RESUMEN
Molecular chaperones are critical for protein homeostasis and are implicated in several human pathologies such as neurodegeneration and cancer. While the binding of chaperones to nascent and misfolded proteins has been studied in great detail, the direct interaction between chaperones and RNA has not been systematically investigated. Here, we provide the evidence for widespread interaction between chaperones and RNA in human cells. We show that the major chaperone heat shock protein 70 (HSP70) binds to non-coding RNA transcribed by RNA polymerase III (RNA Pol III) such as tRNA and 5S rRNA. Global chromatin profiling revealed that HSP70 binds genomic sites of transcription by RNA Pol III. Detailed biochemical analyses showed that HSP70 alleviates the inhibitory effect of cognate tRNA transcript on tRNA gene transcription. Thus, our study uncovers an unexpected role of HSP70-RNA interaction in the biogenesis of a specific class of non-coding RNA with wider implications in cancer therapeutics.
Asunto(s)
Proteínas HSP70 de Choque Térmico , Neoplasias , Humanos , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , ARN , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , ARN de Transferencia/genética , ARN no Traducido/genéticaRESUMEN
The Central Dogma has been a useful conceptualization of the transfer of genetic information, and our understanding of the detailed mechanisms involved in that transfer continues to evolve. Here, we speak to several scientists about their research, how it influences our understanding of information transfer, and questions for the future.
RESUMEN
In vitro-transcribed (IVT) mRNAs are modalities that can combat human disease, exemplified by their use as vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). IVT mRNAs are transfected into target cells, where they are translated into recombinant protein, and the biological activity or immunogenicity of the encoded protein exerts an intended therapeutic effect1,2. Modified ribonucleotides are commonly incorporated into therapeutic IVT mRNAs to decrease their innate immunogenicity3-5, but their effects on mRNA translation fidelity have not been fully explored. Here we demonstrate that incorporation of N1-methylpseudouridine into mRNA results in +1 ribosomal frameshifting in vitro and that cellular immunity in mice and humans to +1 frameshifted products from BNT162b2 vaccine mRNA translation occurs after vaccination. The +1 ribosome frameshifting observed is probably a consequence of N1-methylpseudouridine-induced ribosome stalling during IVT mRNA translation, with frameshifting occurring at ribosome slippery sequences. However, we demonstrate that synonymous targeting of such slippery sequences provides an effective strategy to reduce the production of frameshifted products. Overall, these data increase our understanding of how modified ribonucleotides affect the fidelity of mRNA translation, and although there are no adverse outcomes reported from mistranslation of mRNA-based SARS-CoV-2 vaccines in humans, these data highlight potential off-target effects for future mRNA-based therapeutics and demonstrate the requirement for sequence optimization.
Asunto(s)
Sistema de Lectura Ribosómico , Seudouridina , ARN Mensajero , Animales , Humanos , Ratones , Vacuna BNT162/efectos adversos , Vacuna BNT162/genética , Vacuna BNT162/inmunología , Sistema de Lectura Ribosómico/genética , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Seudouridina/análogos & derivados , Seudouridina/metabolismo , Ribosomas/metabolismo , Biosíntesis de ProteínasRESUMEN
During the translation surveillance mechanism known as ribosome-associated quality control, the ASC-1 complex (ASCC) disassembles ribosomes stalled on the mRNA. Here, we show that there are two distinct classes of stalled ribosome. Ribosomes stalled by translation elongation inhibitors or methylated mRNA are short lived in human cells because they are split by the ASCC. In contrast, although ultraviolet light and 4-nitroquinoline 1-oxide induce ribosome stalling by damaging mRNA, and the ASCC is recruited to these stalled ribosomes, we found that they are refractory to the ASCC. Consequently, unresolved UV- and 4NQO-stalled ribosomes persist in human cells. We show that ribosome stalling activates cell-cycle arrest, partly through ZAK-p38MAPK signaling, and that this cell-cycle delay is prolonged when the ASCC cannot resolve stalled ribosomes. Thus, we propose that the sensitivity of stalled ribosomes to the ASCC influences the kinetics of stall resolution, which in turn controls the adaptive stress response.
Asunto(s)
Daño del ADN , Ribosomas , Humanos , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
Einstein et al. (2021) uncover a novel role for the RNA-binding protein YTHDF2, one of the m6A reader proteins, in TNBC proliferation and survival. This study demonstrates the clinical potential of targeting a specific reader protein in the treatment of breast cancer.
