RESUMEN
While the centrality of posttranscriptional modifications to RNA biology has long been acknowledged, the function of the vast majority of modified sites remains to be discovered. Illustrative of this, there is not yet a discrete biological role assigned for one of the most highly conserved modifications, 5-methyluridine at position 54 in tRNAs (m5U54). Here, we uncover contributions of m5U54 to both tRNA maturation and protein synthesis. Our mass spectrometry analyses demonstrate that cells lacking the enzyme that installs m5U in the T-loop (TrmA in Escherichia coli, Trm2 in Saccharomyces cerevisiae) exhibit altered tRNA modification patterns. Furthermore, m5U54-deficient tRNAs are desensitized to small molecules that prevent translocation in vitro. This finding is consistent with our observations that relative to wild-type cells, trm2Δ cell growth and transcriptome-wide gene expression are less perturbed by translocation inhibitors. Together our data suggest a model in which m5U54 acts as an important modulator of tRNA maturation and translocation of the ribosome during protein synthesis.
Asunto(s)
Escherichia coli , ARN de Transferencia , Ribosomas , Saccharomyces cerevisiae , Uridina , ARN de Transferencia/metabolismo , ARN de Transferencia/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Ribosomas/metabolismo , Uridina/metabolismo , Escherichia coli/metabolismo , Escherichia coli/genética , Procesamiento Postranscripcional del ARN , Biosíntesis de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , ARNt Metiltransferasas/metabolismo , ARNt Metiltransferasas/genéticaRESUMEN
Pseudouridine (Ψ) is a ubiquitous RNA modification incorporated by pseudouridine synthase (Pus) enzymes into hundreds of noncoding and protein-coding RNA substrates. Here, we determined the contributions of substrate structure and protein sequence to binding and catalysis by pseudouridine synthase 7 (Pus7), one of the principal messenger RNA (mRNA) modifying enzymes. Pus7 is distinct among the eukaryotic Pus proteins because it modifies a wider variety of substrates and shares limited homology with other Pus family members. We solved the crystal structure of Saccharomyces cerevisiae Pus7, detailing the architecture of the eukaryotic-specific insertions thought to be responsible for the expanded substrate scope of Pus7. Additionally, we identified an insertion domain in the protein that fine-tunes Pus7 activity both in vitro and in cells. These data demonstrate that Pus7 preferentially binds substrates possessing the previously identified UGUAR (R = purine) consensus sequence and that RNA secondary structure is not a strong requirement for Pus7-binding. In contrast, the rate constants and extent of Ψ incorporation are more influenced by RNA structure, with Pus7 modifying UGUAR sequences in less-structured contexts more efficiently both in vitro and in cells. Although less-structured substrates were preferred, Pus7 fully modified every transfer RNA, mRNA, and nonnatural RNA containing the consensus recognition sequence that we tested. Our findings suggest that Pus7 is a promiscuous enzyme and lead us to propose that factors beyond inherent enzyme properties (e.g., enzyme localization, RNA structure, and competition with other RNA-binding proteins) largely dictate Pus7 substrate selection.
