RESUMEN
RNA structure influences numerous processes in all organisms. In bacteria, these processes include transcription termination and attenuation, small RNA and protein binding, translation initiation, and mRNA stability, and can be regulated via metabolite availability and other stresses. Here we use Structure-seq2 to probe the in vivo RNA structurome of Bacillus subtilis grown in the presence and absence of amino acids. Our results reveal that amino acid starvation results in lower overall dimethyl sulfate (DMS) reactivity of the transcriptome, indicating enhanced protection owing to protein binding or RNA structure. Starvation-induced changes in DMS reactivity correlated inversely with transcript abundance changes. This correlation was particularly pronounced in genes associated with the stringent response and CodY regulons, which are involved in adaptation to nutritional stress, suggesting that RNA structure contributes to transcript abundance change in regulons involved in amino acid metabolism. Structure-seq2 accurately reported on four known amino acid-responsive riboswitches: T-box, SAM, glycine, and lysine riboswitches. Additionally, we discovered a transcription attenuation mechanism that reduces yfmG expression when amino acids are added to the growth medium. We also found that translation of a leader peptide (YfmH) encoded just upstream of yfmG regulates yfmG expression. Our results are consistent with a model in which a slow rate of yfmH translation caused by limitation of the amino acids encoded in YfmH prevents transcription termination in the yfmG leader region by favoring formation of an overlapping antiterminator structure. This novel RNA switch offers a way to simultaneously monitor the levels of multiple amino acids.
Asunto(s)
Aminoácidos/genética , Bacillus subtilis/genética , Proteínas Bacterianas/genética , ARN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica/genética , Conformación de Ácido Nucleico , Estabilidad del ARN/genética , Transcripción Genética/genética , Transcriptoma/genéticaRESUMEN
In vivo genome-wide RNA structure probing provides a global view of RNA structure as it occurs in the cell and can assist in elucidating important functional aspects of RNA structure. Structure-seq2 provides high-quality data on transcriptome-wide RNA structure in vivo but contains numerous steps that require technical precision. In this chapter we present the steps needed to produce high-quality structural data with a focus on controls and troubleshooting. Structure-seq2 can be applied to numerous organisms including plants, humans, and bacteria and is amenable to a wide variety of RNA-modifying chemicals including DMS, glyoxal, and SHAPE reagents. Notably, the data generated by the method highlighted here can be readily analyzed using our StructureFold2 computational pipeline.
Asunto(s)
Arabidopsis/genética , Genoma de Planta , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Sondas ARN/química , ARN de Planta/química , Análisis de Secuencia de ARN/métodos , Conformación de Ácido Nucleico , ARN de Planta/genética , Ésteres del Ácido Sulfúrico/químicaRESUMEN
BACKGROUND: The RNA helicase eIF4A1 is a key component of the translation initiation machinery and is required for the translation of many pro-oncogenic mRNAs. There is increasing interest in targeting eIF4A1 therapeutically in cancer, thus understanding how this protein leads to the selective re-programming of the translational landscape is critical. While it is known that eIF4A1-dependent mRNAs frequently have long GC-rich 5'UTRs, the details of how 5'UTR structure is resculptured by eIF4A1 to enhance the translation of specific mRNAs are unknown. RESULTS: Using Structure-seq2 and polysome profiling, we assess global mRNA structure and translational efficiency in MCF7 cells, with and without eIF4A inhibition with hippuristanol. We find that eIF4A inhibition does not lead to global increases in 5'UTR structure, but rather it leads to 5'UTR remodeling, with localized gains and losses of structure. The degree of these localized structural changes is associated with 5'UTR length, meaning that eIF4A-dependent mRNAs have greater localized gains of structure due to their increased 5'UTR length. However, it is not solely increased localized structure that causes eIF4A-dependency but the position of the structured regions, as these structured elements are located predominantly at the 3' end of the 5'UTR. CONCLUSIONS: By measuring changes in RNA structure following eIF4A inhibition, we show that eIF4A remodels local 5'UTR structures. The location of these structural elements ultimately determines the dependency on eIF4A, with increased structure just upstream of the CDS being the major limiting factor in translation, which is overcome by eIF4A activity.
