RESUMO
The SARS-CoV-2 genome has been shown to be m6A methylated at several positions inâ vivo. Strikingly, a DRACH motif, the recognition motif for adenosine methylation, resides in the core of the transcriptional regulatory leader sequence (TRS-L) at position A74, which is highly conserved and essential for viral discontinuous transcription. Methylation at position A74 correlates with viral pathogenicity. Discontinuous transcription produces a set of subgenomic mRNAs that function as templates for translation of all structural and accessory proteins. A74 is base-paired in the short stem-loop structure 5'SL3 that opens during discontinuous transcription to form long-range RNA-RNA interactions with nascent (-)-strand transcripts at complementary TRS-body sequences. A74 can be methylated by the human METTL3/METTL14 complex inâ vitro. Here, we investigate its impact on the structural stability of 5'SL3 and the long-range TRS-leader:TRS-body duplex formation necessary for synthesis of subgenomic mRNAs of all four viral structural proteins. Methylation uniformly destabilizes 5'SL3 and long-range duplexes and alters their relative equilibrium populations, suggesting that the m6A74 modification acts as a regulator for the abundance of viral structural proteins due to this destabilization.
Assuntos
Adenosina , Metiltransferases , RNA Mensageiro , RNA Viral , SARS-CoV-2 , Transcrição Gênica , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/química , RNA Viral/química , RNA Viral/metabolismo , RNA Viral/genética , Metiltransferases/química , Metiltransferases/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Humanos , Metilação , Adenosina/química , Adenosina/análogos & derivados , Conformação de Ácido Nucleico , Genoma ViralRESUMO
More than 170 post-transcriptional modifications of RNAs have currently been identified. Detailed biophysical investigations of these modifications have been limited since large RNAs containing these post-transcriptional modifications are difficult to produce. Further, adequate readout of spectroscopic fingerprints are important, necessitating additional labeling procedures beyond the naturally occurring RNA modifications. Here, we report the chemo-enzymatic synthesis of RNA modifications and several structurally similar fluorine-modified analogs further optimizing a recently developed methodology.[1] This chemo-enzymatic method allows synthesis of also large RNAs. We were able to incorporate 16 modified nucleotides and 6 19 F-labeled nucleotides. To showcase the applicability of such modified large RNAs, we incorporated a 19 F-labeled cytidine into the aptamer domain of the 2'dG sensing riboswitch (2'dG-sw) from Mesoplasma florum, enabling characterizing RNA fold, ligand binding and kinetics. Thanks to the large chemical shift dispersion of 19 F, we can detect conformational heterogeneity in the apo state of the riboswitch.
Assuntos
Nucleotídeos , Riboswitch , Nucleotídeos/química , Conformação de Ácido Nucleico , RNA/química , Biossíntese de ProteínasRESUMO
H/ACA Box ribonucleoprotein complexes (RNPs) play a major role in modification of rRNA and snRNA, catalyzing the sequence specific pseudouridylation in eukaryotes and archaea. This enzymatic reaction takes place on a substrate RNA recruited via base pairing to an internal loop of the snoRNA. Eukaryotic snoRNPs contain the four proteins Nop10, Cbf5, Gar1 and Nhp2, with Cbf5 as the catalytic subunit. In contrast to archaeal H/ACA RNPs, eukaryotic snoRNPs contain several conserved features in both the snoRNA as well as the protein components. Here, we reconstituted the eukaryotic H/ACA RNP containing snR81 as a guide RNA in vitro and report on the effects of these eukaryote specific features on complex assembly and enzymatic activity. We compare their contribution to pseudouridylation activity for stand-alone hairpins versus the bipartite RNP. Using single molecule FRET spectroscopy, we investigated the role of the different eukaryote-specific proteins and domains on RNA folding and complex assembly, and assessed binding of substrate RNA to the RNP. Interestingly, we found diverging effects for the two hairpins of snR81, suggesting hairpin-specific requirements for folding and RNP formation. Our results for the first time allow assessing interactions between the individual hairpin RNPs in the context of the full, bipartite snoRNP.