Asunto(s)
Secuencia de Aminoácidos , Sitios de Unión , Modelos Moleculares , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Dominio Catalítico , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , ARN de Hongos/química , ARN de Hongos/genética , ARN Mensajero/química , ARN Mensajero/genética , Estrés Fisiológico , Relación Estructura-Actividad , Especificidad por Sustrato , Temperatura , TermodinámicaRESUMEN
Ribosome speed is dictated by multiple factors including substrate availability, cellular conditions, and product (peptide) formation. Translation slows during the synthesis of cationic peptide sequences, potentially influencing the expression of thousands of proteins. Available evidence suggests that ionic interactions between positively charged nascent peptides and the negatively charged ribosome exit tunnel impede translation. However, this hypothesis was difficult to test directly because of inability to decouple the contributions of amino acid charge from mRNA sequence and tRNA identity/abundance in cells. Furthermore, it is unclear if other components of the translation system central to ribosome function (e.g., RNA modification) influence the speed and accuracy of positively charged peptide synthesis. In this study, we used a fully reconstituted Escherichia coli translation system to evaluate the effects of peptide charge, mRNA sequence, and RNA modification status on the translation of lysine-rich peptides. Comparison of translation reactions on poly(lysine)-encoding mRNAs conducted with either Lys-tRNALys or Val-tRNALys reveals that that amino acid charge, while important, only partially accounts for slowed translation on these transcripts. We further find that in addition to peptide charge, mRNA sequence and both tRNA and mRNA modification status influence the rates of amino acid addition and the ribosome's ability to maintain frame (instead of entering the -2, -1, and +1 frames) during poly(lysine) peptide synthesis. Our observations lead us to expand the model for explaining how the ribosome slows during poly(lysine) peptide synthesis and suggest that posttranscriptional RNA modifications can provide cells a mechanism to precisely control ribosome movements along an mRNA.
Asunto(s)
Biosíntesis de Péptidos , Polilisina , ARN Mensajero , ARN de Transferencia , Ribosomas , Péptidos/metabolismo , Polilisina/metabolismo , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , ARN de Transferencia de Lisina/metabolismo , Ribosomas/metabolismoRESUMEN
Light is crucial for many biological activities of most organisms, including vision, resetting of circadian rhythm, photosynthesis, and DNA repair. The cryptochrome/photolyase family (CPF) represents an ancient group of UV-A/blue light sensitive proteins that perform different functions such as DNA repair, circadian photoreception, and transcriptional regulation. The CPF is widely distributed throughout all organisms, including marine prokaryotes. The bacterium Vibrio cholerae was previously shown to have a CPD photolyase that repairs UV-induced thymine dimers and two CRY-DASHs that repair UV-induced single-stranded DNA damage. Here, we characterize a hypothetical gene Vca0809 encoding a new member of CPF in this organism. The spectroscopic analysis of the purified protein indicated that this enzyme possessed a catalytic cofactor, FAD, and photoantenna chromophore 6,7-dimethyl 8-ribityl-lumazin. With a slot blot-based DNA repair assay, we showed that it possessed (6-4) photolyase activity. Further phylogenetic and computational analyses enabled us to classify this gene as a member of the family of iron-sulfur bacterial cryptochromes and photolyases (FeS-BCP). Therefore, we named this gene Vc(6-4) FeS-BCP.
Asunto(s)
Proteínas Bacterianas/química , Desoxirribodipirimidina Fotoliasa/química , Vibrio cholerae/enzimología , Agrobacterium tumefaciens/enzimología , Secuencia de Aminoácidos , Arabidopsis/enzimología , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Criptocromos/química , Criptocromos/aislamiento & purificación , Criptocromos/metabolismo , ADN/química , ADN/efectos de la radiación , Desoxirribodipirimidina Fotoliasa/aislamiento & purificación , Desoxirribodipirimidina Fotoliasa/metabolismo , Escherichia coli/enzimología , Flavina-Adenina Dinucleótido/química , Flavina-Adenina Dinucleótido/metabolismo , Filogenia , Unión Proteica , Pteridinas/química , Pteridinas/metabolismo , Rhodobacter sphaeroides/enzimología , Alineación de Secuencia , Rayos UltravioletaRESUMEN
Drought stress is one of the main environmental factors that affects growth and productivity of crop plants, including lentil. To gain insights into the genome-wide transcriptional regulation in lentil root and leaf under short- and long-term drought conditions, we performed RNA-seq on a drought-sensitive lentil cultivar (Lens culinaris Medik. cv. Sultan). After establishing drought conditions, lentil samples were subjected to de novo RNA-seq-based transcriptome analysis. The 207,076 gene transcripts were successfully constructed by de novo assembly from the sequences obtained from root, leaf, and stems. Differentially expressed gene (DEG) analysis on these transcripts indicated that period of drought stress had a greater impact on the transcriptional regulation in lentil root. The numbers of DEGs were 2915 under short-term drought stress while the numbers of DEGs were increased to 18,327 under long-term drought stress condition in the root. Further, Gene Ontology analysis revealed that the following biological processes were differentially regulated in response to long-term drought stress: protein phosphorylation, embryo development seed dormancy, DNA replication, and maintenance of root meristem identity. Additionally, DEGs, which play a role in circadian rhythm and photoreception, were downregulated suggesting that drought stress has a negative effect on the internal oscillators which may have detrimental consequences on plant growth and survival. Collectively, this study provides a detailed comparative transcriptome response of drought-sensitive lentil strain under short- and long-term drought conditions in root and leaf. Our finding suggests that not only the regulation of genes in leaves is important but also genes regulated in roots are important and need to be considered for improving drought tolerance in lentil.
