RESUMEN
Translation regulation and localized translation are essential for protein synthesis, controlling all aspects of cellular function in health and disease [...].
RESUMEN
Aminoacyl tRNA synthetases (aaRSs) are a well-studied family of enzymes with a canonical role in charging tRNAs with a specific amino acid. These proteins appear to also have non-canonical roles, including post-transcriptional regulation of mRNA expression. Many aaRSs were found to bind mRNAs and regulate their translation into proteins. However, the mRNA targets, mechanism of interaction, and regulatory consequences of this binding are not fully resolved. Here, we focused on yeast cytosolic threonine tRNA synthetase (ThrRS) to decipher its impact on mRNA binding. Affinity purification of ThrRS with its associated mRNAs followed by transcriptome analysis revealed a preference for mRNAs encoding RNA polymerase subunits. An mRNA that was significantly bound compared to all others was the mRNA encoding RPC10, a small subunit of RNA polymerase III. Structural modeling suggested that this mRNA includes a stem-loop element that is similar to the anti-codon stem loop (ASL) structure of ThrRS cognate tRNA (tRNAThr). We introduced random mutations within this element and found that almost every change from the normal sequence leads to reduced binding by ThrRS. Furthermore, point mutations at six key positions that abolish the predicted ASL-like structure showed a significant decrease in ThrRS binding with a decrease in RPC10 protein levels. Concomitantly, tRNAThr levels were reduced in the mutated strain. These data suggest a novel regulatory mechanism in which cellular tRNA levels are regulated through a mimicking element within an RNA polymerase III subunit in a manner that involves the tRNA cognate aaRS.
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ARN Polimerasa III , Aminoacil-ARNt Sintetasas/genética , Codón , Ligasas/genética , ARN Polimerasa III/genética , ARN Mensajero/genética , ARN de Transferencia/metabolismo , ARN de Transferencia de Treonina/metabolismo , Saccharomyces cerevisiae/genética , Treonina/genética , Treonina/metabolismo , Treonina-ARNt Ligasa/química , Treonina-ARNt Ligasa/genética , Treonina-ARNt Ligasa/metabolismoRESUMEN
Nuclear-encoded mitochondrial protein mRNAs have been found to be localized and locally translated within neuronal processes. However, the mechanism of transport for those mRNAs to distal locations is not fully understood. Here, we describe axonal co-transport of Cox7c with mitochondria. Fractionation analysis and single-molecule fluorescence in situ hybridization (smFISH) assay revealed that endogenous mRNA encoding Cox7c was preferentially associated with mitochondria in a mouse neuronal cell line and within mouse primary motor neuron axons, whereas other mRNAs that do not encode mitochondrial protein were much less associated. Live-cell imaging of MS2-tagged Cox7c mRNA further confirmed the preferential colocalization and co-transport of Cox7c mRNA with mitochondria in motor neuron axons. Intriguingly, the coding region, rather than the 3' untranslated region (UTR), was the key domain for the co-transport. Our results reveal that Cox7c mRNA can be transported with mitochondria along significant distances and that its coding region is a major recognition feature. This is consistent with the idea that mitochondria can play a vital role in spatial regulation of the axonal transcriptome at distant neuronal sites.
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Axones , Complejo IV de Transporte de Electrones/metabolismo , Mitocondrias , Regiones no Traducidas 3'/genética , Animales , Axones/metabolismo , Hibridación Fluorescente in Situ , Ratones , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
A new environmental study has discovered marine phages containing deoxyuridine instead of deoxythymidine in their DNA. The newly isolated viruses are phylogenetically distinct from any currently known double-stranded DNA phages.
