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T-box riboswitches are noncoding RNA elements involved in genetic regulation of most Gram-positive bacteria. They regulate amino acid metabolism by assessing the aminoacylation status of tRNA, subsequently affecting the transcription or translation of downstream amino acid metabolism-related genes. Here we present single-molecule FRET studies of the Mycobacterium tuberculosis IleS T-box riboswitch, a paradigmatic translational T-box. Results support a two-step binding model, where the tRNA anticodon is recognized first, followed by interactions with the NCCA sequence. Furthermore, after anticodon recognition, tRNA can transiently dock into the discriminator domain even in the absence of the tRNA NCCA-discriminator interactions. Establishment of the NCCA-discriminator interactions significantly stabilizes the fully bound state. Collectively, the data suggest high conformational flexibility in translational T-box riboswitches; and supports a conformational selection model for NCCA recognition. These findings provide a kinetic framework to understand how specific RNA elements underpin the binding affinity and specificity required for gene regulation.
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Anticodón , Mycobacterium tuberculosis , Conformación de Ácido Nucleico , ARN Bacteriano , ARN de Transferencia , Riboswitch , Riboswitch/genética , ARN de Transferencia/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/química , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/genética , Anticodón/metabolismo , Anticodón/genética , ARN Bacteriano/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/química , Transferencia Resonante de Energía de Fluorescencia , Biosíntesis de Proteínas , Regulación Bacteriana de la Expresión Génica , CinéticaRESUMEN
RNA molecules often play critical roles in assisting the formation of membraneless organelles in eukaryotic cells. Yet, little is known about the organization of RNAs within membraneless organelles. Here, using super-resolution imaging and nuclear speckles as a model system, we demonstrate that different sequence domains of RNA transcripts exhibit differential spatial distributions within speckles. Specifically, we image transcripts containing a region enriched in binding motifs of serine/arginine-rich (SR) proteins and another region enriched in binding motifs of heterogeneous nuclear ribonucleoproteins (hnRNPs). We show that these transcripts localize to the outer shell of speckles, with the SR motif-rich region localizing closer to the speckle center relative to the hnRNP motif-rich region. Further, we identify that this intra-speckle RNA organization is driven by the strength of RNA-protein interactions inside and outside speckles. Our results hint at novel functional roles of nuclear speckles and likely other membraneless organelles in organizing RNA substrates for biochemical reactions.
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Stress-induced condensation of mRNA and proteins into stress granules is conserved across eukaryotes, yet the function, formation mechanisms, and relation to well-studied conserved transcriptional responses remain largely unresolved. Stress-induced exposure of ribosome-free mRNA following translational shutoff is thought to cause condensation by allowing new multivalent RNA-dependent interactions, with RNA length and associated interaction capacity driving increased condensation. Here we show that, in striking contrast, virtually all mRNA species condense in response to multiple unrelated stresses in budding yeast, length plays a minor role, and instead, stress-induced transcripts are preferentially excluded from condensates, enabling their selective translation. Using both endogenous genes and reporter constructs, we show that translation initiation blockade, rather than resulting ribosome-free RNA, causes condensation. These translation initiation-inhibited condensates (TIICs) are biochemically detectable even when stress granules, defined as microscopically visible foci, are absent or blocked. TIICs occur in unstressed yeast cells, and, during stress, grow before the appearance of visible stress granules. Stress-induced transcripts are excluded from TIICs primarily due to the timing of their expression, rather than their sequence features. Together, our results reveal a simple system by which cells redirect translational activity to newly synthesized transcripts during stress, with broad implications for cellular regulation in changing conditions.