Asunto(s)
Proteínas de Unión al ARN , Proteínas de Unión al ARN/genéticaRESUMEN
Wagner et al. (2020), Bohlen et al. (2020), and Lin et al. (2020) use Sel-TCP-seq or selective ribosome profiling to gain insights into mRNA translation initiation, highlighting distinctions between yeast and higher eukaryotes and a role for eIF3 in elongation.
Asunto(s)
Factor 3 de Iniciación Eucariótica , Ribosomas , Iniciación de la Cadena Peptídica Traduccional , Saccharomyces cerevisiaeRESUMEN
Although the subcellular dynamics of RNA and proteins are key determinants of cell homeostasis, their characterization is still challenging. Here we present an integrative framework to simultaneously interrogate the dynamics of the transcriptome and proteome at subcellular resolution by combining two methods: localization of RNA (LoRNA) and a streamlined density-based localization of proteins by isotope tagging (dLOPIT) to map RNA and protein to organelles (nucleus, endoplasmic reticulum and mitochondria) and membraneless compartments (cytosol, nucleolus and cytosolic granules). Interrogating all RNA subcellular locations at once enables system-wide quantification of the proportional distribution of RNA. We obtain a cell-wide overview of localization dynamics for 31,839 transcripts and 5,314 proteins during the unfolded protein response, revealing that endoplasmic reticulum-localized transcripts are more efficiently recruited to cytosolic granules than cytosolic RNAs, and that the translation initiation factor eIF3d is key to sustaining cytoskeletal function. Overall, we provide the most comprehensive overview so far of RNA and protein subcellular localization dynamics.
Asunto(s)
Retículo Endoplásmico , ARN , ARN/genética , ARN/metabolismo , Fracciones Subcelulares/metabolismo , Retículo Endoplásmico/metabolismo , Proteoma/análisisRESUMEN
RNA binding proteins drive proliferation and tumorigenesis by regulating the translation and stability of specific subsets of messenger RNAs (mRNAs). We have investigated the role of eukaryotic initiation factor 4B (eIF4B) in this process and identify 10-fold more RNA binding sites for eIF4B in tumour cells from patients with diffuse large B-cell lymphoma compared to control B cells and, using individual-nucleotide resolution UV cross-linking and immunoprecipitation, find that eIF4B binds the entire length of mRNA transcripts. eIF4B stimulates the helicase activity of eIF4A, thereby promoting the unwinding of RNA structure within the 5' untranslated regions of mRNAs. We have found that, in addition to its well-documented role in mRNA translation, eIF4B additionally interacts with proteins associated with RNA turnover, including UPF1 (up-frameshift protein 1), which plays a key role in histone mRNA degradation at the end of S phase. Consistent with these data, we locate an eIF4B binding site upstream of the stem-loop structure in histone mRNAs and show that decreased eIF4B expression alters histone mRNA turnover and delays cell cycle progression through S phase. Collectively, these data provide insight into how eIF4B promotes tumorigenesis.
RESUMEN
Characterising RNA-protein interaction dynamics is fundamental to understand how bacteria respond to their environment. In this study, we have analysed the dynamics of 91% of the Escherichia coli expressed proteome and the RNA-interaction properties of 271 RNA-binding proteins (RBPs) at different growth phases. We find that 68% of RBPs differentially bind RNA across growth phases and characterise 17 previously unannotated proteins as bacterial RBPs including YfiF, a ncRNA-binding protein. While these new RBPs are mostly present in Proteobacteria, two of them are orthologs of human mitochondrial proteins associated with rare metabolic disorders. Moreover, we reveal novel RBP functions for proteins such as the chaperone HtpG, a new stationary phase tRNA-binding protein. For the first time, the dynamics of the bacterial RBPome have been interrogated, showcasing how this approach can reveal the function of uncharacterised proteins and identify critical RNA-protein interactions for cell growth which could inform new antimicrobial therapies.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , ARN Bacteriano , Proteínas de Unión al ARN , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , ARN Bacteriano/metabolismo , ARN Bacteriano/genética , Proteoma/metabolismo , Unión Proteica , Regulación Bacteriana de la Expresión Génica , HumanosRESUMEN
The intratumor microenvironment generates phenotypically distinct but interconvertible malignant cell subpopulations that fuel metastatic spread and therapeutic resistance. Whether different microenvironmental cues impose invasive or therapy-resistant phenotypes via a common mechanism is unknown. In melanoma, low expression of the lineage survival oncogene microphthalmia-associated transcription factor (MITF) correlates with invasion, senescence, and drug resistance. However, how MITF is suppressed in vivo and how MITF-low cells in tumors escape senescence are poorly understood. Here we show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B. ATF4, a key transcription mediator of the integrated stress response, also activates AXL and suppresses senescence to impose the MITF-low/AXL-high drug-resistant phenotype observed in human tumors. However, unexpectedly, without translation reprogramming an ATF4-high/MITF-low state is insufficient to drive invasion. Importantly, translation reprogramming dramatically enhances tumorigenesis and is linked to a previously unexplained gene expression program associated with anti-PD-1 immunotherapy resistance. Since we show that inhibition of eIF2B also drives neural crest migration and yeast invasiveness, our results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome.
