RESUMO
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.
Assuntos
RNA , Transcrição Reversa , RNA/genética , RNA/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sítios de Ligação/genética , Ligação ProteicaRESUMO
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.
Assuntos
Biossíntese de Proteínas , Ribossomos , Ribossomos/metabolismo , Entropia , Movimento (Física)RESUMO
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.
Assuntos
Ubiquitina-Proteína Ligases/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Humanos , Modelos Moleculares , Transdução de Sinais , Estresse Fisiológico , Ubiquitina-Proteína Ligases/química , UbiquitinaçãoRESUMO
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.
Assuntos
Glycine max , Mariposas , Animais , Agricultura , Alimentos , Máquina de Vetores de SuporteRESUMO
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.
Assuntos
Adenosina/análogos & derivados , DNA Catalítico/química , RNA/química , Adenosina/análise , Adenosina/química , Células HeLa , Humanos , MetilaçãoRESUMO
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.
Assuntos
Bactérias/citologia , Imunofluorescência/métodos , Hibridização in Situ Fluorescente/métodos , Anticorpos/química , Bactérias/ultraestrutura , Permeabilidade da Membrana Celular , Desoxirribonuclease I/química , Escherichia coli/citologia , Escherichia coli/ultraestrutura , Fixadores/química , Microscopia de Fluorescência , Muramidase/química , Imagem Óptica/métodos , Fixação de Tecidos/métodosRESUMO
Nuclear speckles are self-assembled organelles composed of RNAs and proteins. They are proposed to act as structural domains that control distinct steps in gene expression, including transcription, splicing and mRNA export. Earlier studies identified differential localization of a few components within the speckles. It was speculated that the spatial organization of speckle components might contribute directly to the order of operations that coordinate distinct processes. Here, by performing multi-color structured illumination microscopy, we characterized the multilayer organization of speckles at a higher resolution. We found that SON and SC35 (also known as SRSF2) localize to the central region of the speckle, whereas MALAT1 and small nuclear (sn)RNAs are enriched at the speckle periphery. Coarse-grained simulations indicate that the non-random organization arises due to the interplay between favorable sequence-encoded intermolecular interactions of speckle-resident proteins and RNAs. Finally, we observe positive correlation between the total amount of RNA present within a speckle and the speckle size. These results imply that speckle size may be regulated to accommodate RNA accumulation and processing. Accumulation of RNA from various actively transcribed speckle-associated genes could contribute to the observed speckle size variations within a single cell.
Assuntos
Núcleo Celular/genética , Proteínas de Ligação a DNA/genética , Antígenos de Histocompatibilidade Menor/genética , Organelas/genética , RNA Longo não Codificante/genética , Fatores de Processamento de Serina-Arginina/genética , Núcleo Celular/ultraestrutura , Regulação da Expressão Gênica , Células HeLa , Humanos , Organelas/ultraestrutura , Proteínas/genética , RNA/genética , RNA Nucleolar Pequeno/genéticaRESUMO
Single-cell fluorescence imaging is a powerful technique for studying inherently heterogeneous biological processes. To correlate a genotype or phenotype to a specific cell, images containing a population of cells must first be properly segmented. However, a proper segmentation with minimal user input becomes challenging when cells are clustered or overlapping in three dimensions. We introduce a new analysis package, Seg-3D, for the segmentation of bacterial cells in three-dimensional (3D) images, based on local thresholding, shape analysis, concavity-based cluster splitting, and morphology-based 3D reconstruction. The reconstructed cell volumes allow us to directly quantify the fluorescent signals from biomolecules of interest within individual cells. We demonstrate the application of this analysis package in 3D segmentation of individual bacterial pathogens invading host cells. We believe Seg-3D can be an efficient and simple program that can be used to analyze a wide variety of single-cell images, especially for biological systems involving random 3D orientation and clustering behavior, such as bacterial infection or colonization.
Assuntos
Bactérias/ultraestrutura , Imageamento Tridimensional/métodos , Imagem Óptica/métodos , Análise de Célula Única/métodos , Animais , Automação , Simulação por Computador , Proteínas de Fluorescência Verde/análise , Interações Hospedeiro-Patógeno , Análise dos Mínimos Quadrados , Macrófagos/microbiologia , Camundongos , Salmonella/química , Salmonella/ultraestrutura , Interface Usuário-ComputadorRESUMO
The RNase P-mediated endonucleolytic cleavage plays a crucial role in the 3' end processing and cellular accumulation of MALAT1, a nuclear-retained long noncoding RNA that promotes malignancy. The regulation of this cleavage event is largely undetermined. Here we characterize a broadly expressed natural antisense transcript at the MALAT1 locus, designated as TALAM1, that positively regulates MALAT1 levels by promoting the 3' end cleavage and maturation of MALAT1 RNA. TALAM1 RNA preferentially localizes at the site of transcription, and also interacts with MALAT1 RNA. Depletion of TALAM1 leads to defects in the 3' end cleavage reaction and compromises cellular accumulation of MALAT1. Conversely, overexpression of TALAM1 facilitates the cleavage reaction in trans Interestingly, TALAM1 is also positively regulated by MALAT1 at the level of both transcription and RNA stability. Together, our data demonstrate a novel feed-forward positive regulatory loop that is established to maintain the high cellular levels of MALAT1, and also unravel the existence of sense-antisense mediated regulatory mechanism for cellular lncRNAs that display RNase P-mediated 3' end processing.