Asunto(s)
Regiones no Traducidas 5' , Factor 4A Eucariótico de Iniciación/metabolismo , ARN Mensajero/metabolismo , Codón Iniciador , Humanos , Células MCF-7 , EsterolesRESUMEN
The heat shock response is crucial for organism survival in natural environments. RNA structure is known to influence numerous processes related to gene expression, but there have been few studies on the global RNA structurome as it prevails in vivo. Moreover, how heat shock rapidly affects RNA structure genome-wide in living systems remains unknown. We report here in vivo heat-regulated RNA structuromes. We applied Structure-seq chemical [dimethyl sulfate (DMS)] structure probing to rice (Oryza sativa L.) seedlings with and without 10 min of 42 °C heat shock and obtained structural data on >14,000 mRNAs. We show that RNA secondary structure broadly regulates gene expression in response to heat shock in this essential crop species. Our results indicate significant heat-induced elevation of DMS reactivity in the global transcriptome, revealing RNA unfolding over this biological temperature range. Our parallel Ribo-seq analysis provides no evidence for a correlation between RNA unfolding and heat-induced changes in translation, in contrast to the paradigm established in prokaryotes, wherein melting of RNA thermometers promotes translation. Instead, we find that heat-induced DMS reactivity increases correlate with significant decreases in transcript abundance, as quantified from an RNA-seq time course, indicating that mRNA unfolding promotes transcript degradation. The mechanistic basis for this outcome appears to be mRNA unfolding at both 5' and 3'-UTRs that facilitates access to the RNA degradation machinery. Our results thus reveal unexpected paradigms governing RNA structural changes and the eukaryotic RNA life cycle.
Asunto(s)
Genoma de Planta , Respuesta al Choque Térmico , Oryza/fisiología , ARN Mensajero/metabolismo , ARN de Planta/genética , Calor , Oryza/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , ARN Mensajero/genética , ARN de Planta/metabolismo , TranscriptomaRESUMEN
The secondary structure of an RNA is often implicit to its function. Recently, various high-throughput RNA structure probing techniques have been developed to elucidate important RNA structure-function relationships genome-wide. These techniques produce unwieldy experimental data sets that require evaluation with unique computational pipelines. Herein, we present StructureFold2, a user-friendly set of analysis tools that makes precise data processing and detailed downstream analyses of such data sets both available and practical. StructureFold2 processes high-throughput reads sequenced from libraries prepared after experimental probing for reverse transcription (RT) stops generated by chemical modification of RNA at solvent accessible residues. This pipeline is able to analyze reads generated from a variety of structure-probing chemicals (e.g. DMS, glyoxal, SHAPE). Notably, StructureFold2 offers a new fully featured suite of utilities and tools to guide a user through multiple types of analyses. A particular emphasis is placed on analyzing the reactivity patterns of transcripts, complementing their use as folding restraints for predicting RNA secondary structure. StructureFold2 is hosted as a Github repository and is available at (https://github.com/StructureFold2/StructureFold2).
Asunto(s)
Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Conformación de Ácido Nucleico , ARN/química , Análisis de Secuencia de ARN/métodos , Algoritmos , Biología Computacional/instrumentación , Perfilación de la Expresión Génica/instrumentación , Genoma , Secuenciación de Nucleótidos de Alto Rendimiento/instrumentación , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN/genética , Análisis de Secuencia de ARN/instrumentación , Programas Informáticos , Transcriptoma/genéticaRESUMEN
Elucidation of the folded structures that RNA forms in vivo is vital to understanding its functions. Chemical reagents that modify the Watson-Crick (WC) face of unprotected nucleobases are particularly useful in structure elucidation. Dimethyl sulfate penetrates cell membranes and informs on RNA base-pairing and secondary structure but only modifies the WC face of adenines and cytosines. We present glyoxal, methylglyoxal, and phenylglyoxal as potent in vivo reagents that target the WC face of guanines as well as cytosines and adenines. Tests on rice (Oryza sativa) 5.8S rRNA in vitro read out by reverse transcription and gel electrophoresis demonstrate specific modification of almost all guanines in a time- and pH-dependent manner. Subsequent in vivo tests on rice, a eukaryote, and Bacillus subtilis and Escherichia coli, Gram-positive and Gram-negative bacteria, respectively, showed that all three reagents enter living cells without prior membrane permeabilization or pH adjustment of the surrounding media and specifically modify solvent-exposed guanine, cytosine, and adenine residues.
