RESUMEN
In general, mRNAs are assumed to be loaded with ribosomes instantly upon entry into the cytoplasm. To measure ribosome density (RD) on nascent mRNA, we developed nascent Ribo-Seq by combining Ribo-Seq with progressive 4-thiouridine labeling. In mouse macrophages, we determined experimentally the lag between the appearance of nascent mRNA and its association with ribosomes, which was calculated to be 20-22 min for bulk mRNA. In mouse embryonic stem cells, nRibo-Seq revealed an even stronger lag of 35-38 min in ribosome loading. After stimulation of macrophages with lipopolysaccharide, the lag between cytoplasmic and translated mRNA leads to uncoupling between input and ribosome-protected fragments, which gives rise to distorted RD measurements under conditions where mRNA amounts are far from steady-state expression. As a result, we demonstrate that transcriptional changes affect RD in a passive way.
Asunto(s)
Biosíntesis de Proteínas , Ribosomas/genética , Ribosomas/metabolismo , Análisis de Secuencia de ARN/métodos , Animales , Citoplasma/genética , Cinética , Lipopolisacáridos/farmacología , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/fisiología , Células RAW 264.7 , ARN Mensajero/genética , Proteínas Ribosómicas/biosíntesis , Proteínas Ribosómicas/genética , Ribosomas/efectos de los fármacos , Factores de TiempoRESUMEN
The autogenous regulation of ribosomal protein (r-protein) synthesis plays a key role in maintaining the stoichiometry of ribosomal components in bacteria. In this work, taking the rpsO gene as a classic example, we addressed for the first time the in vivo regulation of r-protein synthesis in the mycobacteria M. smegmatis (Msm) and M. tuberculosis (Mtb). We used a strategy based on chromosomally integrated reporters under the control of the rpsO regulatory regions and the ectopic expression of Msm S15 to measure its impact on the reporter expression. Because the use of E. coli as a host appeared inefficient, a fluorescent reporter system was developed by inserting Msm or Mtb rpsO-egfp fusions into the Msm chromosome and expressing Msm S15 or E. coli S15 in trans from a novel replicative shuttle vector, pAMYC. The results of the eGFP expression measurements in Msm cells provided evidence that the rpsO gene in Msm and Mtb was feedback-regulated at the translation level. The mutagenic analysis showed that the folding of Msm rpsO 5'UTR in a pseudoknot appeared crucial for repression by both Msm S15 and E. coli S15, thus indicating a striking resemblance of the rpsO feedback control in mycobacteria and in E. coli.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Mycobacterium/fisiología , Biosíntesis de Proteínas , Proteínas Ribosómicas/biosíntesis , Secuencia de Aminoácidos , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Humanos , Conformación de Ácido Nucleico , Operón , Plásmidos/genética , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
Delivery of putative compounds of therapeutic value to the brain is limited by brain barriers: the blood-brain barrier located in the endothelium of the brain microvessels (BrMVs) and the blood-cerebrospinal fluid barrier located in the epithelium of the choroid plexus (ChP). Understanding their function and modulation by the circadian clock may enhance the efficacy of brain-targeting therapies. The aim of the present study was to evaluate the stability of 10 reference genes in the BrMV and ChP, isolated from male and female rats at six time points (ZT1, 5, 9, 13, 17, and 21). Gene evaluations were performed by qPCR, analyzed by RefFinder tool, and verified by analyzing the expression of the brain and muscle ARNT-like 1 (Bmal1) using the qPCR and digital PCR methods. We identified as the most stable genes for circadian studies tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (Ywhaz) and apolipoprotein E (Apoe) for BrMV, and beta actin (Actb) and hypoxanthine-guanine phosphoribosyltransferase (Hprt1) for ChP. After verification, ribosomal protein (Rps18) was also included as a sufficient reference gene. Additionally, the observed gender difference in the Bmal1 oscillations in both BrMV and ChP suggests that separate studies for each gender are recommended.