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Bacteriófagos , Bacteriófagos/genética , Biología , Genoma ViralRESUMEN
Aminoacyl-tRNA synthetases (aaRSs) are a conserved family of enzymes with an essential role in protein synthesis: ligating amino acids to cognate tRNA molecules for translation. In addition to their role in tRNA charging, aaRSs have acquired non-canonical functions, including post-transcriptional regulation of mRNA expression. Yet, the extent and mechanisms of these post-transcriptional functions are largely unknown. Herein, we performed a comprehensive transcriptome analysis to define the mRNAs that are associated with almost all aaRSs present in S. cerevisiae cytosol. Nineteen (out of twenty) isogenic strains of GFP-tagged cytosolic aaRSs were subjected to immunoprecipitation with anti-GFP beads along with an untagged control. mRNAs associated with each aaRS were then identified by RNA-seq. The extent of mRNA association varied significantly between aaRSs, from MetRS in which none appeared to be statistically significant, to PheRS that binds hundreds of different mRNAs. Interestingly, many target mRNAs are bound by multiple aaRSs, suggesting co-regulation by this family of enzymes. Gene Ontology analyses for aaRSs with a considerable number of target mRNAs discovered an enrichment for pathways of amino acid metabolism and of ribosome biosynthesis. Furthermore, sequence and structure motif analysis revealed for some aaRSs an enrichment for motifs that resemble the anticodon stem loop of cognate tRNAs. These data suggest that aaRSs coordinate mRNA expression in response to amino acid availability and may utilize RNA elements that mimic their canonical tRNA binding partners.
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Aminoacil-ARNt Sintetasas/metabolismo , Citosol/enzimología , Regulación Fúngica de la Expresión Génica , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Aminoacil-ARNt Sintetasas/genética , ARN Mensajero/genética , ARN de Transferencia/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Recent studies underscore RNA modifications as a novel mechanism to coordinate expression and function of different genes. While modifications on the sugar or base moieties of tRNA are well known, their roles in mRNA regulation are only starting to emerge. Interestingly, some modifications are present in both tRNA and mRNA, and here we discuss the functional significance of these common features. We describe key modifications that are present in both RNA types, elaborate on proteins that interact with them, and indicate recent works that identify roles in communicating tRNA processes and mRNA regulation. We propose that as tools are developed, the shortlist of features that are common between types of RNA will greatly expand and proteins that interact with them will be identified. In conclusion, the presence of the same modification in both RNA types provides an intersect between tRNA processes and mRNA regulation and implies a novel mechanism for connecting diverse cellular processes.
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Procesamiento Postranscripcional del ARN/genética , ARN Mensajero/genética , ARN de Transferencia/genética , ARN/genética , Epigénesis Genética/ética , Epigénesis Genética/genética , Biosíntesis de Proteínas/genética , ARN/biosíntesisRESUMEN
Modification of nucleotides within an mRNA emerges as a key path for gene expression regulation. Pseudouridine is one of the most common RNA modifications; however, only a few mRNA modifiers have been identified to date, and no one mRNA pseudouridine reader is known. Here, we applied a novel genome-wide approach to identify mRNA regions that are bound by yeast methionine aminoacyl tRNAMet synthetase (MetRS). We found a clear enrichment to regions that were previously described to contain pseudouridine (Ψ). Follow-up in vitro and in vivo analyses on a prime target (position 1074 within YEF3 mRNA) demonstrated the importance of pseudouridine for MetRS binding. Furthermore, polysomal and protein analyses revealed that Ψ1074 mediates translation. Modification of this site occurs presumably by Pus6, a pseudouridine synthetase known to modify MetRS cognate tRNA. Consistently, the deletion of Pus6 leads to a decrease in MetRS association with both tRNAMet and YEF3 mRNA. Furthermore, while global protein synthesis decreases in pus6Δ, translation of YEF3 increases. Together, our data imply that Pus6 'writes' modifications on tRNA and mRNA, and both types of RNAs are 'read' by MetRS for translation regulation purposes. This represents a novel integrated path for writing and reading modifications on both tRNA and mRNA, which may lead to coordination between global and gene-specific translational responses.
