RESUMO
Argonaute-bound microRNAs silence mRNA expression in a dynamic and regulated manner to control organismal development, physiology, and disease. We employed metabolic small RNA sequencing for a comprehensive view on intracellular microRNA kinetics in Drosophila. Based on absolute rate of biogenesis and decay, microRNAs rank among the fastest produced and longest-lived cellular transcripts, disposing up to 105 copies per cell at steady-state. Mature microRNAs are produced within minutes, revealing tight intracellular coupling of biogenesis that is selectively disrupted by pre-miRNA-uridylation. Control over Argonaute protein homeostasis generates a kinetic bottleneck that cooperates with non-coding RNA surveillance to ensure faithful microRNA loading. Finally, regulated small RNA decay enables the selective rapid turnover of Ago1-bound microRNAs, but not of Ago2-bound small interfering RNAs (siRNAs), reflecting key differences in the robustness of small RNA silencing pathways. Time-resolved small RNA sequencing opens new experimental avenues to deconvolute the timescales, molecular features, and regulation of small RNA silencing pathways in living cells.
Assuntos
Proteínas Argonautas/metabolismo , Proteínas de Drosophila/metabolismo , Homeostase/fisiologia , MicroRNAs/metabolismo , Análise de Sequência de RNA , Animais , Proteínas Argonautas/genética , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , MicroRNAs/genéticaRESUMO
Members of the 3'-5' RNA polymerase family, comprised of tRNAHis guanylyltransferase (Thg1) and Thg1-like proteins (TLPs), catalyze templated synthesis of RNA in the reverse direction to all other known 5'-3' RNA and DNA polymerases. Discovery of enzymes capable of this reaction raised the possibility of exploiting 3'-5' polymerases for post-transcriptional incorporation of nucleotides to the 5'-end of nucleic acids without ligation, and instead by templated polymerase addition. To date, studies of these enzymes have focused on nucleotide addition to highly structured RNAs, such as tRNA and other non-coding RNA. Consequently, general principles of RNA substrate recognition and nucleotide preferences that might enable broader application of 3'-5' polymerases have not been elucidated. Here, we investigated the feasibility of using Thg1 or TLPs for multiple nucleotide incorporation to the 5'-end of a short duplex RNA substrate, using a templating RNA oligonucleotide provided in trans to guide 5'-end addition of specific sequences. Using optimized assay conditions, we demonstrated a remarkable capacity of certain TLPs to accommodate short RNA substrate-template duplexes of varying lengths with significantly high affinity, resulting in the ability to incorporate a desired nucleotide sequence of up to 8 bases to 5'-ends of the model RNA substrates in a template-dependent manner. This work has further advanced our goals to develop this atypical enzyme family as a versatile nucleic acid 5'-end labeling tool.
RESUMO
Tissue dissociation, a crucial step in single-cell sample preparation, can alter the transcriptional state of a sample through the intrinsic cellular stress response. Here we demonstrate a general approach for measuring transcriptional response during sample preparation. In our method, transcripts made during dissociation are labeled for later identification upon sequencing. We found general as well as cell-type-specific dissociation response programs in zebrafish larvae, and we observed sample-to-sample variation in the dissociation response of mouse cardiomyocytes despite well-controlled experimental conditions. Finally, we showed that dissociation of the mouse hippocampus can lead to the artificial activation of microglia. In summary, our approach facilitates experimental optimization of dissociation procedures as well as computational removal of transcriptional perturbation response.
Assuntos
RNA , Transcriptoma , Camundongos , Animais , Peixe-Zebra/genética , Análise de Sequência de RNA/métodos , Microglia , Análise de Célula Única , Perfilação da Expressão Gênica/métodosRESUMO
Metabolic labeling of newly transcribed RNAs coupled with RNA-seq is being increasingly used for genome-wide analysis of RNA dynamics. Methods including standard biochemical enrichment and recent nucleotide conversion protocols each require special experimental and computational treatment. Despite their immediate relevance, these technologies have not yet been assessed and benchmarked, and no data are currently available to advance reproducible research and the development of better inference tools. Here, we present a systematic evaluation and comparison of four RNA labeling protocols: 4sU-tagging biochemical enrichment, including spike-in RNA controls, SLAM-seq, TimeLapse-seq and TUC-seq. All protocols are evaluated based on practical considerations, conversion efficiency and wet lab requirements to handle hazardous substances. We also compute decay rate estimates and confidence intervals for each protocol using two alternative statistical frameworks, pulseR and GRAND-SLAM, for over 11 600 human genes and evaluate the underlying computational workflows for their robustness and ease of use. Overall, we demonstrate a high inter-method reliability across eight use case scenarios. Our results and data will facilitate reproducible research and serve as a resource contributing to a fuller understanding of RNA biology.