Asunto(s)
Circulación Cerebrovascular , Plexo Coroideo/metabolismo , Microcirculación , Proteínas 14-3-3/metabolismo , Factores de Transcripción ARNTL/metabolismo , Actinas/metabolismo , Algoritmos , Animales , Apolipoproteínas E/metabolismo , Encéfalo/metabolismo , Ritmo Circadiano , Femenino , Regulación de la Expresión Génica , Hipoxantina Fosforribosiltransferasa/biosíntesis , Masculino , Chaperonas Moleculares/metabolismo , Oscilometría , Reacción en Cadena de la Polimerasa , Ratas , Ratas Wistar , Proteínas Ribosómicas/biosíntesis , Factores SexualesRESUMEN
Cytosolic ribosomes (cytoribosomes) are macromolecular ribonucleoprotein complexes that are assembled from ribosomal RNA and ribosomal proteins, which are essential for protein biosynthesis. Mitochondrial ribosomes (mitoribosomes) perform translation of the proteins essential for the oxidative phosphorylation system. The biogenesis of cytoribosomes and mitoribosomes includes ribosomal RNA processing, modification and binding to ribosomal proteins and is assisted by numerous biogenesis factors. This is a major energy-consuming process in the cell and, therefore, is highly coordinated and sensitive to several cellular stressors. In mitochondria, the regulation of mitoribosome biogenesis is essential for cellular respiration, a process linked to cell growth and proliferation. This review briefly overviews the key stages of cytosolic and mitochondrial ribosome biogenesis; summarizes the main steps of ribosome biogenesis alterations occurring during tumorigenesis, highlighting the changes in the expression level of cytosolic ribosomal proteins (CRPs) and mitochondrial ribosomal proteins (MRPs) in different types of tumors; focuses on the currently available information regarding the extra-ribosomal functions of CRPs and MRPs correlated to cancer; and discusses the role of CRPs and MRPs as biomarkers and/or molecular targets in cancer treatment.
Asunto(s)
Transformación Celular Neoplásica , Neoplasias/metabolismo , Biogénesis de Organelos , Ribosomas , Animales , Apoptosis , Autofagia , Ciclo Celular , Movimiento Celular , Nucléolo Celular/metabolismo , Citosol/metabolismo , Reparación del ADN , Estrés del Retículo Endoplásmico , Células Eucariotas/metabolismo , Células Eucariotas/ultraestructura , Regulación Neoplásica de la Expresión Génica , Terapia Genética/métodos , Humanos , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/terapia , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN Mitocondrial/metabolismo , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/biosíntesis , Proteínas Ribosómicas/fisiología , Ribosomas/fisiologíaRESUMEN
Mitochondrial misreading, conferred by mutation V338Y in mitoribosomal protein Mrps5, in-vivo is associated with a subtle neurological phenotype. Brain mitochondria of homozygous knock-in mutant Mrps5V338Y/V338Y mice show decreased oxygen consumption and reduced ATP levels. Using a combination of unbiased RNA-Seq with untargeted metabolomics, we here demonstrate a concerted response, which alleviates the impaired functionality of OXPHOS complexes in Mrps5 mutant mice. This concerted response mitigates the age-associated decline in mitochondrial gene expression and compensates for impaired respiration by transcriptional upregulation of OXPHOS components together with anaplerotic replenishment of the TCA cycle (pyruvate, 2-ketoglutarate).