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Regulación Fúngica de la Expresión Génica , Metionina-ARNt Ligasa/metabolismo , Factores de Elongación de Péptidos/biosíntesis , Biosíntesis de Proteínas , Seudouridina/fisiología , ARN de Hongos/genética , ARN Mensajero/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sistemas CRISPR-Cas , Metionina/metabolismo , Factores de Elongación de Péptidos/genética , Polirribosomas/metabolismo , Unión Proteica , Procesamiento Postranscripcional del ARN , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Mitochondria contain a complete translation machinery that is used to translate its internally transcribed mRNAs. This machinery uses a distinct set of tRNAs that are charged with cognate amino acids inside the organelle. Interestingly, charging is executed by aminoacyl tRNA synthetases (aaRS) that are encoded by the nuclear genome, translated in the cytosol, and need to be imported into the mitochondria. Here, we review import mechanisms of these enzymes with emphasis on those that are localized to both mitochondria and cytosol. Furthermore, we describe RNA recognition features of these enzymes and their interaction with tRNA and non-tRNA molecules. The dual localization of mitochondria-destined aaRSs and their association with various RNA types impose diverse impacts on cellular physiology. Yet, the breadth and significance of these functions are not fully resolved. We highlight here possibilities for future explorations.
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Aminoacil-ARNt Sintetasas/metabolismo , Citosol/enzimología , Mitocondrias/enzimología , ARN/metabolismo , Animales , HumanosRESUMEN
A major challenge in cell and developmental biology is the automated identification and quantitation of cells in complex multilayered tissues. We developed CytoCensus: an easily deployed implementation of supervised machine learning that extends convenient 2D 'point-and-click' user training to 3D detection of cells in challenging datasets with ill-defined cell boundaries. In tests on such datasets, CytoCensus outperforms other freely available image analysis software in accuracy and speed of cell detection. We used CytoCensus to count stem cells and their progeny, and to quantify individual cell divisions from time-lapse movies of explanted Drosophila larval brains, comparing wild-type and mutant phenotypes. We further illustrate the general utility and future potential of CytoCensus by analysing the 3D organisation of multiple cell classes in Zebrafish retinal organoids and cell distributions in mouse embryos. CytoCensus opens the possibility of straightforward and robust automated analysis of developmental phenotypes in complex tissues.
There are around 200 billion cells in the human brain that are generated by a small pool of rapidly dividing stem cells. For the brain to develop correctly, these stem cells must produce an appropriate number of each type of cell in the right place, at the right time. However, it remains unclear how individual stem cells in the brain know when and where to divide. To answer this question, Hailstone et al. studied the larvae of fruit flies, which use similar genes and mechanisms as humans to control brain development. This involved devising a new method for extracting the brains of developing fruit flies and keeping the intact tissue alive for up to 24 hours while continuously imaging individual cells in three dimensions. Manually tracking the division of each cell across multiple frames of a time-lapse is extremely time consuming. To tackle this problem, Hailstone et al. created a tool called CytoCensus, which uses machine learning to automatically identify stem cells from three-dimensional images and track their rate of division over time. Using the CytoCensus tool, Hailstone et al. identified a gene that controls the diverse rates at whichstem cells divide in the brain. Earlier this year some of the same researchers also published a study showing that this gene regulates a well-known cancer-related protein using an unconventional mechanism. CytoCensus was also able to detect cells in other developing tissues, including the embryos of mice. In the future, this tool could aid research into diseases that affect complex tissues, such as neurodegenerative disorders and cancer.