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Nuclear speckles, a type of membraneless nuclear organelle in higher eukaryotic cells, play a vital role in gene expression regulation. Using the reverse transcription-based RNA-binding protein binding sites sequencing (ARTR-seq) method, we study human transcripts associated with nuclear speckles. We identify three gene groups whose transcripts demonstrate different speckle localization properties and dynamics: stably enriched in nuclear speckles, transiently enriched in speckles at the pre-mRNA stage, and not enriched in speckles. Specifically, we find that stably-enriched transcripts contain inefficiently spliced introns. We show that nuclear speckles specifically facilitate splicing of speckle-enriched transcripts. We further reveal RNA sequence features contributing to transcript speckle localization, underscoring a tight interplay between genome organization, RNA cis-elements, and transcript speckle enrichment, and connecting transcript speckle localization with splicing efficiency. Finally, we show that speckles can act as hubs for the regulated retention of introns during cellular stress. Collectively, our data highlight a role of nuclear speckles in both co- and post-transcriptional splicing regulation.
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RNA fate and function are affected by their structures and interactomes. However, how RNA and RNA-binding proteins (RBPs) assemble into higher-order structures and how RNA molecules may interact with each other to facilitate functions remain largely unknown. Here we present KARR-seq, which uses N3-kethoxal labeling and multifunctional chemical crosslinkers to covalently trap and determine RNA-RNA interactions and higher-order RNA structures inside cells, independent of local protein binding to RNA. KARR-seq depicts higher-order RNA structure and detects widespread intermolecular RNA-RNA interactions with high sensitivity and accuracy. Using KARR-seq, we show that translation represses mRNA compaction under native and stress conditions. We determined the higher-order RNA structures of respiratory syncytial virus (RSV) and vesicular stomatitis virus (VSV) and identified RNA-RNA interactions between the viruses and the host RNAs that potentially regulate viral replication.
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RNA-binding proteins (RBPs) regulate diverse cellular processes by dynamically interacting with RNA targets. However, effective methods to capture both stable and transient interactions between RBPs and their RNA targets are still lacking, especially when the interaction is dynamic or samples are limited. Here we present an assay of reverse transcription-based RBP binding site sequencing (ARTR-seq), which relies on in situ reverse transcription of RBP-bound RNAs guided by antibodies to identify RBP binding sites. ARTR-seq avoids ultraviolet crosslinking and immunoprecipitation, allowing for efficient and specific identification of RBP binding sites from as few as 20 cells or a tissue section. Taking advantage of rapid formaldehyde fixation, ARTR-seq enables capturing the dynamic RNA binding by RBPs over a short period of time, as demonstrated by the profiling of dynamic RNA binding of G3BP1 during stress granule assembly on a timescale as short as 10 minutes.
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ARN , Transcripción Reversa , ARN/genética , ARN/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Sitios de Unión/genética , Unión ProteicaRESUMEN
Multiple paralogous ABCF ATPases are encoded in most genomes, but the physiological functions remain unknown for most of them. We herein compare the four Escherichia coli K12 ABCFs - EttA, Uup, YbiT, and YheS - using assays previously employed to demonstrate EttA gates the first step of polypeptide elongation on the ribosome dependent on ATP/ADP ratio. A Δ uup knockout, like Δ ettA , exhibits strongly reduced fitness when growth is restarted from long-term stationary phase, but neither Δ ybiT nor Δ yheS exhibits this phenotype. All four proteins nonetheless functionally interact with ribosomes based on in vitro translation and single-molecule fluorescence resonance energy transfer experiments employing variants harboring glutamate-to-glutamine active-site mutations (EQ 2 ) that trap them in the ATP-bound conformation. These variants all strongly stabilize the same global conformational state of a ribosomal elongation complex harboring deacylated tRNA Val in the P site. However, EQ 2 -Uup uniquely exchanges on/off the ribosome on a second timescale, while EQ 2 -YheS-bound ribosomes uniquely sample alternative global conformations. At sub-micromolar concentrations, EQ 2 -EttA and EQ 2 -YbiT fully inhibit in vitro translation of an mRNA encoding luciferase, while EQ 2 -Uup and EQ 2 -YheS only partially inhibit it at ~10-fold higher concentrations. Moreover, tripeptide synthesis reactions are not inhibited by EQ 2 -Uup or EQ 2 -YheS, while EQ 2 -YbiT inhibits synthesis of both peptide bonds and EQ 2 -EttA specifically traps ribosomes after synthesis of the first peptide bond. These results support the four E. coli ABCF paralogs all having different activities on translating ribosomes, and they suggest that there remains a substantial amount of functionally uncharacterized "dark matter" involved in mRNA translation.