Assuntos
Hidroliases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , RNA Nucleolar Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Catálise , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Expressão Gênica , Hidroliases/genética , Técnicas In Vitro , Sequências Repetidas Invertidas , Proteínas Associadas aos Microtúbulos/genética , Modelos Moleculares , Proteínas Nucleares/genética , Domínios Proteicos , Dobramento de RNA , RNA Guia de Cinetoplastídeos , RNA Nucleolar Pequeno/genética , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The outbreak of COVID-19 in December 2019 required the formation of international consortia for a coordinated scientific effort to understand and combat the virus. In this Viewpoint Article, we discuss how the NMR community has gathered to investigate the genome and proteome of SARS-CoV-2 and tested them for binding to low-molecular-weight binders. External factors including extended lockdowns due to the global pandemic character of the viral infection triggered the transition from locally focused collaborative research conducted within individual research groups to digital exchange formats for immediate discussion of unpublished results and data analysis, sample sharing, and coordinated research between more than 50 groups from 18 countries simultaneously. We discuss key lessons that might pertain after the end of the pandemic and challenges that we need to address.
Assuntos
COVID-19 , Humanos , SARS-CoV-2 , Controle de Doenças Transmissíveis , Espectroscopia de Ressonância Magnética , Imageamento por Ressonância MagnéticaRESUMO
Temperature-dependent alternative splicing was recently demonstrated for intron 2 of the gene coding for heat shock factor HsfA2 of the tomato plant Solanum lycopersicum, but the molecular mechanism regulating the abundance of such temperature-dependent splice variants is still unknown. We report here on regulatory pre-mRNA structures that could function as regulators by controlling the use of splice sites in a temperature-dependent manner. We investigate pre-mRNA structures at the splice sites of intron 2 of the gene coding for HsfA2 from S. lycopersicum using NMR- and CD-spectroscopy as well as in-line probing. The pre-mRNA undergoes conformational changes between two different secondary structures at the 3' splice site of the intron in a temperature-dependent manner. Previously, it was shown that three single nucleotide polymorphisms (SNPs) in intron 2 of the HsfA2 pre-mRNA affect the splicing efficiency of its pre-mRNA and are linked to the thermotolerance in different tomato species. By comparing pre-mRNA fragments of the tomato species S. lycopersicum and S. peruvianum, we show that these SNPs result in substantial structural differences between the pre-mRNAs of the two species.
Assuntos
Processamento Alternativo , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição de Choque Térmico/genética , Precursores de RNA/genética , Solanum lycopersicum/genética , Temperatura , Espectroscopia de Ressonância Magnética , Ligação Proteica , Sítios de Splice de RNARESUMO
The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5' end, the ribosomal frameshift segment and the 3'-untranslated region (3'-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.
Assuntos
COVID-19/prevenção & controle , Espectroscopia de Ressonância Magnética/métodos , Conformação de Ácido Nucleico , RNA Viral/química , SARS-CoV-2/genética , Regiões 3' não Traduzidas/genética , Sequência de Bases , COVID-19/epidemiologia , COVID-19/virologia , Mudança da Fase de Leitura do Gene Ribossômico/genética , Genoma Viral/genética , Humanos , Modelos Moleculares , Pandemias , SARS-CoV-2/fisiologiaRESUMO
H/ACA ribonucleoproteins catalyse the sequence-dependent pseudouridylation of ribosomal and spliceosomal RNAs. Here, we reconstitute site-specifically fluorophore labelled H/ACA complexes and analyse their structural dynamics using single-molecule FRET spectroscopy. Our results show that the guide RNA is distorted into a substrate-binding competent conformation by specific protein interactions. Analysis of the reaction pathway using atomic mutagenesis establishes a new model how individual protein domains contribute to catalysis. Taken together, these results identify and characterize individual roles for all accessory proteins on the assembly and function of H/ACA RNPs.