Asunto(s)
Plasticidad de la Célula/genética , Reprogramación Celular/genética , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/genética , Melanoma/genética , Factor de Transcripción Asociado a Microftalmía/genética , Biosíntesis de Proteínas/genética , Animales , Microambiente Celular , Evolución Molecular , Retroalimentación Fisiológica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glutamina/farmacología , Humanos , Inmunoterapia , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Invasividad Neoplásica/genética , Cresta Neural/citología , Fenotipo , Factores de Transcripción/metabolismo , Pez Cebra/embriologíaRESUMEN
The molecular events in response to severe hyperthermia are not fully understood, and research has focused mainly on the effects of cooling at temperatures between 28°C and 35°C. In a new study, Fischl et al have analysed human cardiomyocytes at lower temperatures (8°C, 18°C and 28°C) and identified a novel mechanism by which hypothermia synchronises the circadian clock: cooling induces nuclear accumulation of transcripts that encode negative regulators of the circadian clock, which are released into the cytoplasm upon rewarming allowing synthesis of specific clock proteins.
Asunto(s)
Relojes Circadianos , Ritmo Circadiano , Proteínas CLOCK , Cromatina , Relojes Circadianos/genética , Ritmo Circadiano/genética , Humanos , ARN MensajeroRESUMEN
Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies.
Asunto(s)
Técnicas Biosensibles/métodos , Prueba de COVID-19/métodos , COVID-19/virología , Mediciones Luminiscentes/métodos , Péptido Hidrolasas/análisis , SARS-CoV-2/enzimología , Proteínas Virales/análisis , COVID-19/diagnóstico , Línea Celular , Humanos , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/fisiología , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
The assessment of transcriptome-wide ribosome binding to mRNAs is useful for studying the dynamic regulation of protein synthesis. Two methods frequently applied in eukaryotic cells that operate at different levels of resolution are polysome profiling, which reveals the distribution of ribosome loads across the transcriptome, and ribosome footprinting (also termed ribosome profiling or Ribo-Seq), which when combined with appropriate data on mRNA expression can reveal ribosome densities on individual transcripts. In this study we develop methods for relating the information content of these two methods to one another, by reconstructing theoretical polysome profiles from ribosome footprinting data. Our results validate both approaches as experimental tools. Although we show that both methods can yield highly consistent data, some published ribosome footprinting datasets give rise to reconstructed polysome profiles with non-physiological features. We trace these aberrant features to inconsistencies in RNA and Ribo-Seq data when compared to datasets yielding physiological polysome profiles, thereby demonstrating that modelled polysomes are useful for assessing global dataset properties such as its quality in a simple, visual approach. Aside from using polysome profile reconstructions on published datasets, we propose that this also provides a useful tool for validating new ribosome footprinting datasets in early stages of analyses.
Asunto(s)
Biosíntesis de Proteínas , Ribosomas , Ribosomas/genética , Ribosomas/metabolismo , Polirribosomas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , TranscriptomaRESUMEN
In tumourigenesis, oncogenes or dysregulated tumour suppressor genes alter the canonical translation machinery leading to a reprogramming of the translatome that, in turn, promotes the translation of selected mRNAs encoding proteins involved in proliferation and metastasis. It is therefore unsurprising that abnormal expression levels and activities of eukaryotic initiation factors (eIFs), elongation factors (eEFs) or termination factors (eRFs) are associated with poor outcome for patients with a wide range of cancers. In this review we discuss how RNA binding proteins (RBPs) within the canonical translation factor machinery are dysregulated in cancers and how targeting such proteins is leading to new therapeutic avenues.