Asunto(s)
Envejecimiento/metabolismo , Encéfalo/metabolismo , Regulación de la Expresión Génica , Mitocondrias/metabolismo , Proteínas Mitocondriales/biosíntesis , Mutación Missense , Biosíntesis de Proteínas , Proteínas Ribosómicas/biosíntesis , Adenosina Trifosfato/metabolismo , Envejecimiento/genética , Envejecimiento/patología , Animales , Encéfalo/patología , Ciclo del Ácido Cítrico/genética , Técnicas de Sustitución del Gen , Ratones , Ratones Transgénicos , Mitocondrias/genética , Mitocondrias/patología , Proteínas Mitocondriales/genética , Proteínas Ribosómicas/genéticaRESUMEN
Free radicals, or reactive oxygen species, have been implicated as one of the primary causes of myocardial pathologies elicited by chronic diseases and age. The imbalance between pro-oxidants and antioxidants, termed "oxidative stress", involves several pathological changes in mouse hearts, including hypertrophy and cardiac dysfunction. However, the molecular mechanisms and adaptations of the hearts in mice lacking cytoplasmic superoxide dismutase (Sod1KO) have not been investigated. We used echocardiography to characterize cardiac function and morphology in vivo. Protein expression and enzyme activity of Sod1KO were confirmed by targeted mass spectrometry and activity gel. The heart weights of the Sod1KO mice were significantly increased compared with their wildtype peers. The increase in heart weights was accompanied by concentric hypertrophy, posterior wall thickness of the left ventricles (LV), and reduced LV volume. Activated downstream pathways in Sod1KO hearts included serine-threonine kinase and ribosomal protein synthesis. Notably, the reduction in LV volume was compensated by enhanced systolic function, measured by increased ejection fraction and fractional shortening. A regulatory sarcomeric protein, troponin I, was hyper-phosphorylated in Sod1KO, while the vinculin protein was upregulated. In summary, mice lacking cytoplasmic superoxide dismutase were associated with an increase in heart weights and concentric hypertrophy, exhibiting a pathological adaptation of the hearts to oxidative stress.
Asunto(s)
Miocardio/patología , Estrés Oxidativo , Sístole , Animales , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Fibrosis , Hipertrofia , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Tamaño de los Órganos , Oxidación-Reducción , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Ribosómicas/biosíntesis , Ribosomas/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Superóxido Dismutasa/metabolismo , Troponina I/metabolismoRESUMEN
Periods of inactivity experienced by older adults induce nutrient anabolic resistance creating a cascade of skeletal muscle transcriptional and translational aberrations contributing to muscle dysfunction. The purpose of this study was to identify how inactivity alters leucine-stimulated translation of molecules and pathways within the skeletal muscle of older adults. We performed ribosomal profiling alongside RNA sequencing from skeletal muscle biopsies taken from older adults (n = 8; ~72 years; 6 F/2 M) in response to a leucine bolus before (Active) and after (Reduced Activity) 2 weeks of reduced physical activity. At both visits, muscle biopsies were taken at baseline, 60 minutes (early response), and 180 minutes (late response) after leucine ingestion. Previously identified inactivity-related gene transcription changes (PFKFB3, GADD45A, NMRK2) were heightened by leucine with corresponding changes in translation. In contrast, leucine also stimulated translational efficiency of several transcripts in a manner not explained by corresponding changes in mRNA abundance ("uncoupled translation"). Inactivity eliminated this uncoupled translational response for several transcripts, and reduced the translation of most mRNAs encoding for ribosomal proteins. Ingenuity Pathway Analysis identified discordant circadian translation and transcription as a result of inactivity such as translation changes to PER2 and PER3 despite unchanged transcription. We demonstrate inactivity alters leucine-stimulated "uncoupled translation" of ribosomal proteins and circadian regulators otherwise not detectable by traditional RNA sequencing. Innovative techniques such as ribosomal profiling continues to further our understanding of how physical activity mediates translational regulation, and will set a path toward therapies that can restore optimal protein synthesis on the transcript-specific level to combat negative consequences of inactivity on aging muscle.
Asunto(s)
Ejercicio Físico , Músculo Esquelético , Proteínas Ribosómicas , Anciano , Femenino , Humanos , Leucina/farmacología , Masculino , Músculo Esquelético/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/metabolismo , Proteínas Ribosómicas/biosíntesis , RibosomasRESUMEN
Coagulase-negative staphylococci and Staphylococcus aureus colonize similar niches in mammals and conceivably compete for space and nutrients. Here, we report that a coagulase-negative staphylococcus, Staphylococcus chromogenes ATCC43764, synthesizes and secretes 6-thioguanine (6-TG), a purine analog that suppresses S. aureus growth by inhibiting de novo purine biosynthesis. We identify a 6-TG biosynthetic gene cluster in S. chromogenes and other coagulase-negative staphylococci including S. epidermidis, S. pseudintermedius and S. capitis. Recombinant S. aureus strains harbouring this operon produce 6-TG and, when used in subcutaneous co-infections in mice with virulent S. aureus USA300, protect the host from necrotic lesion formation. Used prophylactically, 6-TG reduces necrotic skin lesions in mice infected with USA300, and this effect is mediated by abrogation of toxin production. RNAseq analyses reveal that 6-TG downregulates expression of genes coding for purine biosynthesis, the accessory gene regulator (agr) and ribosomal proteins in S. aureus, providing an explanation for its effect on toxin production.