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Biomolecular machines are complex macromolecular assemblies that utilize thermal and chemical energy to perform essential, multistep, cellular processes. Despite possessing different architectures and functions, an essential feature of the mechanisms of action of all such machines is that they require dynamic rearrangements of structural components. Surprisingly, biomolecular machines generally possess only a limited set of such motions, suggesting that these dynamics must be repurposed to drive different mechanistic steps. Although ligands that interact with these machines are known to drive such repurposing, the physical and structural mechanisms through which ligands achieve this remain unknown. Using temperature-dependent, single-molecule measurements analyzed with a time-resolution-enhancing algorithm, here, we dissect the free-energy landscape of an archetypal biomolecular machine, the bacterial ribosome, to reveal how its dynamics are repurposed to drive distinct steps during ribosome-catalyzed protein synthesis. Specifically, we show that the free-energy landscape of the ribosome encompasses a network of allosterically coupled structural elements that coordinates the motions of these elements. Moreover, we reveal that ribosomal ligands which participate in disparate steps of the protein synthesis pathway repurpose this network by differentially modulating the structural flexibility of the ribosomal complex (i.e., the entropic component of the free-energy landscape). We propose that such ligand-dependent entropic control of free-energy landscapes has evolved as a general strategy through which ligands may regulate the functions of all biomolecular machines. Such entropic control is therefore an important driver in the evolution of naturally occurring biomolecular machines and a critical consideration for the design of synthetic molecular machines.
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Biosíntesis de Proteínas , Ribosomas , Ribosomas/metabolismo , Entropía , Movimiento (Física)RESUMEN
Soybean plays an important role in food, medicine, and industry. The quality inspection of soybean is essential for soybean yield and the agricultural economy. However, soybean pest is an important factor that seriously affects soybean yield, among which leguminivora glycinivorella matsumura is the most frequent pest. Aiming at the problem that the traditional detection methods have low accuracy and need a large number of samples to train the model, this paper proposed a detection method for leguminivora glycinivorella matsumura based on an A-ResNet (Attention-ResNet) meta-learning model. In this model, the ResNet network was combined with Attention to obtain the feature vectors that can better express the samples, so as to improve the performance of the model. As well, the classifier was designed as a multi-class support vector machine (SVM) to reduce over-fitting. Furthermore, in order to improve the training stability of the model and the prediction performance on the testing set, the traditional Batch Normalization was replaced by the Layer Normalization, and the Label Smooth method was used to punish the original loss. The experimental results showed that the accuracy of the A-ResNet meta-learning model reached 94.57 ± 0.19%, which can realize rapid and accurate nondestructive detection, and provides theoretical support for the intelligent detection of soybean pests.
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Glycine max , Mariposas Nocturnas , Animales , Agricultura , Alimentos , Máquina de Vectores de SoporteRESUMEN
The N6-methyladenosine (m6A) modification is deposited to nascent transcripts on chromatin, but its site-specificity mechanism is mostly unknown. Here we model the m6A deposition to pre-mRNA by iM6A (intelligent m6A), a deep learning method, demonstrating that the site-specific m6A methylation is primarily determined by the flanking nucleotide sequences. iM6A accurately models the m6A deposition (AUROC = 0.99) and uncovers surprisingly that the cis-elements regulating the m6A deposition preferentially reside within the 50 nt downstream of the m6A sites. The m6A enhancers mostly include part of the RRACH motif and the m6A silencers generally contain CG/GT/CT motifs. Our finding is supported by both independent experimental validations and evolutionary conservation. Moreover, our work provides evidences that mutations resulting in synonymous codons can affect the m6A deposition and the TGA stop codon favors m6A deposition nearby. Our iM6A deep learning modeling enables fast paced biological discovery which would be cost-prohibitive and unpractical with traditional experimental approaches, and uncovers a key cis-regulatory mechanism for m6A site-specific deposition.
