-coupling reactions of halogenated nucleosides, nucleotides and nucleoside triphosphates derived from 5-iodocytosine and 7-iodo-7-deazaadenine with methyl-, benzyl- and tritylsufanylphenylboronic acids gave the corresponding alkylsulfanylphenyl derivatives of nucleosides and nucleotides. The modified nucleoside triphosphates were incorporated into DNA by primer extension by using Vent(exo-) polymerase. The electrochemical behaviour of the alkylsulfanylphenyl nucleosides indicated formation of compact layers on the electrode. Modified nucleotides and DNA with incorporated benzyl- or tritylsulfanylphenyl moieties produced signals in [Co(NH(3))(6)](3+) ammonium buffer, attributed to the Brdicka catalytic response, depending on the negative potential applied. Repeated constant current chronopotentiometric scans in this medium showed increased Brdicka catalytic response, which suggests the deprotection of the alkylsulfanyl derivatives to free thiols under the conditions.