Asunto(s)
Infecciones Cutáneas Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus/genética , Staphylococcus/metabolismo , Tioguanina/metabolismo , Animales , Proteínas Bacterianas/biosíntesis , Coagulasa/deficiencia , Femenino , Ratones , Ratones Endogámicos BALB C , Purinas/biosíntesis , Proteínas Ribosómicas/biosíntesis , Staphylococcus aureus/patogenicidad , Staphylococcus capitis/metabolismo , Staphylococcus epidermidis/metabolismo , Tioguanina/farmacología , Transactivadores/biosíntesisRESUMEN
Diamond-Blackfan anemia (DBA) is mainly caused by pathogenic variants in ribosomal proteins and 22 responsible genes have been identified to date. The most common causative gene of DBA is RPS19 [NM_001022.4]. Nearly 180 RPS19 variants have been reported, including three deep intronic variants outside the splicing consensus sequence (c.72-92A > G, c.356 + 18G > C, and c.411 + 6G > C). We also identified one case with a c.412-3C > G intronic variant. Without conducting transcript analysis, the pathogenicity of these variants is unknown. However, it is difficult to assess transcripts because of their fragility. In such cases, in vitro functional splicing assays can be used to assess pathogenicity. Here, we report functional splicing analysis results of four RPS19 deep intronic variants identified in our case and in previously reported cases. One splicing consensus variant (c.411 + 1G > A) was also examined as a positive control. Aberrant splicing with a 2-bp insertion between exons 5 and 6 was identified in the patient samples and minigene assay results also identified exon 6 skipping in our case. The exon 6 skipping transcript was confirmed by further evaluation using quantitative RT-PCR. Additionally, minigene assay analysis of three reported deep intronic variants revealed that none of them showed aberrant splicing and that these variants were not considered to be pathogenic. In conclusion, the minigene assay is a useful method for functional splicing analysis of inherited disease.
Asunto(s)
Anemia de Diamond-Blackfan , Mutación , Empalme del ARN , Proteínas Ribosómicas , Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/metabolismo , Humanos , Recién Nacido , Masculino , Proteínas Ribosómicas/biosíntesis , Proteínas Ribosómicas/genéticaRESUMEN
Ribosome biogenesis in eukaryotes requires the coordinated production and assembly of 80 ribosomal proteins and four ribosomal RNAs (rRNAs), and its rate must be synchronized with cellular growth. Here, we showed that the Microprocessor complex, which mediates the first step of microRNA processing, potentiated the transcription of ribosomal protein genes by eliminating DNA/RNA hybrids known as R-loops. Nutrient deprivation triggered the nuclear export of Drosha, a key component of the Microprocessor complex, and its subsequent degradation by the E3 ubiquitin ligase Nedd4, thereby reducing ribosomal protein production and protein synthesis. In mouse erythroid progenitors, conditional deletion of Drosha led to the reduced production of ribosomal proteins, translational inhibition of the mRNA encoding the erythroid transcription factor Gata1, and impaired erythropoiesis. This phenotype mirrored the clinical presentation of human "ribosomopathies." Thus, the Microprocessor complex plays a pivotal role in synchronizing protein synthesis capacity with cellular growth rate and is a potential drug target for anemias caused by ribosomal insufficiency.