Asunto(s)
Neoplasias , Biosíntesis de Proteínas , Humanos , Eucariontes/genética , Eucariontes/metabolismo , Carcinogénesis/genética , Factores Eucarióticos de Iniciación/genética , Factores Eucarióticos de Iniciación/metabolismo , Neoplasias/genética , Neoplasias/patología , Transformación Celular Neoplásica/genéticaRESUMEN
Cell division, differentiation and function are largely dependent on accurate proteome composition and regulated gene expression. To control this, protein synthesis is an intricate process governed by upstream signalling pathways. Eukaryotic translation is a multistep process and can be separated into four distinct phases: initiation, elongation, termination and recycling of ribosomal subunits. Translation initiation, the focus of this article, is highly regulated to control the activity and/or function of eukaryotic initiation factors (eIFs) and permit recruitment of mRNAs to the ribosomes. In this Cell Science at a Glance and accompanying poster, we outline the mechanisms by which tumour cells alter the process of translation initiation and discuss how this benefits tumour formation, proliferation and metastasis.
Asunto(s)
Neoplasias , Ribosomas , Factores Eucarióticos de Iniciación/metabolismo , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
Diagnostic testing continues to be an integral component of the strategy to contain the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) global pandemic, the causative agent of Coronavirus Disease 2019 (COVID-19). The SARS-CoV-2 genome encodes the 3C-like protease (3CLpro) which is essential for coronavirus replication. This study adapts an in vitro colorimetric gold nanoparticle (AuNP) based protease assay to specifically detect the activity of SARS-CoV-2 3CLpro as a purified recombinant protein and as a cellular protein exogenously expressed in HEK293T human cells. We also demonstrate that the specific sensitivity of the assay for SARS-CoV-2 3CLpro can be improved by use of an optimised peptide substrate and through hybrid dimerisation with inactive 3CLpro mutant monomers. These findings highlight the potential for further development of the AuNP protease assay to detect SARS-CoV-2 3CLpro activity as a novel, accessible and cost-effective diagnostic test for SARS-CoV-2 infection at the point-of-care. Importantly, this versatile assay could also be easily adapted to detect specific protease activity associated with other viruses or diseases conditions.
Asunto(s)
COVID-19 , Nanopartículas del Metal , Antivirales , COVID-19/diagnóstico , Colorimetría , Proteasas 3C de Coronavirus , Oro , Células HEK293 , Humanos , Péptido Hidrolasas , Inhibidores de Proteasas , SARS-CoV-2RESUMEN
The mammalian target of rapamycin (mTOR) is a critical regulator of cell growth, integrating multiple signalling cues and pathways. Key among the downstream activities of mTOR is the control of the protein synthesis machinery. This is achieved, in part, via the co-ordinated regulation of mRNAs that contain a terminal oligopyrimidine tract (TOP) at their 5'ends, although the mechanisms by which this occurs downstream of mTOR signalling are still unclear. We used RNA-binding protein (RBP) capture to identify changes in the protein-RNA interaction landscape following mTOR inhibition. Upon mTOR inhibition, the binding of LARP1 to a number of mRNAs, including TOP-containing mRNAs, increased. Importantly, non-TOP-containing mRNAs bound by LARP1 are in a translationally-repressed state, even under control conditions. The mRNA interactome of the LARP1-associated protein PABPC1 was found to have a high degree of overlap with that of LARP1 and our data show that PABPC1 is required for the association of LARP1 with its specific mRNA targets. Finally, we demonstrate that mRNAs, including those encoding proteins critical for cell growth and survival, are translationally repressed when bound by both LARP1 and PABPC1.