Asunto(s)
Deshidratación/genética , Sequías , Lens (Planta)/genética , Hojas de la Planta/genética , Proteínas de Plantas/genética , Raíces de Plantas/genética , Estrés Fisiológico , Regulación de la Expresión Génica de las Plantas , Ontología de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Lens (Planta)/crecimiento & desarrollo , Hojas de la Planta/crecimiento & desarrollo , Raíces de Plantas/crecimiento & desarrollo , TranscriptomaRESUMEN
Previous studies have demonstrated that deletion of cryptochrome (Cry) genes protects p53-/- mutant mice from the early onset of cancer and extends their median life-span by about 1.5-fold. Subsequent in vitro studies had revealed that deletion of Crys enhances apoptosis in response to UV damage through activation of p73 and inactivation of GSK3ß. However, it was not known at the transcriptome-wide level how deletion of Crys delays the onset of cancer in p53-/- mutant mice. In this study, the RNA-seq approach was taken to uncover the differentially expressed genes (DEGs) and pathways following UV-induced DNA damage in p53-/- and p53-/-Cry1-/-Cry2-/- mouse skin fibroblasts. Gene set enrichment analysis with the DEGs demonstrated enrichment in immune surveillance-associated genes regulated by IFN-γ and genes involved in TNFα signaling via NF-κB. Furthermore, protein network analysis enabled identification of DEGs p21, Sirt1, and Jun as key players, along with their interacting partners. It was also observed that the DEGs contained a high ratio of non-coding transcripts. Collectively, the present study suggests new genes in NF-κB regulation and IFN-γ response, as well as non-coding RNAs, may contribute to delaying the onset of cancer in p53-/-Cry1-/-Cry2-/- mice and increasing the life-span of these animals compared to p53-/- mice.
Asunto(s)
Apoptosis , Carcinogénesis/patología , Criptocromos/fisiología , Daño del ADN , Neoplasias Experimentales/patología , Transcriptoma , Proteína p53 Supresora de Tumor/fisiología , Animales , Carcinogénesis/metabolismo , Carcinogénesis/efectos de la radiación , Fibroblastos/metabolismo , Fibroblastos/patología , Fibroblastos/efectos de la radiación , Ratones , Ratones Noqueados , Neoplasias Experimentales/etiología , Neoplasias Experimentales/metabolismo , Piel/metabolismo , Piel/patología , Piel/efectos de la radiación , Rayos UltravioletaRESUMEN
Light is one of the main environmental cues that affects the physiology and behavior of many organisms. The effect of light on genome-wide transcriptional regulation has been well-studied in green algae and plants, but not in red algae. Cyanidioschyzon merolae is used as a model red algae, and is suitable for studies on transcriptomics because of its compact genome with a relatively small number of genes. In addition, complete genome sequences of the nucleus, mitochondrion, and chloroplast of this organism have been determined. Together, these attributes make C. merolae an ideal model organism to study the response to light stimuli at the transcriptional and the systems biology levels. Previous studies have shown that light significantly affects cell signaling in this organism, but there are no reports on its blue light- and red light-mediated transcriptional responses. We investigated the direct effects of blue and red light at the transcriptional level using RNA-seq. Blue and red lights were found to regulate 35 % of the total genes in C. merolae. Blue light affected the transcription of genes involved in protein synthesis while red light specifically regulated the transcription of genes involved in photosynthesis and DNA repair. Blue or red light regulated genes involved in carbon metabolism and pigment biosynthesis. Overall, our data showed that red and blue light regulate the majority of the cellular, cell division, and repair processes in C. merolae.