Asunto(s)
Biosíntesis de Proteínas , Proteínas Ribosómicas/biosíntesis , Ribosomas , Animales , Eritropoyesis , Ratones , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/genética , Ribosomas/metabolismoRESUMEN
The impact of resistance exercise frequency on muscle protein synthesis rates remains unknown. The aim of this study was to compare daily myofibrillar protein synthesis rates over a 7-day period of low-frequency (LF) versus high-frequency (HF) resistance exercise training. Nine young men (21 ± 2 years) completed a 7-day period of habitual physical activity (BASAL). This was followed by a 7-day exercise period of volume-matched, LF (10 × 10 repetitions at 70% one-repetition maximum, once per week) or HF (2 × 10 repetitions at â¼70% one-repetition maximum, five times per week) resistance exercise training. The participants had one leg randomly allocated to LF and the other to HF. Skeletal muscle biopsies and daily saliva samples were collected to determine myofibrillar protein synthesis rates using 2H2O, with intracellular signaling determined using Western blotting. The myofibrillar protein synthesis rates did not differ between the LF (1.46 ± 0.26%/day) and HF (1.48 ± 0.33%/day) conditions over the 7-day exercise training period (p > .05). There were no significant differences between the LF and HF conditions over the first 2 days (1.45 ± 0.41%/day vs. 1.25 ± 0.46%/day) or last 5 days (1.47 ± 0.30%/day vs. 1.50 ± 0.41%/day) of the exercise training period (p > .05). Daily myofibrillar protein synthesis rates were not different from BASAL at any time point during LF or HF (p > .05). The phosphorylation status and total protein content of selected proteins implicated in skeletal muscle ribosomal biogenesis were not different between conditions (p > .05). Under the conditions of the present study, resistance exercise training frequency did not modulate daily myofibrillar protein synthesis rates in young men.
Asunto(s)
Proteínas Musculares/biosíntesis , Miofibrillas/metabolismo , Entrenamiento de Fuerza , Actigrafía/estadística & datos numéricos , Biopsia , Óxido de Deuterio/metabolismo , Dieta , Ingestión de Energía , Humanos , Pierna , Masculino , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/metabolismo , Fosforilación , Distribución Aleatoria , Proteínas Ribosómicas/biosíntesis , Transducción de Señal , Factores de Tiempo , Adulto JovenRESUMEN
Ribosomes are multicomponent molecular machines that synthesize all of the proteins of living cells. Most of the genes that encode the protein components of ribosomes are therefore essential. A reduction in gene dosage is often viable albeit deleterious and is associated with human syndromes, which are collectively known as ribosomopathies1-3. The cell biological basis of these pathologies has remained unclear. Here, we model human ribosomopathies in Drosophila and find widespread apoptosis and cellular stress in the resulting animals. This is not caused by insufficient protein synthesis, as reasonably expected. Instead, ribosomal protein deficiency elicits proteotoxic stress, which we suggest is caused by the accumulation of misfolded proteins that overwhelm the protein degradation machinery. We find that dampening the integrated stress response4 or autophagy increases the harm inflicted by ribosomal protein deficiency, suggesting that these activities could be cytoprotective. Inhibition of TOR activity-which decreases ribosomal protein production, slows down protein synthesis and stimulates autophagy5-reduces proteotoxic stress in our ribosomopathy model. Interventions that stimulate autophagy, combined with means of boosting protein quality control, could form the basis of a therapeutic strategy for this class of diseases.
Asunto(s)
Mutación/genética , Proteínas/toxicidad , Ribosomas/genética , Ribosomas/patología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Alelos , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Drosophila melanogaster/efectos de los fármacos , Drosophila melanogaster/metabolismo , Células HEK293 , Heterocigoto , Humanos , Discos Imaginales/efectos de los fármacos , Discos Imaginales/metabolismo , Agregado de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Proteómica , Proteínas Ribosómicas/biosíntesis , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Alas de Animales/efectos de los fármacos , Alas de Animales/metabolismoRESUMEN
Ribosomal protein synthesis is an important target in antibacterial drug discovery. Numerous natural products have served as starting points for the development of antibiotics. We report here the total synthesis of xenocoumacin 1, a natural product that binds to 16S ribosomal RNA at a highly conserved region, as well as analogues thereof. Preliminary structure-activity relationship studies were aimed at understanding and modulating the selectivity between eukaryotic and prokaryotic ribosomes. Modifications were mainly tolerated in the aromatic region. Whole-cell activity against Gram-negative bacteria is limited by efflux and penetration, as demonstrated in genetically modified strains of E. coli. Analogues with high selectivity for eukaryotic ribosomes were identified, but it was not possible to obtain inhibitors selective for bacterial protein synthesis. Achieving high selectivity (albeit not the desired one) was thus possible despite the high homology between eukaryotic and prokaryotic ribosomes in the binding region.