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División Celular , Procesamiento de Imagen Asistido por Computador , Aprendizaje Automático , Microscopía por Video , Imagen de Lapso de Tiempo , Animales , Animales Modificados Genéticamente , Automatización , Encéfalo/embriología , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Embrión de Mamíferos/citología , Femenino , Larva/citología , Masculino , Ratones , Mutación , Organoides/citología , Fenotipo , Reproducibilidad de los Resultados , Retina/citología , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Pez CebraRESUMEN
During Drosophila and vertebrate brain development, the conserved transcription factor Prospero/Prox1 is an important regulator of the transition between proliferation and differentiation. Prospero level is low in neural stem cells and their immediate progeny, but is upregulated in larval neurons and it is unknown how this process is controlled. Here, we use single molecule fluorescent in situ hybridisation to show that larval neurons selectively transcribe a long prospero mRNA isoform containing a 15â kb 3' untranslated region, which is bound in the brain by the conserved RNA-binding protein Syncrip/hnRNPQ. Syncrip binding increases the stability of the long prospero mRNA isoform, which allows an upregulation of Prospero protein production. Adult flies selectively lacking the long prospero isoform show abnormal behaviour that could result from impaired locomotor or neurological activity. Our findings highlight a regulatory strategy involving alternative polyadenylation followed by differential post-transcriptional regulation.This article has an associated First Person interview with the first author of the paper.
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Proteínas de Drosophila/genética , Drosophila/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Proteínas Nucleares/genética , Poliadenilación , ARN Mensajero/genética , Factores de Transcripción/genética , Regiones no Traducidas 3' , Animales , Proteínas de Drosophila/metabolismo , Inmunohistoquímica , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Especificidad de Órganos/genética , Estabilidad del ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The yeast S. cerevisiae serves as a model organism for many decades. Numerous molecular tools have been developed throughout the years to engineer its genome. Specifically, homologous recombination protocols allowed gene deletion, replacement and tagging of almost every S. cerevisiae gene, thus enabling mechanistic understanding of various cellular processes. Recently, CRISPR/Cas9-based approaches have been adapted to the yeast system, simplifying the protocols to manipulate this organism. In CRISPR/Cas9 systems, guide-RNA directs a site-specific double-strand DNA cleavage by the Cas9 nuclease. The directed cleavage enhances homologous recombination events, thereby facilitating changes to desired genomic loci. The use of a single vector to express both guide-RNA and Cas9 enzyme may simplify genomic manipulations and was used to introduce double-strand breaks at artificial sites (Anand et al. in Nature 544(7650):377-380, 2017. https://doi.org/10.1038/nature22046) or within selection markers (Ryan et al. in Cold Spring Harbor Protoc, 2014. https://doi.org/10.1101/pdb.prot086827). Here, we generalize this approach to demonstrate its utility in modifying natural genomic loci. We devise vectors to perform common genetic manipulations in S. cerevisiae, including gene deletion, single-base mutations, introduction of site-specific polymorphism and tag insertion. Notably, a vector that efficiently cleaves within GFP was generated, allowing replacing a GFP tag with other sequences. This vector may be of utility for replacing any gene tagged with GFP by a sequence of choice. Importantly, we demonstrate the efficiency of chemically synthesized 80-mer homologous DNA as a substrate for recombination, alleviating the need for PCR steps in the procedure. In all presented applications, high efficiency of the expected gene alteration and no other change in the genomic loci were obtained. Overall, this work expands the repertoire of single-plasmid CRISPR/cas9 approaches and provides a facile alternative to manipulate the yeast genome.
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Sistemas CRISPR-Cas , Ingeniería Genética/métodos , Genoma Fúngico , Recombinación Homóloga , Plásmidos/genética , Saccharomyces cerevisiae/genética , Vectores Genéticos , ARN Guía de Kinetoplastida/genéticaRESUMEN
Aminoacyl tRNA synthetases (aaRS) are well studied for their roles in tRNA charging with cognate amino acid. Nevertheless, numerous lines of evidence indicate that these proteins have roles other than tRNA charging. These include coordination of cellular signaling cascades, induction of cytokines outside the cell and transcription regulation. Herein, we focus on their roles in post-transcriptional regulation of mRNA expression. We describe functions that are related to antitermination of transcription, RNA splicing and mRNA translation. Cases were recognition of mRNA by the aaRS involves recognition of tRNA-like structures are described. Such recognition may be achieved by repurposing tRNA-binding domains or through domains added to the aaRS later in evolution. Furthermore, we describe cases in which binding by aaRS is implicated in autogenous regulation of expression. Overall, we propose RNA-mimicry as a common mode of interaction between aaRS and mRNA which allows efficient expression regulation. This article is categorized under: RNA Processing > tRNA Processing RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications Translation > Translation Regulation.