Assuntos
Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , RNA/genética , Coloração e Rotulagem/métodos , Linhagem Celular , Humanos , RNA/metabolismo , Estabilidade de RNA , Fluxo de TrabalhoRESUMO
RNA splicing, the removal of introns and ligation of exons, is a crucial process during mRNA maturation. Group II introns are large ribozymes that self-catalyze their splicing, as well as their transposition. They are living fossils of spliceosomal introns and eukaryotic retroelements. The yeast mitochondrial Sc.ai5γ is the first identified and best-studied self-splicing group II intron. A combination of biochemical, biophysical, and computational tools enables studying its catalytic properties, structure, and dynamics, while also serving to develop new therapeutic and biotechnological tools. We survey the history of group II intron studies paralleling the trends in RNA methodology with Sc.ai5γ in the spotlight.
Assuntos
Biotecnologia , Mitocôndrias , Íntrons , Biofísica , CatáliseRESUMO
Site-specific introduction of bioorthogonal handles into RNAs is in high demand for decorating RNAs with fluorophores, affinity labels or other modifications. Aldehydes represent attractive functional groups for post-synthetic bioconjugation reactions. Here, we report a ribozyme-based method for the synthesis of aldehyde-functionalized RNA by directly converting a purine nucleobase. Using the methyltransferase ribozyme MTR1 as an alkyltransferase, the reaction is initiated by site-specific N1 benzylation of purine, followed by nucleophilic ring opening and spontaneous hydrolysis under mild conditions to yield a 5-amino-4-formylimidazole residue in good yields. The modified nucleotide is accessible to aldehyde-reactive probes, as demonstrated by the conjugation of biotin or fluorescent dyes to short synthetic RNAs and tRNA transcripts. Upon fluorogenic condensation with a 2,3,3-trimethylindole, a novel hemicyanine chromophore was generated directly on the RNA. This work expands the MTR1 ribozyme's area of application from a methyltransferase to a tool for site-specific late-stage functionalization of RNA.
Assuntos
RNA Catalítico , RNA , RNA/química , RNA Catalítico/química , Catálise , Aldeídos , Metiltransferases , Corantes Fluorescentes/químicaRESUMO
RNA transport and localization represent important post-transcriptional mechanisms to determine the subcellular localization of protein synthesis. Plants have the capacity to transport messenger (m)RNA molecules beyond the cell boundaries through plasmodesmata and over long distances in the phloem. RNA viruses exploit these transport pathways to disseminate their infections and represent important model systems to investigate RNA transport in plants. Here, we present an in vivo plant RNA-labeling system based on the Escherichia coli RNA-binding protein BglG. Using the detection of RNA in mobile RNA particles formed by viral movement protein (MP) as a model, we demonstrate the efficiency and specificity of mRNA detection by the BglG system as compared with MS2 and λN systems. Our observations show that MP mRNA is specifically associated with MP in mobile MP particles but hardly with MP localized at plasmodesmata. MP mRNA is clearly absent from MP accumulating along microtubules. We show that the in vivo BglG labeling of the MP particles depends on the presence of the BglG-binding stem-loop aptamers within the MP mRNA and that the aptamers enhance the coprecipitation of BglG by MP, thus demonstrating the presence of an MP:MP mRNA complex. The BglG system also allowed us to monitor the cell-to-cell transport of the MP mRNA, thus linking the observation of mobile MP mRNA granules with intercellular MP mRNA transport. Given its specificity demonstrated here, the BglG system may be widely applicable for studying mRNA transport and localization in plants.
Assuntos
Proteínas de Bactérias , RNA Mensageiro/ultraestrutura , RNA de Plantas/ultraestrutura , Proteínas de Ligação a RNA , Escherichia coli , Proteínas de Escherichia coli , Proteínas de Fluorescência Verde , Imunoprecipitação , Microscopia de Fluorescência , Epiderme Vegetal/metabolismo , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Nicotiana/genéticaRESUMO
The introduction of chemical modifications into long RNA molecules at specific positions for visualization, biophysical investigations, diagnostic and therapeutic applications still remains challenging. In this review, we present recent approaches for covalent internal labeling of long RNAs. Topics included are the assembly of large modified RNAs via enzymatic ligation of short synthetic oligonucleotides and synthetic biology approaches preparing site-specifically modified RNAs via inâ vitro transcription using an expanded genetic alphabet. Moreover, recent approaches to employ deoxyribozymes (DNAzymes) and ribozymes for RNA labeling and RNA methyltransferase based labeling strategies are presented. We discuss the potentials and limits of the individual methods, their applicability for RNAs with several hundred to thousands of nucleotides in length and indicate future directions in the field.