Asunto(s)
Antibacterianos/farmacología , Benzopiranos/farmacología , Escherichia coli/efectos de los fármacos , Proteínas Ribosómicas/antagonistas & inhibidores , Antibacterianos/química , Benzopiranos/química , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Escherichia coli/metabolismo , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Proteínas Ribosómicas/biosíntesis , Relación Estructura-ActividadRESUMEN
Tumor cells require nominal increases in protein synthesis in order to maintain high proliferation rates. As such, tumor cells must acquire enhanced ribosome production. How the numerous mutations in tumor cells ultimately achieve this aberrant production is largely unknown. The gene encoding ARF is the most commonly deleted gene in human cancer. ARF plays a significant role in regulating ribosomal RNA synthesis and processing, ribosome export into the cytoplasm, and global protein synthesis. Utilizing ribosome profiling, we show that ARF is a major suppressor of 5'-terminal oligopyrimidine mRNA translation. Genes with increased translational efficiency following loss of ARF include many ribosomal proteins and translation factors. Knockout of p53 largely phenocopies ARF loss, with increased protein synthesis and expression of 5'-TOP encoded proteins. The 5'-TOP regulators eIF4G1 and LARP1 are upregulated in Arf- and p53-null cells.
Asunto(s)
Factor 1 de Ribosilacion-ADP/genética , Neoplasias/genética , Proteínas Ribosómicas/genética , Proteína p53 Supresora de Tumor/genética , Autoantígenos/genética , Proliferación Celular/genética , Factor 4G Eucariótico de Iniciación/genética , Humanos , Neoplasias/patología , Biosíntesis de Proteínas/genética , Ribonucleoproteínas/genética , Proteínas Ribosómicas/biosíntesis , Ribosomas/genética , Activación Transcripcional/genética , Antígeno SS-BRESUMEN
While expression of ribosomal protein genes (RPGs) in the budding yeast has been extensively studied, a longstanding enigma persists regarding their co-regulation under fluctuating growth conditions. Most RPG promoters display one of two distinct arrangements of a core set of transcription factors (TFs) and are further differentiated by the presence or absence of the HMGB protein Hmo1. However, a third group of promoters appears not to be bound by any of these proteins, raising the question of how the whole suite of genes is co-regulated. We demonstrate here that all RPGs are regulated by two distinct, but complementary mechanisms driven by the TFs Ifh1 and Sfp1, both of which are required for maximal expression in optimal conditions and coordinated downregulation upon stress. At the majority of RPG promoters, Ifh1-dependent regulation predominates, whereas Sfp1 plays the major role at all other genes. We also uncovered an unexpected protein homeostasis-dependent binding property of Hmo1 at RPG promoters. Finally, we show that the Ifh1 paralog Crf1, previously described as a transcriptional repressor, can act as a constitutive RPG activator. Our study provides a more complete picture of RPG regulation and may serve as a paradigm for unravelling RPG regulation in multicellular eukaryotes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas Ribosómicas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Estrés Fisiológico/genética , Transactivadores/metabolismo , Transcripción Genética , Secuenciación de Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/genética , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Proteínas del Grupo de Alta Movilidad/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas Ribosómicas/biosíntesis , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Sirolimus/farmacología , Estrés Fisiológico/efectos de los fármacos , Transactivadores/genéticaRESUMEN
Translation of mitochondrial messenger RNA (mt-mRNA) is performed by distinct mitoribosomes comprising at least 36 mitochondria-specific proteins. How these mitoribosomal proteins assist in the binding of mt-mRNA and to what extent they are involved in the translocation of transfer RNA (mt-tRNA) is unclear. To visualize the process of translation in human mitochondria, we report ~3.0 Å resolution structure of the human mitoribosome, including the L7/L12 stalk, and eight structures of its functional complexes with mt-mRNA, mt-tRNAs, recycling factor and additional trans factors. The study reveals a transacting protein module LRPPRC-SLIRP that delivers mt-mRNA to the mitoribosomal small subunit through a dedicated platform formed by the mitochondria-specific protein mS39. Mitoribosomal proteins of the large subunit mL40, mL48, and mL64 coordinate translocation of mt-tRNA. The comparison between those structures shows dynamic interactions between the mitoribosome and its ligands, suggesting a sequential mechanism of conformational changes.