Asunto(s)
Autoantígenos/fisiología , Proteína I de Unión a Poli(A)/fisiología , Polirribosomas/metabolismo , Biosíntesis de Proteínas/fisiología , ARN Mensajero/metabolismo , Ribonucleoproteínas/fisiología , Serina-Treonina Quinasas TOR/fisiología , Regiones no Traducidas 5'/genética , Autoantígenos/genética , Regulación de la Expresión Génica , Genes Reporteros , Células HeLa , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Diana Mecanicista del Complejo 2 de la Rapamicina/antagonistas & inhibidores , Mutagénesis Sitio-Dirigida , Mutación Missense , Naftiridinas/farmacología , Mutación Puntual , Biosíntesis de Proteínas/genética , Interferencia de ARN , ARN Mensajero/genética , Proteínas de Unión al ARN/aislamiento & purificación , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas/genética , Antígeno SS-BRESUMEN
We show that a common polymorphic variant in the ERCC5 5' untranslated region (UTR) generates an upstream ORF (uORF) that affects both the background expression of this protein and its ability to be synthesized following exposure to agents that cause bulky adduct DNA damage. Individuals that harbor uORF1 have a marked resistance to platinum-based agents, illustrated by the significantly reduced progression-free survival of pediatric ependymoma patients treated with such compounds. Importantly, inhibition of DNA-PKcs restores sensitivity to platinum-based compounds by preventing uORF1-dependent ERCC5 expression. Our data support a model in which a heritable 5' noncoding mRNA element influences individuals' responses to platinum-based chemotherapy.
Asunto(s)
Regiones no Traducidas 5'/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Resistencia a Antineoplásicos/genética , Endonucleasas/genética , Endonucleasas/metabolismo , Ependimoma/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Sistemas de Lectura Abierta/genética , Polimorfismo Genético/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Proteínas de Unión al Calcio/metabolismo , Línea Celular , Línea Celular Tumoral , Cisplatino/farmacología , Cisplatino/uso terapéutico , Daño del ADN , Ependimoma/tratamiento farmacológico , Ependimoma/mortalidad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Células HeLa , HumanosRESUMEN
Protein synthesis is dysregulated in the majority of cancers and this process therefore provides a good therapeutic target. Many novel anti-cancer agents are directed to target the initiation stage of translation, however, translation elongation also holds great potential as a therapeutic target. The elongation factor eIF5A that assists the formation of peptidyl bonds during the elongation process is of considerable interest in this regard. Overexpression of eIF5A has been linked with the development of a variety of cancers and inhibitors of the molecule have been proposed for anti-cancer clinical applications. eIF5A is the only protein in the cell that contains the post-translational modification hypusine. Hypusination is a two-step enzymatic process catalysed by the Deoxyhypusine Synthase (DHPS) and Deoxyhypusine Hydroxylase (DOHH). In addition, eIF5A can be acetylated by p300/CBP-associated factor (PCAF) which leads to translocation of the protein to the nucleus and its deactivation. In addition to the nucleus, eIF5A has been found in the mitochondria and the endoplasmic reticulum (ER) with eIF5A localisation related to function from regulation of mitochondrial activity and apoptosis to maintenance of ER integrity and control of the unfolded protein response (UPR). Given the pleiotropic functions of eIF5A and by extension the hypusination enzymes, this system is being considered as a target for a range of cancers including multiple myeloma, B-Cell lymphoma, and neuroblastoma. In this review, we explore the role of eIF5A and discuss the therapeutic strategies that are currently developing both in the pre- and the clinical stage.
Asunto(s)
Neoplasias , Factores de Iniciación de Péptidos , Factores de Iniciación de Péptidos/genética , Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , Apoptosis , Neoplasias/tratamiento farmacológicoRESUMEN
Posttranscriptional modifications in transfer RNA (tRNA) are often critical for normal development because they adapt protein synthesis rates to a dynamically changing microenvironment. However, the precise cellular mechanisms linking the extrinsic stimulus to the intrinsic RNA modification pathways remain largely unclear. Here, we identified the cytosine-5 RNA methyltransferase NSUN2 as a sensor for external stress stimuli. Exposure to oxidative stress efficiently repressed NSUN2, causing a reduction of methylation at specific tRNA sites. Using metabolic profiling, we showed that loss of tRNA methylation captured cells in a distinct catabolic state. Mechanistically, loss of NSUN2 altered the biogenesis of tRNA-derived noncoding fragments (tRFs) in response to stress, leading to impaired regulation of protein synthesis. The intracellular accumulation of a specific subset of tRFs correlated with the dynamic repression of global protein synthesis. Finally, NSUN2-driven RNA methylation was functionally required to adapt cell cycle progression to the early stress response. In summary, we revealed that changes in tRNA methylation profiles were sufficient to specify cellular metabolic states and efficiently adapt protein synthesis rates to cell stress.