Asunto(s)
Fotosíntesis/genética , Filogenia , Rhodophyta/genética , Transcriptoma/genética , Carbono/metabolismo , Cloroplastos/genética , Cloroplastos/efectos de la radiación , Extremófilos/genética , Extremófilos/efectos de la radiación , Genoma de Planta/efectos de la radiación , Secuenciación de Nucleótidos de Alto Rendimiento , Luz , Mitocondrias/genética , Fotosíntesis/efectos de la radiación , Pigmentos Biológicos/biosíntesis , Rhodophyta/efectos de la radiación , Transcriptoma/efectos de la radiaciónRESUMEN
While the centrality of post-transcriptional modifications to RNA biology has long been acknowledged, the function of the vast majority of modified sites remains to be discovered. Illustrative of this, there is not yet a discrete biological role assigned for one the most highly conserved modifications, 5-methyluridine at position 54 in tRNAs (m 5 U54). Here, we uncover contributions of m 5 U54 to both tRNA maturation and protein synthesis. Our mass spectrometry analyses demonstrate that cells lacking the enzyme that installs m 5 U in the T-loop (TrmA in E. coli , Trm2 in S. cerevisiae ) exhibit altered tRNA modifications patterns. Furthermore, m 5 U54 deficient tRNAs are desensitized to small molecules that prevent translocation in vitro. This finding is consistent with our observations that, relative to wild-type cells, trm2 Δ cell growth and transcriptome-wide gene expression are less perturbed by translocation inhibitors. Together our data suggest a model in which m 5 U54 acts as an important modulator of tRNA maturation and translocation of the ribosome during protein synthesis.
RESUMEN
Post-transcriptional modifications to messenger RNAs (mRNAs) have the potential to alter the biological function of this important class of biomolecules. The study of mRNA modifications is a rapidly emerging field, and the full complement of chemical modifications in mRNAs is not yet established. We sought to identify and quantify the modifications present in yeast mRNAs using an ultra-high performance liquid chromatography tandem mass spectrometry method to detect 40 nucleoside variations in parallel. We observe six modified nucleosides with high confidence in highly purified mRNA samples (N7-methylguanosine, N6-methyladenosine, 2'-O-methylguanosine, 2'-O-methylcytidine, N4-acetylcytidine, and 5-formylcytidine) and identify the yeast protein responsible for N4-acetylcytidine incorporation in mRNAs (Rra1). In addition, we find that mRNA modification levels change in response to heat shock, glucose starvation, and/or oxidative stress. This work expands the repertoire of potential chemical modifications in mRNAs and highlights the value of integrating mass spectrometry tools in the mRNA modification discovery and characterization pipeline.