Assuntos
Núcleo Celular/metabolismo , RNA Antissenso/genética , RNA Longo não Codificante/antagonistas & inibidores , Sequência de Bases , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Células HCT116 , Células HeLa , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Clivagem do RNA , Estabilidade de RNA , RNA Antissenso/química , RNA Antissenso/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Transdução de SinaisRESUMO
There are several sources of fluctuations in gene expression. Here we study the effects of time-dependent DNA replication, itself a tightly controlled process, on noise in mRNA levels. Stochastic simulations of constitutive and regulated gene expression are used to analyze the time-averaged mean and variation in each case. The simulations demonstrate that to capture mRNA distributions correctly, chromosome replication must be realistically modeled. Slow relaxation of mRNA from the low copy number steady state before gene replication to the high steady state after replication is set by the transcript's half-life and contributes significantly to the shape of the mRNA distribution. Consequently both the intrinsic kinetics and the gene location play an important role in accounting for the mRNA average and variance. Exact analytic expressions for moments of the mRNA distributions that depend on the DNA copy number, gene location, cell doubling time, and the rates of transcription and degradation are derived for the case of constitutive expression and subsequently extended to provide approximate corrections for regulated expression and RNA polymerase variability. Comparisons of the simulated models and analytical expressions to experimentally measured mRNA distributions show that they better capture the physics of the system than previous theories.
Assuntos
Algoritmos , Replicação do DNA , Regulação da Expressão Gênica , Modelos Genéticos , RNA Mensageiro/genética , DNA/genética , DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Dosagem de Genes , Cinética , RNA Mensageiro/metabolismo , Processos Estocásticos , Fatores de TempoRESUMO
The bacterial small RNA (sRNA) SgrS has been a fruitful model for discovery of novel RNA-based regulatory mechanisms and new facets of bacterial physiology and metabolism. SgrS is one of only a few characterized dual-function sRNAs. SgrS can control gene expression posttranscriptionally via sRNA-mRNA base-pairing interactions. Its second function is coding for the small protein SgrT. Previous work demonstrated that both functions contribute to relief of growth inhibition caused by glucose-phosphate stress, a condition characterized by disrupted glycolytic flux and accumulation of sugar phosphates. The base-pairing activity of SgrS has been the subject of numerous studies, but the activity of SgrT is less well characterized. Here, we provide evidence that SgrT acts to specifically inhibit the transport activity of the major glucose permease PtsG. Superresolution microscopy demonstrated that SgrT localizes to the cell membrane in a PtsG-dependent manner. Mutational analysis determined that residues in the N-terminal domain of PtsG are important for conferring sensitivity to SgrT-mediated inhibition of transport activity. Growth assays support a model in which SgrT-mediated inhibition of PtsG transport activity reduces accumulation of nonmetabolizable sugar phosphates and promotes utilization of alternative carbon sources by modulating carbon catabolite repression. The results of this study expand our understanding of a basic and well-studied biological problem, namely, how cells coordinate carbohydrate transport and metabolism. Further, this work highlights the complex activities that can be carried out by sRNAs and small proteins in bacteria.IMPORTANCE Sequencing, annotation and investigation of hundreds of bacterial genomes have identified vast numbers of small RNAs and small proteins, the majority of which have no known function. In this study, we explore the function of a small protein that acts in tandem with a well-characterized small RNA during metabolic stress to help bacterial cells maintain balanced metabolism and continue growing. Our results indicate that this protein acts on the glucose transport system, inhibiting its activity under stress conditions in order to allow cells to utilize alternative carbon sources. This work sheds new light on a key biological problem: how cells coordinate carbohydrate transport and metabolism. The study also expands our understanding of the functional capacities of small proteins.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Glucose/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Transporte Biológico , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Regiões Promotoras GenéticasRESUMO
One of the most challenging unanswered questions regarding the structural biology of biomolecular machines such as the two-subunit ribosome is whether and how these machines coordinate seemingly independent and random conformational fluctuations to maximize and regulate their functional efficiencies. To address this question, we have used ribosome mutagenesis or a ribosome-targeting antibiotic to predictably perturb the dynamics of intersubunit rotation, a structural rearrangement of the ribosome that is essential for the translocation and ejection of ribosome-bound tRNAs during translation. Concomitantly, we have used single-molecule fluorescence resonance energy transfer (smFRET) to characterize the effects of these perturbations on the dynamics of ribosomal L1 stalk movements and ribosome-bound tRNA reconfigurations, conformational changes that are likewise essential for the translocation and ejection of tRNAs during translation. Together with the results of complementary biochemical studies, our smFRET studies demonstrate that the ribosome uses cooperative conformational changes to maximize and regulate the efficiency with which it translocates and ejects tRNAs during translation. We propose that the ribosome employs cooperative conformational changes to efficiently populate global conformational states that are productive for translation, that translation factors exploit this cooperativity as part of their mechanisms of action, and that antibiotics exploit it to maximize the potency with which they inhibit translation. It is likely that similar cooperative conformational changes underlie the function and regulation of other biomolecular machines.