Assuntos
Proteínas Arqueais/metabolismo , Pseudouridina/metabolismo , Pyrococcus furiosus/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , RNA Nucleolar Pequeno/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Proteínas Arqueais/genética , Pareamento de Bases , Catálise , Pseudouridina/genética , Pyrococcus furiosus/genética , RNA Guia de Cinetoplastídeos/genética , RNA Nucleolar Pequeno/genética , Ribonucleoproteínas Nucleolares Pequenas/genética , SpliceossomosRESUMO
SARS-CoV-2 contains a positive single-stranded RNA genome of approximately 30 000 nucleotides. Within this genome, 15 RNA elements were identified as conserved between SARS-CoV and SARS-CoV-2. By nuclear magnetic resonance (NMR) spectroscopy, we previously determined that these elements fold independently, in line with data from in vivo and ex-vivo structural probing experiments. These elements contain non-base-paired regions that potentially harbor ligand-binding pockets. Here, we performed an NMR-based screening of a poised fragment library of 768 compounds for binding to these RNAs, employing three different 1 H-based 1D NMR binding assays. The screening identified common as well as RNA-element specific hits. The results allow selection of the most promising of the 15 RNA elements as putative drug targets. Based on the identified hits, we derive key functional units and groups in ligands for effective targeting of the RNA of SARS-CoV-2.
Assuntos
Genoma , RNA Viral/metabolismo , SARS-CoV-2/genética , Bibliotecas de Moléculas Pequenas/metabolismo , Avaliação Pré-Clínica de Medicamentos , Ligantes , Estrutura Molecular , Conformação de Ácido Nucleico , Espectroscopia de Prótons por Ressonância Magnética , RNA Viral/química , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Post-translational modifications of proteins are widespread in eukaryotes. To elucidate the functional role of these modifications, detection methods need to be developed that provide information at atomic resolution. Here, we report on the development of a novel Arg-specific NMR experiment that detects the methylation status and symmetry of each arginine side chain even in highly repetitive RGG amino acid sequence motifs found in numerous proteins within intrinsically disordered regions. The experiment relies on the excellent resolution of the backbone H,N correlation spectra even in these low complexity sequences. It requires 13C, 15N labeled samples.
Assuntos
Arginina/química , Espectroscopia de Ressonância Magnética/métodos , Proteínas/química , Humanos , MetilaçãoRESUMO
N6-methyladenosine (m6A) is the most abundant modification in mRNA. The core of the human N6-methyltransferase complex (MTC) is formed by a heterodimer consisting of METTL3 and METTL14, which specifically catalyzes m6A formation within an RRACH sequence context. Using recombinant proteins in a site-specific methylation assay that allows determination of quantitative methylation yields, our results show that this complex methylates its target RNAs not only sequence but also secondary structure dependent. Furthermore, we demonstrate the role of specific protein domains on both RNA binding and substrate turnover, focusing on postulated RNA binding elements. Our results show that one zinc finger motif within the complex is sufficient to bind RNA, however, both zinc fingers are required for methylation activity. We show that the N-terminal domain of METTL3 alters the secondary structure dependence of methylation yields. Our results demonstrate that a cooperative effect of all RNA-binding elements in the METTL3-METTL14 complex is required for efficient catalysis, and that binding of further proteins affecting the NTD of METTL3 may regulate substrate specificity.
Assuntos
Metiltransferases/metabolismo , RNA Mensageiro/metabolismo , RNA/metabolismo , Humanos , Metilação , Metiltransferases/química , Metiltransferases/genética , Conformação de Ácido Nucleico , RNA/química , RNA Mensageiro/química , RNA Mensageiro/genéticaRESUMO
Nature relies on reading and synthesizing the genetic code with high fidelity. Nucleic acid building blocks that are orthogonal to the canonical A-T and G-C base-pairs are therefore uniquely suitable to facilitate position-specific labeling of nucleic acids. Here, we employ the orthogonal kappa-xanthosine-base-pair for in vitro transcription of labeled RNA. We devised an improved synthetic route to obtain the phosphoramidite of the deoxy-version of the kappa nucleoside in solid phase synthesis. From this DNA template, we demonstrate the reliable incorporation of xanthosine during in vitro transcription. Using NMR spectroscopy, we show that xanthosine introduces only minor structural changes in an RNA helix. We furthermore synthesized a clickable 7-deaza-xanthosine, which allows to site-specifically modify transcribed RNA molecules with fluorophores or other labels.