Assuntos
DNA Catalítico/metabolismo , RNA Catalítico/metabolismo , RNA Longo não Codificante/metabolismo , Coloração e Rotulagem , Humanos , RNA Longo não Codificante/análiseRESUMO
Labeled RNAs are invaluable probes for investigation of RNA function and localization. However, mRNA labeling remains challenging. Here, we developed an improved method for 3'-end labeling of inâ vitro transcribed RNAs. We synthesized novel adenosine 3',5'-bisphosphate analogues modified at the N6 or C2 position of adenosine with an azide-containing linker, fluorescent label, or biotin and assessed these constructs as substrates for RNA labeling directly by T4 ligase or via postenzymatic strain-promoted alkyne-azide cycloaddition (SPAAC). All analogues were substrates for T4 RNA ligase. Analogues containing bulky fluorescent labels or biotin showed better overall labeling yields than postenzymatic SPAAC. We successfully labeled uncapped RNAs, NAD-capped RNAs, and 5'-fluorescently labeled m7 Gp3 Am -capped mRNAs. The obtained highly homogenous dually labeled mRNA was translationally active and enabled fluorescence-based monitoring of decapping. This method will facilitate the use of various functionalized mRNA-based probes.
Assuntos
Azidas , RNA , Alcinos , Reação de Cicloadição , RNA Mensageiro/genéticaRESUMO
The COVID-19 pandemic caused by SARS-CoV-2 coronavirus deeply affected the world community. It gave a strong impetus to the development of not only approaches to diagnostics and therapy, but also fundamental research of the molecular biology of this virus. Fluorescence microscopy is a powerful technology enabling detailed investigation of virus-cell interactions in fixed and live samples with high specificity. While spatial resolution of conventional fluorescence microscopy is not sufficient to resolve all virus-related structures, super-resolution fluorescence microscopy can solve this problem. In this paper, we review the use of fluorescence microscopy to study SARS-CoV-2 and related viruses. The prospects for the application of the recently developed advanced methods of fluorescence labeling and microscopy-which in our opinion can provide important information about the molecular biology of SARS-CoV-2-are discussed.
Assuntos
Microscopia de Fluorescência , SARS-CoV-2/fisiologia , COVID-19/patologia , COVID-19/virologia , Endocitose , Corantes Fluorescentes/química , Genes Reporter , Humanos , RNA Viral/química , RNA Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Internalização do VírusRESUMO
The mRNA modification N6 -methyladenosine (m6 A) is associated with multiple roles in cell function and disease. The methyltransferases METTL3-METTL14 and METTL16 act as "writers" for different target transcripts and sequence motifs. The modification is perceived by dedicated "reader" and "eraser" proteins, but not by polymerases. We report that METTL3-14 shows remarkable cosubstrate promiscuity, enabling sequence-specific internal labeling of RNA without additional guide RNAs. The transfer of ortho-nitrobenzyl and 6-nitropiperonyl groups allowed enzymatic photocaging of RNA in the consensus motif, which impaired polymerase-catalyzed primer extension in a reversible manner. METTL16 was less promiscuous but suitable for chemo-enzymatic labeling using different types of click chemistry. Since both enzymes act on distinct sequence motifs, their combination allowed orthogonal chemo-enzymatic modification of different sites in a single RNA.
Assuntos
Metiltransferases/metabolismo , RNA Mensageiro/metabolismo , Adenosina/metabolismo , Cromatografia Líquida de Alta Pressão , Química Click , Humanos , Luz , Fotólise , RNA Mensageiro/química , S-Adenosilmetionina/química , Especificidade por Substrato , Espectrometria de Massas em TandemRESUMO
Inâ vitro selected ribozymes are promising tools for site-specific labeling of RNA. Previously known nucleic acid catalysts attached fluorescently labeled adenosine or guanosine derivatives through 2',5'-branched phosphodiester bonds to the RNA of interest. Herein, we report new ribozymes that use orthogonal substrates, derived from the antiviral drug tenofovir, and attach bioorthogonal functional groups, as well as affinity handles and fluorescent reporter units through a hydrolytically more stable phosphonate ester linkage. The tenofovir transferase ribozymes were identified by inâ vitro selection and are orthogonal to nucleotide transferase ribozymes. As genetically encodable functional RNAs, these ribozymes may be developed for potential cellular applications. The orthogonal ribozymes addressed desired target sites in large RNAs inâ vitro, as shown by fluorescent labeling of E.â coli 16S and 23S rRNAs in total cellular RNA.