Asunto(s)
Mitocondrias/ultraestructura , Proteínas Mitocondriales/biosíntesis , Ribosomas Mitocondriales/ultraestructura , Biosíntesis de Proteínas , Proteínas Ribosómicas/biosíntesis , Células HEK293 , Humanos , Mitocondrias/fisiología , Proteínas Mitocondriales/genética , Ribosomas Mitocondriales/fisiología , Modelos Moleculares , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , ARN Mensajero/metabolismo , ARN Mitocondrial/metabolismo , ARN de Transferencia/metabolismo , Proteínas de Unión al ARN/biosíntesis , Proteínas de Unión al ARN/genética , Proteínas Ribosómicas/genéticaRESUMEN
The mechanism of hair loss caused by aging is related to mitochondrial dysfunction. Pep-1-mediated mitochondrial transplantation is a potential therapeutic application for mitochondrial disorders, but its efficacy against hair aging remains unknown. This study compared platelet-rich plasma (PRP) therapy with mitochondrial transplantation for hair restoration and examined the related regulation in naturally aging mice. After dorsal hair removal, 100-week-old mice received weekly unilateral injections of 200 µg of allogeneic mitochondria-labeled 5-bromo-2'-deoxyuridine with (P-Mito) or without Pep-1 conjugation (Mito) or human PRP with a stamp-type electric injector for 1 month. The contralateral sides were used as corresponding sham controls. Compared with the control and corresponding sham groups, all treatments stimulated hair regrowth, and the effectiveness of P-Mito was equal to that of PRP. However, histology revealed that only P-Mito maintained hair length until day 28 and yielded more anagen follicles with abundant dermal collagen equivalent to that of the PRP group. Mitochondrial transplantation increased the thickness of subcutaneous fat compared with the control and PRP groups, and only P-Mito consistently increased mitochondria in the subcutaneous muscle and mitochondrial DNA copies in the skin layer. Therefore, P-Mito had a higher penetrating capacity than Mito did. Moreover, P-Mito treatment was as effective as PRP treatment in comprehensively reducing the expression of aging-associated gene markers, such as IGF1R and MRPS5, and increasing antiaging Klotho gene expression. This study validated the efficacy of mitochondrial therapy in the restoration of aging-related hair loss and demonstrated the distinct effects of PRP treatment.
Asunto(s)
Envejecimiento/fisiología , Cabello/crecimiento & desarrollo , Mitocondrias/trasplante , Plasma Rico en Plaquetas , Trasplante Autólogo/instrumentación , Trasplante Autólogo/métodos , Envejecimiento/genética , Alopecia/fisiopatología , Animales , Bromodesoxiuridina/farmacología , Cisteamina/análogos & derivados , Cisteamina/química , Cisteamina/farmacología , ADN Mitocondrial/biosíntesis , ADN Mitocondrial/genética , Expresión Génica , Glucuronidasa/biosíntesis , Glucuronidasa/genética , Humanos , Proteínas Klotho , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Mitocondriales/biosíntesis , Proteínas Mitocondriales/genética , Agujas , Péptidos/química , Péptidos/farmacología , Receptor IGF Tipo 1/biosíntesis , Receptor IGF Tipo 1/genética , Proteínas Ribosómicas/biosíntesis , Proteínas Ribosómicas/genéticaRESUMEN
Different structures and cell types of the periodontium respond to orthodontic tooth movement (OTM) individually. Cementoblasts (OC/CM) located in the immediate vicinity of the fibroblasts on the cement have found way to the centre of actual research. Here, we identify and validate possible reference genes for OC/CM cells by RT-qPCR with and without static compressive loading. We investigated the suitability of 3 reference genes in an in vitro model of cementoblast cells using four different algorithms (Normfinder, geNorm, comparative delta-Ct method and BestKeeper) under different confluences and time. Comparable to our previous publications about reference genes in OTM in rats and human periodontal ligament fibroblasts (hPDLF), Rpl22 in murine OC/CM cells appears as the least regulated gene so that it represents the most appropriate reference gene. Furthermore, unlike to the expression of our recommended reference genes, the expression of additionally investigated target genes changes with confluence and under loading compression. Based on our findings for future RT-qPCR analyses in OC/CM cells, Rpl22 or the combination Rpl22/Tbp should be favored as reference gene. According to our results, although many publications propose the use of Gapdh, it does not seem to be the most suitable approach.