Asunto(s)
Nucleósidos/análisis , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina/análogos & derivados , Adenosina/análisis , Adenosina/metabolismo , Citidina/análogos & derivados , Citidina/análisis , Citidina/metabolismo , Glucosa/metabolismo , Guanosina/análogos & derivados , Guanosina/análisis , Guanosina/metabolismo , Respuesta al Choque Térmico , Nucleósidos/metabolismo , Estrés Oxidativo , ARN de Hongos/química , ARN Mensajero/química , Saccharomyces cerevisiae/químicaRESUMEN
Blue light (BL) is a major environmental factor that affects the physiology, behavior, and infectivity of bacteria as it contributes to the generation of reactive oxygen species (ROS) while increasing photo-oxidative stress in cells. However, precise photo-oxidative response mechanism in non-phototrophic bacteria is yet to be elucidated. In this study, we investigated the effect of BL in Vibrio cholerae by using genetics and transcriptome profiling. Genome-wide analysis revealed that transcription of 6.3% of V. cholerae genes were regulated by BL. We further showed that BL enhances ROS production, which is generated through the oxidative phosphorylation. To understand signaling mechanisms, we generated several knockouts and analyzed their transcriptome under BL exposure. Studies with a double-knockout confirm an anti-sigma factor (ChrR) and putative metalloregulatory-like protein (MerR) are responsible for the genome-wide regulation to BL response in V. cholerae. Collectively, these results demonstrate that MerR-like proteins, in addition to ChrR, are required for V. cholerae to mount an appropriate response against photo-oxidative stress induced by BL. Outside its natural host, V. cholerae can survive for extended periods in natural aquatic environments. Therefore, the regulation of light response for V. cholerae may be a critical cellular process for its survival in these environments.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Luz , Estrés Oxidativo/efectos de la radiación , Factor sigma/metabolismo , Vibrio cholerae/genética , Proteínas Bacterianas/genética , Criptocromos/metabolismo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Perfilación de la Expresión Génica , Genoma Bacteriano , Especies Reactivas de Oxígeno/metabolismo , Factor sigma/deficiencia , Factor sigma/genética , Transcripción Genética/efectos de la radiación , Vibrio cholerae/crecimiento & desarrollo , Vibrio cholerae/metabolismoRESUMEN
Light is a very important environmental factor that governs many cellular responses in organisms. As a consequence, organisms possess different kinds of light-sensing photoreceptors to regulate their physiological variables and adapt to a given habitat. The cryptochrome/photolyase family (CPF) includes photoreceptors that perform different functions in different organisms. Photolyases repair ultraviolet-induced DNA damage by a process known as photoreactivation using photons absorbed from the blue end of the light spectrum. On the other hand, cryptochromes act as blue light circadian photoreceptors in plants and Drosophila to regulate growth and development. In mammals, cryptochromes have light-independent functions and are very important transcriptional regulators that act at the molecular level as negative transcriptional regulators of the circadian clock. In this review, we highlight current knowledge concerning the structural and functional relationships of CPF members.
Asunto(s)
Criptocromos/metabolismo , Reparación del ADN , Desoxirribodipirimidina Fotoliasa/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética , Animales , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Ritmo Circadiano , Cristalografía por Rayos X , Desoxirribodipirimidina Fotoliasa/química , Drosophila , Proteínas de Drosophila/metabolismo , Conformación Proteica , Relación Estructura-Actividad , Rayos UltravioletaRESUMEN
Damage to the mitochondrial genome (mtDNA) can lead to diseases for which there are no clearly effective treatments. Since mitochondrial function and biogenesis are controlled by the nutrient environment of the cell, it is possible that perturbation of conserved, nutrient-sensing pathways may successfully treat mitochondrial disease. We found that restricting glucose or otherwise reducing the activity of the protein kinase A (PKA) pathway can lead to improved proliferation of Saccharomyces cerevisiae cells lacking mtDNA and that the transcriptional response to mtDNA loss is reduced in cells with diminished PKA activity. We have excluded many pathways and proteins from being individually responsible for the benefits provided to cells lacking mtDNA by PKA inhibition, and we found that robust import of mitochondrial polytopic membrane proteins may be required in order for cells without mtDNA to receive the full benefits of PKA reduction. Finally, we have discovered that the transcription of genes involved in arginine biosynthesis and aromatic amino acid catabolism is altered after mtDNA damage. Our results highlight the potential importance of nutrient detection and availability on the outcome of mitochondrial dysfunction.