Assuntos
Antivirais/farmacologia , Biocatálise , Reposicionamento de Medicamentos , RNA Catalítico/metabolismo , Tenofovir/farmacologia , Transferases/metabolismoRESUMO
BACKGROUND: Mutations in the transcription factor, KLF1, are common within certain populations of the world. Heterozygous missense mutations in KLF1 mostly lead to benign phenotypes, but a heterozygous mutation in a DNA-binding residue (E325K in human) results in severe Congenital Dyserythropoietic Anemia type IV (CDA IV); i.e. an autosomal-dominant disorder characterized by neonatal hemolysis. RESULTS: To investigate the biochemical and genetic mechanism of CDA IV, we generated murine erythroid cell lines that harbor tamoxifen-inducible (ER™) versions of wild type and mutant KLF1 on a Klf1-/- genetic background. Nuclear translocation of wild type KLF1 results in terminal erythroid differentiation, whereas mutant KLF1 results in hemolysis without differentiation. The E to K variant binds poorly to the canonical 9 bp recognition motif (NGG-GYG-KGG) genome-wide but binds at high affinity to a corrupted motif (NGG-GRG-KGG). We confirmed altered DNA-binding specificity by quantitative in vitro binding assays of recombinant zinc-finger domains. Our results are consistent with previously reported structural data of KLF-DNA interactions. We employed 4sU-RNA-seq to show that a corrupted transcriptome is a direct consequence of aberrant DNA binding. CONCLUSIONS: Since all KLF/SP family proteins bind DNA in an identical fashion, these results are likely to be generally applicable to mutations in all family members. Importantly, they explain how certain mutations in the DNA-binding domain of transcription factors can generate neomorphic functions that result in autosomal dominant disease.
Assuntos
Anemia Diseritropoética Congênita/genética , DNA/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Mutação Puntual , Animais , Linhagem Celular , DNA/química , Regulação da Expressão Gênica , Camundongos , Motivos de Nucleotídeos , Ligação Proteica , Transcrição GênicaRESUMO
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
Our ability to observe biochemical events with high spatial and temporal resolution is essential for understanding the functioning of living systems. Intrinsically fluorescent proteins such as the green fluorescent protein (GFP) have revolutionized the way biologists study cells and organisms. The fluorescence toolbox has been recently extended with new fluorescent reporters composed of a genetically encoded tag that binds endogenously present or exogenously applied fluorogenic chromophores (so-called fluorogens) and activates their fluorescence. This review presents the toolbox of fluorogen-based reporters and biosensors available to biologists. Various applications are detailed to illustrate the possible uses and opportunities offered by this new generation of fluorescent probes and sensors for advanced bioimaging.
Assuntos
Técnicas Biossensoriais , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/química , Imagem Óptica , Animais , HumanosRESUMO
The replication of a virus within its host cell involves numerous interactions between viral and cellular factors, which have to be tightly controlled in space and time. The intricate interplay between viral exploitation of cellular pathways and the intrinsic host defense mechanisms is difficult to unravel by traditional bulk approaches. In recent years, novel fluorescence microscopy techniques and single virus tracking have transformed the investigation of dynamic virus-host interactions. A prerequisite for the application of these imaging-based methods is the attachment of a fluorescent label to the structure of interest. However, their small size, limited coding capacity and multifunctional proteins render viruses particularly challenging targets for fluorescent labeling approaches. Click chemistry in conjunction with genetic code expansion provides virologists with a novel toolbox for site-specific, minimally invasive labeling of virion components, whose potential has just recently begun to be exploited. Here, we summarize recent achievements, current developments and future challenges for the labeling of viral nucleic acids, proteins, glycoproteins or lipids using click chemistry in order to study dynamic processes in virus-cell interactions.