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Aminoacil-ARNt Sintetasas/metabolismo , ARN/química , ARN/metabolismo , Humanos , ARN/genética , Procesamiento Postranscripcional del ARNRESUMEN
PUF proteins, named for Drosophila Pumilio (PUM) and Caenorhabditis elegans fem-3-binding factor (FBF), recognize specific sequences in the mRNAs they bind and control. RNA binding by classical PUF proteins is mediated by a characteristic PUM homology domain (PUM-HD). The Puf1 and Puf2 proteins possess a distinct architecture and comprise a highly conserved subfamily among fungal species. Puf1/Puf2 proteins contain two types of RNA-binding domain: a divergent PUM-HD and an RNA recognition motif (RRM). They recognize RNAs containing UAAU motifs, often in clusters. Here, we report a crystal structure of the PUM-HD of a fungal Puf1 in complex with a dual UAAU motif RNA. Each of the two UAAU tetranucleotides are bound by a Puf1 PUM-HD forming a 2:1 protein-to-RNA complex. We also determined crystal structures of the Puf1 RRM domain that identified a dimerization interface. The PUM-HD and RRM domains act in concert to determine RNA-binding specificity: the PUM-HD dictates binding to UAAU, and dimerization of the RRM domain favors binding to dual UAAU motifs rather than a single UAAU. Cooperative action of the RRM and PUM-HD identifies a new mechanism by which multiple RNA-binding modules in a single protein collaborate to create a unique RNA-binding specificity.
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ARN Mensajero/química , Proteínas de Unión al ARN/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Biblioteca de Genes , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Modelos Moleculares , Motivos de Nucleótidos , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Aminoacyl-tRNA synthetases (aaRSs) are well studied for their role in binding and charging tRNAs with cognate amino acids. Recent RNA interactome studies had suggested that these enzymes can also bind polyadenylated RNAs. Here, we explored the mRNA repertoire bound by several yeast aaRSs. RNA immunoprecipitation (RIP) followed by deep sequencing revealed unique sets of mRNAs bound by each aaRS. Interestingly, for every tested aaRSs, a preferential association with its own mRNA was observed, suggesting an autoregulatory process. Self-association of histidyl-tRNA synthetase (HisRS) was found to be mediated primarily through binding to a region predicted to fold into a tRNAHis anticodon-like structure. Introducing point mutations that are expected to disassemble this putative anticodon mimic alleviated self-association, concomitant with increased synthesis of the protein. Finally, we found that increased cellular levels of uncharged tRNAHis lead to reduced self-association and increased HisRS translation, in a manner that depends on the anticodon-like element. Together, these results reveal a novel post-transcriptional autoregulatory mechanism that exploits binding mimicry to control mRNA translation according to tRNA demands.