Assuntos
RNA/química , Ribonucleosídeos/química , Pareamento de Bases , Química Click , Código Genético , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , RNA/metabolismo , Ribonucleosídeos/metabolismo , Técnicas de Síntese em Fase Sólida , XantinasRESUMO
RNA-protein complexes (RNPs) are essential components in a variety of cellular processes, and oftentimes exhibit complex structures and show mechanisms that are highly dynamic in conformation and structure. However, biochemical and structural biology approaches are mostly not able to fully elucidate the structurally and especially conformationally dynamic and heterogeneous nature of these RNPs, to which end single molecule Förster resonance energy transfer (smFRET) spectroscopy can be harnessed to fill this gap. Here we summarize the advantages of strategic smFRET studies to investigate RNP dynamics, complemented by structural and biochemical data. Focusing on recent smFRET studies of three essential biological systems, we demonstrate that investigation of RNPs on a single molecule level can answer important functional questions that remained elusive with structural or biochemical approaches alone: The complex structural rearrangements throughout the splicing cycle, unwinding dynamics of the G-quadruplex (G4) helicase RHAU, and aspects in telomere maintenance regulation and synthesis.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Quadruplex G , RNA/química , Imagem Individual de Molécula , Animais , Bovinos , Análise por Conglomerados , Cristalografia por Raios X , Humanos , Cadeias de Markov , Conformação de Ácido Nucleico , Ligação Proteica , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Splicing de RNA , Ribonucleoproteínas , Spliceossomos/química , Telomerase/química , Telômero/química , Telômero/ultraestruturaRESUMO
At any stage of their lifecycle, mRNAs are coated by specialized proteins. One of few circumstances when free mRNA appears in the cytosol is the disassembly of polysomes during the stress-induced shutdown of protein synthesis. Using quantitative mass spectrometry, we sought to identify the free RNA-interacting cellular machinery in heat-shocked mammalian cells. Free RNA-associated proteins displayed higher disorder and larger size, which supports the role of multivalent interactions during the initial phase of the association with RNAs during stress. Structural features of the free RNA interactors defined them as a subset of RNA-binding proteins. The interaction between these assembled proteins in vivo required RNA. Reconstitution of the association process in vitro indicated a multimolecular basis for increased binding to RNA upon heat shock in the cytosol. Our study represents a step toward understanding how free RNA is processed in the cytosol during proteostasis stress.
Assuntos
Resposta ao Choque Térmico/fisiologia , Biossíntese de Proteínas , Proteostase/fisiologia , RNA Mensageiro/fisiologia , Animais , Citosol/metabolismo , Humanos , Mamíferos , Espectrometria de Massas/métodos , Polirribossomos/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Most single-molecule techniques observing RNA in vitro or in vivo require fluorescent labels that have to be connected to the RNA of interest. In recent years, a plethora of methods has been developed to achieve site-specific labelling, in many cases under near-native conditions. Here, we review chemical as well as enzymatic labelling methods that are compatible with single-molecule fluorescence spectroscopy or microscopy and show how these can be combined to offer a large variety of options to site-specifically place one or more labels in an RNA of interest. By either chemically forming a covalent bond or non-covalent hybridization, these techniques are prerequisites to perform state-of-the-art single-molecule experiments.