Assuntos
Química Click/métodos , Replicação Viral/fisiologia , Humanos , Microscopia de FluorescênciaRESUMO
Live imaging of transcription and RNA dynamics has been successful in cultured cells and tissues of vertebrates but is challenging to accomplish in vivo. The zebrafish offers important advantages to study these processes--optical transparency during embryogenesis, genetic tractability and rapid development. Therefore, to study transcription and RNA dynamics in an intact vertebrate organism, we have adapted the MS2 RNA-labeling system to zebrafish. By using this binary system to coexpress a fluorescent MS2 bacteriophage coat protein (MCP) and an RNA of interest tagged with multiple copies of the RNA hairpin MS2-binding site (MBS), live-cell imaging of RNA dynamics at single RNA molecule resolution has been achieved in other organisms. Here, using a Gateway-compatible MS2 labeling system, we generated stable transgenic zebrafish lines expressing MCP, validated the MBS-MCP interaction and applied the system to investigate zygotic genome activation (ZGA) and RNA localization in primordial germ cells (PGCs) in zebrafish. Although cleavage stage cells are initially transcriptionally silent, we detect transcription of MS2-tagged transcripts driven by the ßactin promoter at â¼ 3-3.5 h post-fertilization, consistent with the previously reported ZGA. Furthermore, we show that MS2-tagged nanos3 3'UTR transcripts localize to PGCs, where they are diffusely cytoplasmic and within larger cytoplasmic accumulations reminiscent of those displayed by endogenous nanos3. These tools provide a new avenue for live-cell imaging of RNA molecules in an intact vertebrate. Together with new techniques for targeted genome editing, this system will be a valuable tool to tag and study the dynamics of endogenous RNAs during zebrafish developmental processes.
Assuntos
RNA/metabolismo , Transcrição Gênica , Peixe-Zebra/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Animais Geneticamente Modificados , Sítios de Ligação , RNA Helicases DEAD-box/metabolismo , Embrião não Mamífero/metabolismo , Genoma , Células Germinativas/citologia , Células Germinativas/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Sinais de Localização Nuclear/metabolismo , Ligação Proteica , Multimerização Proteica , Reprodutibilidade dos Testes , Frações Subcelulares/metabolismo , Proteínas Virais/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Zigoto/metabolismoRESUMO
Modified mRNA (mod-mRNA) has recently been widely studied as the form of RNA useful for therapeutic applications due to its high stability and lowered immune response. Herein, we extend the scope of the recently established RNA-TAG (transglycosylation at guanosine) methodology, a novel approach for genetically encoded site-specific labeling of large mRNA transcripts, by employing mod-mRNA as substrate. As a proof of concept, we covalently attached a fluorescent probe to mCherry encoding mod-mRNA transcripts bearing 5-methylcytidine and/or pseudouridine substitutions with high labeling efficiencies. To provide a versatile labeling methodology with a wide range of possible applications, we employed a two-step strategy for functionalization of the mod-mRNA to highlight the therapeutic potential of this new methodology. We envision that this novel and facile labeling methodology of mod-RNA will have great potential in decorating both coding and noncoding therapeutic RNAs with a variety of diagnostic and functional moieties.
Assuntos
Proteínas de Escherichia coli/química , Pentosiltransferases/química , RNA Mensageiro/química , Coloração e Rotulagem/métodos , Citidina/análogos & derivados , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , RNA Mensageiro/genética , Especificidade por Substrato , Proteína Vermelha FluorescenteRESUMO
RNA-binding proteins (RBPs) are key players in post-transcriptional regulation of gene expression in eukaryotic cells. To be able to unbiasedly identify RBPs in Saccharomyces cerevisiae, we developed a yeast RNA interactome capture protocol which employs RNA labeling, covalent UV crosslinking of RNA and proteins at 365nm wavelength (photoactivatable-ribonucleoside-enhanced crosslinking, PAR-CL) and finally purification of the protein-bound mRNA. The method can be easily implemented in common workflows and takes about 3days to complete. Next to a comprehensive explanation of the method, we focus on our findings about the choice of crosslinking in yeast and discuss the rationale of individual steps in the protocol.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Imunoprecipitação/métodos , RNA Mensageiro/química , Proteínas de Ligação a RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Coloração e Rotulagem/métodos , Anticorpos/química , Sequência de Bases , Sítios de Ligação , Biblioteca Gênica , Espectrometria de Massas/métodos , Processos Fotoquímicos , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleases/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Sequência de RNA/métodos , Radioisótopos de Enxofre , Tiouracila/análogos & derivados , Tiouracila/química , Tiouracila/metabolismo , Transcriptoma , Raios UltravioletaRESUMO
Recent developments in RNA labeling technology have provided viable tools to analyze RNA synthesis, processing and function in cell-free and cellular environments. Notably, emerging methodologies based on posttranscriptional chemical labeling by using bioorthogonal chemistry have enabled the visualization and profiling of exogenous and endogenous RNA transcripts. In this review, we first give an overview of different RNA labeling strategies based on chemical as well as genetically encoded systems. Subsequently, we provided a detailed discussion on methodologies that have been developed to introduce various bioorthogonal reactive groups into RNA transcripts, which are compatible for posttranscriptional functionalization. Finally, the utility of these techniques in imaging and studying the dynamics of RNA production, distribution and decay in complex cellular environment is discussed.