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Aminoacil-ARNt Sintetasas/metabolismo , Anticodón/genética , Biosíntesis de Proteínas , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/genética , Aminoacil-ARNt Sintetasas/química , Secuencia de Bases , Modelos Biológicos , Unión Proteica , Dominios Proteicos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Mitochondria exert their many functions through a repertoire of hundreds of proteins. The vast majority of these proteins are encoded in the nuclear genome, translated in the cytosol and imported into the mitochondria. Current models, derived mainly from work in yeast, suggest that the translation of many of these proteins can occur in close vicinity to the mitochondria outer membrane by localized ribosomes. Here, we applied ribosome-proximity biotin labeling to address this possibility. A clear biotinylation of ribosomes by mitochondrial Tom20-BirA fusion protein was observed in a human cell line. Isolation of these ribosomes revealed their preferred association with mRNAs encoding mitochondrial proteins. Furthermore, knock down of the mitochondrial protein receptor Tom70 resulted in a decrease in ribosomes translating mRNAs encoding proteins predicted to be recognized by Tom70. Intriguingly, levels of ribosomes translating mRNAs encoding targets of Tom20 were increased. We also knocked down the RNA binding protein CLUH that is implicated in regulation of mRNA encoding mitochondrial proteins, and found an increase in association of CLUH targets with mitochondria-proximal ribosomes. This is consistent with a role for CLUH in maintaining mRNAs encoding mitochondrial proteins in the cytosol. Overall, these data shed light on factors that contribute to association of translating ribosomes with human mitochondria and may suggest a co-translational mode of protein import into this organelle.
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Ribosome queuing is a fundamental phenomenon suggested to be related to topics such as genome evolution, synthetic biology, gene expression regulation, intracellular biophysics, and more. However, this phenomenon hasn't been quantified yet at a genomic level. Nevertheless, methodologies for studying translation (e.g. ribosome footprints) are usually calibrated to capture only single ribosome protected footprints (mRPFs) and thus limited in their ability to detect ribosome queuing. On the other hand, most of the models in the field assume and analyze a certain level of queuing. Here we present an experimental-computational approach for studying ribosome queuing based on sequencing of RNA footprints extracted from pairs of ribosomes (dRPFs) using a modified ribosome profiling protocol. We combine our approach with traditional ribosome profiling to generate a detailed profile of ribosome traffic. The data are analyzed using computational models of translation dynamics. The approach was implemented on the Saccharomyces cerevisiae transcriptome. Our data shows that ribosome queuing is more frequent than previously thought: the measured ratio of ribosomes within dRPFs to mRPFs is 0.2-0.35, suggesting that at least one to five translating ribosomes is in a traffic jam; these queued ribosomes cannot be captured by traditional methods. We found that specific regions are enriched with queued ribosomes, such as the 5'-end of ORFs, and regions upstream to mRPF peaks, among others. While queuing is related to higher density of ribosomes on the transcript (characteristic of highly translated genes), we report cases where traffic jams are relatively more severe in lowly expressed genes and possibly even selected for. In addition, our analysis demonstrates that higher adaptation of the coding region to the intracellular tRNA levels is associated with lower queuing levels. Our analysis also suggests that the Saccharomyces cerevisiae transcriptome undergoes selection for eliminating traffic jams. Thus, our proposed approach is an essential tool for high resolution analysis of ribosome traffic during mRNA translation and understanding its evolution.
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Biosíntesis de Proteínas , Ribosomas/fisiología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Calibración , Codón , Biología Computacional , Simulación por Computador , Regulación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Modelos Teóricos , Distribución Normal , Sistemas de Lectura Abierta , Probabilidad , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Análisis de Secuencia de ARN , Programas Informáticos , TranscriptomaRESUMEN
Members of the yeast family of PUF proteins bind unique subsets of mRNA targets that encode proteins with common functions. They therefore became a paradigm for post-transcriptional gene control. To provide new insights into the roles of the seemingly redundant Puf1 and Puf2 members, we monitored the growth rates of their deletions under many different stress conditions. A differential effect was observed at high CaCl2 concentrations, whereby puf1Δ growth was affected much more than puf2Δ, and inhibition was exacerbated in puf1Δpuf2Δ double knockout. Transcriptome analyses upon CaCl2 application for short and long terms defined the transcriptional response to CaCl2 and revealed distinct expression changes for the deletions. Intriguingly, mRNAs known to be bound by Puf1 or Puf2 were affected mainly in the double knockout. We focused on the cell wall regulator Zeo1 and observed that puf1Δpuf2Δ fails to maintain low levels of its mRNA. Complementarily, puf1Δpuf2Δ growth defect in CaCl2 was repaired upon further deletion of the Zeo1 gene. Thus, these proteins probably regulate the cell-wall integrity pathway by regulating Zeo1 post-transcriptionally. This work sheds new light on the roles of Puf proteins during the cellular response to environmental stress.