Assuntos
RNA/isolamento & purificação , Imagem Individual de Molécula , Coloração e Rotulagem/tendências , Química Click , Transferência Ressonante de Energia de Fluorescência/tendências , Corantes Fluorescentes/química , RNA/química , RNA/genéticaRESUMO
The full-length translation-regulating add adenine riboswitch (Asw) from Vibrio vulnificus has a more complex conformational space than its isolated aptamer domain. In addition to the predicted apo (apoA) and holo conformation that feature the conserved three-way junctional purine riboswitch aptamer, it adopts a second apo (apoB) conformation with a fundamentally different secondary structure. Here, we characterized the ligand-dependent conformational dynamics of the full-length add Asw by NMR and by single-molecule FRET (smFRET) spectroscopy. Both methods revealed an adenine-induced secondary structure switch from the apoB-form to the apoA-form that involves no tertiary structural interactions between aptamer and expression platform. This strongly suggests that the add Asw triggers translation by capturing the apoA-form secondary structure in the holo state. Intriguingly, NMR indicated a homogenous, docked aptamer kissing loop fold for apoA and holo, while smFRET showed persistent aptamer kissing loop docking dynamics between comparably stable, undocked and docked substates of the apoA and the holo conformation. Unraveling the folding of large junctional riboswitches thus requires the integration of complementary solution structural techniques such as NMR and smFRET.
Assuntos
Adenina/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Espectroscopia de Ressonância Magnética , Riboswitch , Aptâmeros de Nucleotídeos/química , Pareamento de Bases/genética , Sequência de Bases , Ligantes , Magnésio/farmacologia , Mutação/genética , Conformação de Ácido Nucleico , Imagem Individual de MoléculaRESUMO
Gene repression induced by the formation of transcriptional terminators represents a prime example for the coupling of RNA synthesis, folding, and regulation. In this context, mapping the changes in available conformational space of transcription intermediates during RNA synthesis is important to understand riboswitch function. A majority of riboswitches, an important class of small metabolite-sensing regulatory RNAs, act as transcriptional regulators, but the dependence of ligand binding and the subsequent allosteric conformational switch on mRNA transcript length has not yet been investigated. We show a strict fine-tuning of binding and sequence-dependent alterations of conformational space by structural analysis of all relevant transcription intermediates at single-nucleotide resolution for the I-A type 2'dG-sensing riboswitch from Mesoplasma florum by NMR spectroscopy. Our results provide a general framework to dissect the coupling of synthesis and folding essential for riboswitch function, revealing the importance of metastable states for RNA-based gene regulation.
Assuntos
Riboswitch , Regulação Alostérica , Sítios de Ligação , Expressão Gênica , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Biológicos , Conformação de Ácido Nucleico , Dobramento de RNA , Transcrição GênicaRESUMO
Determination of RNA secondary structures by NMR spectroscopy is a useful tool e.g. to elucidate RNA folding space or functional aspects of regulatory RNA elements. However, current approaches of RNA synthesis and preparation are usually time-consuming and do not provide analysis with single nucleotide precision when applied for a large number of different RNA sequences. Here, we significantly improve the yield and 3' end homogeneity of RNA preparation by in vitro transcription. Further, by establishing a native purification procedure with increased throughput, we provide a shortcut to study several RNA constructs simultaneously. We show that this approach yields µmol quantities of RNA with purities comparable to PAGE purification, while avoiding denaturation of the RNA.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , RNA/química , RNA/metabolismo , Aptâmeros de Nucleotídeos/química , Sequência de Bases , Soluções Tampão , Ligantes , Dados de Sequência Molecular , RNA/isolamento & purificação , Riboswitch , Transcrição GênicaRESUMO
Cellular RNA function is closely linked to RNA structure. It is therefore imperative to develop methods that report on structural stability of RNA and how it is modulated by binding of ions, other osmolytes, and RNA-binding ligands. Here, we present a novel method to analyze the stability of virtually any structured RNA in a highly parallel fashion. This method can easily determine the influence of various additives on RNA stability, and even characterize ligand-induced stabilization of riboswitch RNA. Current approaches to assess RNA stability include thermal melting profiles (absorption or circular dichroism) and differential scanning calorimetry. These techniques, however, require a substantial amount of material and cannot be significantly parallelized. Current fluorescence spectroscopic methods rely on intercalating dyes, which alter the stability of RNA. We employ the commercial fluorescent dye RiboGreen, which discriminates between single-stranded (or unstructured regions) and double-stranded RNA. Binding leads to an increase in fluorescence quantum yield, and thus reports structural changes by a change in fluorescence intensity.