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Aprendizaje Profundo , Secuencia de Bases , Codón de Terminación , Metilación , Secuencias Reguladoras de Ácidos Nucleicos/genéticaRESUMEN
Many RNA-RNA interactions depend on molecular chaperones to form and remain stable in living cells. A prime example is the RNA chaperone Hfq, which is a critical effector involved in regulatory interactions between small RNAs (sRNAs) and cognate target mRNAs in Enterobacteriaceae. While there is a great deal of in vitro biochemical evidence supporting the model that Hfq enhances rates or affinities of sRNA:mRNA interactions, there is little corroborating in vivo evidence. Here we used in vivo tools including reporter genes, co-purification assays, and super-resolution microscopy to analyze the role of Hfq in RyhB-mediated regulation, and we found that Hfq is often unnecessary for efficient RyhB:mRNA complex formation in vivo. Remarkably, our data suggest that a primary function of Hfq is to promote RyhB-induced cleavage of mRNA targets by RNase E. Moreover, our work indicates that Hfq plays a more limited role in dictating regulatory outcomes following sRNAs RybB and DsrA complex formation with specific target mRNAs. Our investigation helps evaluate the roles played by Hfq in some RNA-mediated regulation.
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Small RNAs (sRNAs) are important gene regulators in bacteria. Many sRNAs act post-transcriptionally by affecting translation and degradation of the target mRNAs upon base-pairing interactions. Here we present a general approach combining imaging and mathematical modeling to determine kinetic parameters at different levels of sRNA-mediated gene regulation that contribute to overall regulation efficacy. Our data reveal that certain sRNAs previously characterized as post-transcriptional regulators can regulate some targets co-transcriptionally, leading to a revised model that sRNA-mediated regulation can occur early in an mRNA's lifetime, as soon as the sRNA binding site is transcribed. This co-transcriptional regulation is likely mediated by Rho-dependent termination when transcription-coupled translation is reduced upon sRNA binding. Our data also reveal several important kinetic steps that contribute to the differential regulation of mRNA targets by an sRNA. Particularly, binding of sRNA to the target mRNA may dictate the regulation hierarchy observed within an sRNA regulon.
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Regulación Bacteriana de la Expresión Génica/genética , ARN Bacteriano/genética , ARN Mensajero/metabolismo , ARN Pequeño no Traducido/genética , Bacterias/genética , Bacterias/metabolismo , Fenómenos Fisiológicos Bacterianos , Escherichia coli/genética , ARN Bacteriano/metabolismo , ARN Pequeño no Traducido/metabolismo , Regulón/genéticaRESUMEN
Protein ubiquitination shows remarkable topological and functional diversity through the polymerization of ubiquitin via different linkages. Deciphering the cellular ubiquitin code is of central importance to understand the physiology of the cell. However, our understanding of its function is rather limited due to the lack of specific binders as tools to detect K29-linked polyubiquitin. In this study, we screened and characterized a synthetic antigen-binding fragment, termed sAB-K29, that can specifically recognize K29-linked polyubiquitin using chemically synthesized K29-linked diubiquitin. We further determined the crystal structure of this fragment bound to the K29-linked diubiquitin, which revealed the molecular basis of specificity. Using sAB-K29 as a tool, we uncovered that K29-linked ubiquitination is involved in different kinds of cellular proteotoxic stress response as well as cell cycle regulation. In particular, we showed that K29-linked ubiquitination is enriched in the midbody and downregulation of the K29-linked ubiquitination signal arrests cells in G1/S phase.