Asunto(s)
Glioxal/química , Guanina/química , Sondas ARN/química , Bacillus subtilis , Emparejamiento Base , Escherichia coli , Guanina/metabolismo , Oryza , Sondas ARN/metabolismo , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN de Planta/química , ARN de Planta/metabolismo , Coloración y EtiquetadoRESUMEN
RNA serves many functions in biology such as splicing, temperature sensing, and innate immunity. These functions are often determined by the structure of RNA. There is thus a pressing need to understand RNA structure and how it changes during diverse biological processes both in vivo and genome-wide. Here, we present Structure-seq2, which provides nucleotide-resolution RNA structural information in vivo and genome-wide. This optimized version of our original Structure-seq method increases sensitivity by at least 4-fold and improves data quality by minimizing formation of a deleterious by-product, reducing ligation bias, and improving read coverage. We also present a variation of Structure-seq2 in which a biotinylated nucleotide is incorporated during reverse transcription, which greatly facilitates the protocol by eliminating two PAGE purification steps. We benchmark Structure-seq2 on both mRNA and rRNA structure in rice (Oryza sativa). We demonstrate that Structure-seq2 can lead to new biological insights. Our Structure-seq2 datasets uncover hidden breaks in chloroplast rRNA and identify a previously unreported N1-methyladenosine (m1A) in a nuclear-encoded Oryza sativa rRNA. Overall, Structure-seq2 is a rapid, sensitive, and unbiased method to probe RNA in vivo and genome-wide that facilitates new insights into RNA biology.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Genoma de Planta/genética , Conformación de Ácido Nucleico , ARN de Planta/química , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Modelos Genéticos , Oryza/genética , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Transcripción Reversa/genética , Análisis de Secuencia de ADN , Ésteres del Ácido Sulfúrico/químicaRESUMEN
Gel electrophoresis and subsequent imaging using phosphorimagers is one of the most important and widely used techniques in RNA and DNA analysis. Radiolabeling nucleic acids with 32P and detecting bands using a phoshorimager are useful both in a qualitative sense for nucleic acid detection and in a quantitative sense for structural, kinetic, or binding-based assays. Because of this, good resolution of gel bands based on molecular weight and size of RNA or DNA is essential for analysis. The appearance of blurry gel bands of 32P-labeled RNA and DNA thus represents a serious problem in the laboratory. A quick search on the Internet uncovers numerous reports begrudging the appearance of blurry bands, as well as attempts to fix them without success. Indeed, our laboratories were beset by the intermittent problem of blurry gels for over one year before we found a solution. Herein we describe a simple and cost-effective solution to a problem that we show originates from the phosphorimager cassettes rather than the integrity of the gel itself. We hope that the information provided here will lead to immediate help for other laboratories experiencing similar issues with labeled nucleic acid gel-based assays. The improvement in the clarity of the gels is nothing short of astonishing in many instances and will lead to higher resolution images for analysis and publications.
Asunto(s)
ADN/química , ARN/química , Análisis Costo-Beneficio , Electroforesis en Gel de PoliacrilamidaRESUMEN
Single-stranded RNA molecules fold into extraordinarily complicated secondary and tertiary structures as a result of intramolecular base pairing. In vivo, these RNA structures are not static. Instead, they are remodeled in response to changes in the prevailing physicochemical environment of the cell and as a result of intermolecular base pairing and interactions with RNA-binding proteins. Remarkable technical advances now allow us to probe RNA secondary structure at single-nucleotide resolution and genome-wide, both in vitro and in vivo. These data sets provide new glimpses into the RNA universe. Analyses of RNA structuromes in HIV, yeast, Arabidopsis, and mammalian cells and tissues have revealed regulatory effects of RNA structure on messenger RNA (mRNA) polyadenylation, splicing, translation, and turnover. Application of new methods for genome-wide identification of mRNA modifications, particularly methylation and pseudouridylation, has shown that the RNA "epitranscriptome" both influences and is influenced by RNA structure. In this review, we describe newly developed genome-wide RNA structure-probing methods and synthesize the information emerging from their application.