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Calcio/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Levaduras/genética , Levaduras/metabolismo , Cloruro de Calcio/metabolismo , Cloruro de Calcio/farmacología , Relación Dosis-Respuesta a Droga , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Modelos Biológicos , Estrés Fisiológico , Levaduras/efectos de los fármacosRESUMEN
RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.
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Proteínas de Unión al ARN , Proteínas Portadoras , Fraccionamiento Químico/métodos , Espectrometría de Masas , ARN , ARN Mensajero , Saccharomyces cerevisiaeRESUMEN
BACKGROUND: mRNA binding proteins (RBPs) constitute 10-15% of the eukaryotic proteome and play important part in post-transcriptional regulation of gene expression. Due to the instability of RNA and the transient nature its interaction with RBPs, identification of novel RBPs is a significant challenge. Recently, a novel methodology for RBP purification and identification (termed RaPID) was presented, which allows high affinity purification of RBPs while associated with mRNA in vivo. RESULTS: We performed a RaPID screen for proteins that interact with PMP1 mRNA in order to identify novel mRNA binding proteins. PMP1 mRNA was tagged in its 3' UTR with multiple MS2 loops and co-expressed with MS2-binding protein fused to streptavidin binding protein (SBP). RNA-protein complexes were cross-linked in vivo and isolated through streptavidin beads. The eluted proteins were subjected to mass spectroscopy analysis. The screen identified many proteins, about half of them were previously shown to bind RNA. We focused on eEF3 (YEF3), an essential translation elongation factor that interacts with ribosomes. Purification of TAP-tagged Yef3 with its associated RNAs confirmed that the native PMP1 transcript is associated with it. Intriguingly, high association with Yef3-TAP was observed when purification was performed in the presence of EDTA, and with PMP1 that contains stop codons immediately downstream to the initiation codon. Furthermore, high association was observed with a transcript containing only the 3' UTR of PMP1. Complementary, RaPID isolation of MS2-tagged 3' UTRs with their associated proteins revealed that Yef3 can efficiently interact with these regions. CONCLUSIONS: This study identifies many novel proteins that interact with PMP1 mRNA. Importantly, the elongation factor Yef3 was found to interact with mRNA in non-coding regions and in a translation independent manner. These results suggest an additional, non-elongation function for this factor.
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Proteínas de la Membrana/genética , Factores de Elongación de Péptidos/genética , Proteolípidos/genética , ATPasas de Translocación de Protón/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Regiones no Traducidas 3'/genética , Regulación Fúngica de la Expresión Génica/genética , Proteínas de la Membrana/metabolismo , Unión Proteica/fisiología , Biosíntesis de Proteínas/genética , Proteolípidos/metabolismo , ATPasas de Translocación de Protón/metabolismo , Procesamiento Postranscripcional del ARN , Proteínas de Saccharomyces cerevisiae/metabolismo , Estreptavidina/metabolismoRESUMEN
Local synthesis of proteins near their activity site has been demonstrated in many biological systems, and has diverse contributions to cellular functions. Studies in recent years have revealed that hundreds of mitochondria-destined proteins are synthesized by cytosolic ribosomes near the mitochondrial outer membrane, indicating that localized translation also occurs at this cellular locus. Furthermore, in the last year central factors that are involved in this process were identified in yeast, Drosophila, and human cells. Herein we review the experimental evidence for localized translation on the cytosolic side of the mitochondrial outer membrane; in addition, we describe the factors that are involved in this process and discuss the conservation of this mechanism among various species. We also describe the relationship between localized translation and import into the mitochondria and suggest avenues of study that look beyond cotranslational import. Finally we discuss future challenges in characterizing the mechanisms for localized translation and its physiological significance.