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Ubiquitina-Proteína Ligasas/metabolismo , Ciclo Celular , Línea Celular Tumoral , Humanos , Modelos Moleculares , Transducción de Señal , Estrés Fisiológico , Ubiquitina-Proteína Ligasas/química , UbiquitinaciónRESUMEN
Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current 'state of the art' from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of 'soft recommendations' about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage 'open science' practices.
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Transferencia Resonante de Energía de Fluorescencia/métodos , Biología Molecular/métodos , Imagen Individual de Molécula/métodos , Biología Molecular/instrumentación , Imagen Individual de Molécula/instrumentaciónRESUMEN
Base-pairing interactions mediate many intermolecular target recognition events. Even a single base-pair mismatch can cause a substantial difference in activity but how such changes influence the target search kinetics in vivo is unknown. Here, we use high-throughput sequencing and quantitative super-resolution imaging to probe the mutants of bacterial small RNA, SgrS, and their regulation of ptsG mRNA target. Mutations that disrupt binding of a chaperone protein, Hfq, and are distal to the mRNA annealing region still decrease the rate of target association, kon, and increase the dissociation rate, koff, showing that Hfq directly facilitates sRNA-mRNA annealing in vivo. Single base-pair mismatches in the annealing region reduce kon by 24-31% and increase koff by 14-25%, extending the time it takes to find and destroy the target by about a third. The effects of disrupting contiguous base-pairing are much more modest than that expected from thermodynamics, suggesting that Hfq buffers base-pair disruptions.
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Emparejamiento Base/genética , Estabilidad del ARN , ARN Bacteriano/genética , Secuencia de Bases , Escherichia coli/genética , Dosificación de Gen , Genes Reporteros , Imagenología Tridimensional , Cinética , Mutación/genética , Nucleótidos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de TiempoRESUMEN
RNA-binding proteins play myriad roles in regulating RNAs and RNA-mediated functions. In bacteria, the RNA chaperone Hfq is an important post-transcriptional gene regulator. Using live-cell super-resolution imaging, we can distinguish Hfq binding to different sizes of cellular RNAs. We demonstrate that under normal growth conditions, Hfq exhibits widespread mRNA-binding activity, with the distal face of Hfq contributing mostly to the mRNA binding in vivo. In addition, sRNAs can either co-occupy Hfq with the mRNA as a ternary complex, or displace the mRNA from Hfq in a binding face-dependent manner, suggesting mechanisms through which sRNAs rapidly access Hfq to induce sRNA-mediated gene regulation. Finally, our data suggest that binding of Hfq to certain mRNAs through its distal face can recruit RNase E to promote turnover of these mRNAs in a sRNA-independent manner, and such regulatory function of Hfq can be decoyed by sRNA competitors that bind strongly at the distal face.
Messenger RNAs or mRNAs are molecules that the cell uses to transfer the information stored in the cell's DNA so it can be used to make proteins. Bacteria can regulate their levels of mRNA molecules, and they can therefore control how many proteins are being made, by producing a different type of RNA called small regulatory RNAs or sRNAs. Each sRNA can bind to several specific mRNA targets, and lead to their degradation by an enzyme called RNase E. Certain bacterial RNA-binding proteins, such as Hfq, protect sRNAs from being degraded, and help them find their mRNA targets. Hfq is abundant in bacteria. It is critical for bacterial growth under harsh conditions and it is involved in the process through which pathogenic bacteria infect cells. However, it is outnumbered by the many different RNA molecules in the cell, which compete for binding to the protein. It is not clear how Hfq prioritizes the different RNAs, or how binding to Hfq alters RNA regulation. Park, Prévost et al. imaged live bacterial cells to see how Hfq binds to RNA strands of different sizes. The experiments revealed that, when bacteria are growing normally, Hfq is mainly bound to mRNA molecules, and it can recruit RNase E to speed up mRNA degradation without the need for sRNAs. Park, Prévost et al. also showed that sRNAs could bind to Hfq by either replacing the bound mRNA or co-binding alongside it. The sRNA molecules that strongly bind Hfq can compete against mRNA for binding, and thus slow down the degradation of certain mRNAs. Hfq could be a potential drug target for treating bacterial infections. Understanding how it interacts with other molecules in bacteria could provide help in the development of new therapeutics. These findings suggest that a designed RNA that binds strongly to Hfq could disrupt its regulatory roles in bacteria, killing them. This could be a feasible drug design opportunity to counter the emergence of antibiotic-resistant bacteria.