Assuntos
Biossíntese de Proteínas , Ribossomos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Conformação de Ácido Nucleico , RNA de Transferência/química , Eletricidade EstáticaRESUMO
Ribosomal frameshifting occurs when a ribosome slips a few nucleotides on an mRNA and generates a new sequence of amino acids. Programmed -1 ribosomal frameshifting (-1PRF) is used in various systems to express two or more proteins from a single mRNA at precisely regulated levels. We used single-molecule fluorescence resonance energy transfer (smFRET) to study the dynamics of -1PRF in the Escherichia coli dnaX gene. The frameshifting mRNA (FSmRNA) contained the frameshifting signals: a Shine-Dalgarno sequence, a slippery sequence, and a downstream stem loop. The dynamics of ribosomal complexes translating through the slippery sequence were characterized using smFRET between the Cy3-labeled L1 stalk of the large ribosomal subunit and a Cy5-labeled tRNA(Lys) in the ribosomal peptidyl-tRNA-binding (P) site. We observed significantly slower elongation factor G (EF-G)-catalyzed translocation through the slippery sequence of FSmRNA in comparison with an mRNA lacking the stem loop, ΔSL. Furthermore, the P-site tRNA/L1 stalk of FSmRNA-programmed pretranslocation (PRE) ribosomal complexes exhibited multiple fluctuations between the classical/open and hybrid/closed states, respectively, in the presence of EF-G before translocation, in contrast with ΔSL-programmed PRE complexes, which sampled the hybrid/closed state approximately once before undergoing translocation. Quantitative analysis showed that the stimulatory stem loop destabilizes the hybrid state and elevates the energy barriers corresponding to subsequent substeps of translocation. The shift of the FSmRNA-programmed PRE complex equilibrium toward the classical/open state and toward states that favor EF-G dissociation apparently allows the PRE complex to explore alternative translocation pathways such as -1PRF.
Assuntos
Escherichia coli/fisiologia , Mudança da Fase de Leitura do Gene Ribossômico/fisiologia , Modelos Genéticos , Modelos Moleculares , Conformação Molecular , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Proteínas de Bactérias/genética , DNA Polimerase III/genética , Escherichia coli/genética , Transferência Ressonante de Energia de Fluorescência , Ribossomos/fisiologiaRESUMO
By using single-molecule fluorescence resonance energy transfer (smFRET), we observe the real-time dynamic coupling between the ribosome, labeled at the L1 stalk, and transfer RNA (tRNA). We find that an interaction between the ribosomal L1 stalk and the newly deacylated tRNA is established spontaneously upon peptide bond formation; this event involves coupled movements of the L1 stalk and tRNAs as well as ratcheting of the ribosome. In the absence of elongation factor G, the entire pretranslocation ribosome fluctuates between just two states: a nonratcheted state, with tRNAs in their classical configuration and no L1 stalk-tRNA interaction, and a ratcheted state, with tRNAs in an intermediate hybrid configuration and a direct L1 stalk-tRNA interaction. We demonstrate that binding of EF-G shifts the equilibrium toward the ratcheted state. Real-time smFRET experiments reveal that the L1 stalk-tRNA interaction persists throughout the translocation reaction, suggesting that the L1 stalk acts to direct tRNA movements during translocation.
Assuntos
Conformação de Ácido Nucleico , Elongação Traducional da Cadeia Peptídica , Conformação Proteica , RNA de Transferência , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Carbocianinas/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/metabolismo , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Modelos Moleculares , RNA de Transferência/química , RNA de Transferência/metabolismo , Proteínas Ribossômicas/genética , Ribossomos/química , Ribossomos/metabolismoRESUMO
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.
Assuntos
Anticódon , Mycobacterium tuberculosis , Conformação de Ácido Nucleico , RNA Bacteriano , RNA de Transferência , Riboswitch , Riboswitch/genética , RNA de Transferência/metabolismo , RNA de Transferência/genética , RNA de Transferência/química , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/genética , Anticódon/metabolismo , Anticódon/genética , RNA Bacteriano/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/química , Transferência Ressonante de Energia de Fluorescência , Biossíntese de Proteínas , Regulação Bacteriana da Expressão Gênica , CinéticaRESUMO
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.
RESUMO
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.
RESUMO
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.
RESUMO
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.