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Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteína de Factor 1 del Huésped/metabolismo , ARN Mensajero/metabolismo , ARN Pequeño no Traducido/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteína de Factor 1 del Huésped/genética , Chaperonas Moleculares/metabolismo , Procesamiento Postranscripcional del ARN , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Pequeño no Traducido/genéticaRESUMEN
This chapter describes two mechanical expansion microscopy methods with accompanying step-by-step protocols. The first method, mechanically resolved expansion microscopy, uses non-uniform expansion of partially digested samples to provide the imaging contrast that resolves local mechanical properties. Examining bacterial cell wall with this method, we are able to distinguish bacterial species in mixed populations based on their distinct cell wall rigidity and detect cell wall damage caused by various physiological and chemical perturbations. The second method is mechanically locked expansion microscopy, in which we use a mechanically stable gel network to prevent the original polyacrylate network from shrinking in ionic buffers. This method allows us to use anti-photobleaching buffers in expansion microscopy, enabling detection of novel ultra-structures under the optical diffraction limit through super-resolution single molecule localization microscopy on bacterial cells and whole-mount immunofluorescence imaging in thick animal tissues. We also discuss potential applications and assess future directions.
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Pared Celular , Imagen Individual de Molécula , Animales , Microscopía FluorescenteRESUMEN
N6-Methyladenosine (m6A) is the most prevalent modified base in eukaryotic mRNA and long noncoding RNA. Although candidate sites for the m6A modification are identified at the transcriptomic level, methods for site-specific quantification of absolute m6A modification levels are still limited. Herein, we present a facile method implementing a deoxyribozyme, VMC10, which preferentially cleaves the unmodified RNA. We leveraged reverse transcription and real-time quantitative PCR along with key control experiments to quantify the methylation fraction of specific m6A sites. We validated the accuracy of this method with synthetic RNA in which methylation fractions ranged from 0 to 100% and applied our method to several endogenous sites that were previously identified in sequencing-based studies. This method provides a time- and cost-effective approach for absolute quantification of the m6A fraction at specific loci, with the potential for multiplexed quantifications, expanding the current toolkit for studying RNA modifications.
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Adenosina/análogos & derivados , ADN Catalítico/química , ARN/química , Adenosina/análisis , Adenosina/química , Células HeLa , Humanos , MetilaciónRESUMEN
Immunofluorescence (IF) is widely used to study the cellular localization and organization of proteins. However, steps such as fixation and permeabilization may affect cell morphology and/or introduce artifacts. For bacterial cells, commonly used permeabilization methods for IF include treatment with lysozyme. Here, we demonstrate two potential pitfalls in IF due to specific permeabilization methods: flattening or disruption of the cells caused by lysozyme treatment and inaccessibility of the antibody to the fixed nucleoid region. To solve these issues, we propose an improved IF method for bacterial cells, which includes the combined treatment with 70% ethanol, lysozyme, and DNase I. Treatment with 70% ethanol before the lysozyme permeabilization can better preserve the three-dimensional shape of the cell, and treatment with DNase I after the lysozyme permeabilization can eliminate the inaccessibility of the antibody to the nucleoid region. We further demonstrate that the DNase I treatment does not affect the preservation of the DNA-associated structure or organization of proteins. Finally, the method is also compatible with applications in which IF needs to be combined with RNA fluorescence in situ hybridization.