Asunto(s)
Algoritmos , Cemento Dental/metabolismo , Genes , Ligamento Periodontal/citología , Reacción en Cadena en Tiempo Real de la Polimerasa , Técnicas de Movimiento Dental , Animales , Línea Celular Transformada , Cartilla de ADN , Expresión Génica , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/biosíntesis , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Ratones , Proteínas de Unión al ARN/biosíntesis , Proteínas de Unión al ARN/genética , Proteínas Ribosómicas/biosíntesis , Proteínas Ribosómicas/genética , Estrés Mecánico , Proteína de Unión a TATA-Box/biosíntesis , Proteína de Unión a TATA-Box/genéticaRESUMEN
BACKGROUND: Increasing studies have revealed that circular RNAs (circRNAs) contribute to gastric cancer (GC) progression. The circular RNA ribosomal protein L15 (circ-RPL15) is involved in chronic lymphocytic leukemia. However, its expression and functions in GC remain elusive. METHODS: The expression of circ-RPL15 in human GC tissues and adjacent normal tissues, human gastric cancer cell lines (MGC-803, BGC-823, MGN-28, SGC-7901, AGS) and normal gastric mucosal epithelial cell line (GES-1) were detected by RT-PCR. The relationship between circ-RPL15 level and clinical-pathological indicators were also analyzed. Gain- of function experiments of circ-RPL15 and miR-502-3p were conducted to verify their roles in mediating GC cell proliferation, apoptosis and metastasis. Also, the downstream mechanisms of circ-RPL15 were predicted by bioinformatics analysis, and the interactions between circ-RPL15 and miR-502-3p, miR-502-3p and OLFM4 were verified by dual luciferase reporter gene assay and RNA FISH. RESULTS: circ-RPL15 was upregulated in GC tissues and cell lines, and the overexpressed circ-RPL15 was correlated with poorer survival of GC patients. Functionally, circ-RPL15 upregulation distinctly promoted the proliferation, migration and invasion of GC cells and inhibited apoptosis. Mechanistically, circ-RPL15 functioned as a competitive endogenous RNA via sponging miR-502-3p and activated OLFM4/STAT3 pathway. CONCLUSION: circ-RPL15 promotes GC progression and predicts poor prognosis of GC patients, and regulates the malignant phenotypes of GC cells by mediating the miR-502-3p/OLFM4/STAT3 axis.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/fisiología , Factor Estimulante de Colonias de Granulocitos/metabolismo , MicroARNs/metabolismo , Proteínas Ribosómicas/fisiología , Factor de Transcripción STAT3/metabolismo , Neoplasias Gástricas/fisiopatología , Apoptosis/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas Ribosómicas/biosíntesis , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Análisis de Supervivencia , Regulación hacia ArribaRESUMEN
BACKGROUND: Differences in core or tissue-specific ribosomal protein (Rp) composition within ribosomes contribute to ribosome heterogeneity and functional variability. Yet, the degree to which ribosome heterogeneity modulates development is unknown. The Drosophila melanogaster eRpL22 family contains structurally diverse paralogues, eRpL22 and eRpL22-like. Unlike ubiquitously expressed eRpL22, eRpL22-like expression is tissue-specific, notably within the male germline and the eye. We investigated expression within the developing eye to uncover tissue/cell types where specific paralogue roles might be defined. RESULTS: Immunohistochemistry analysis confirms ubiquitous eRpL22 expression throughout eye development. In larvae, eRpL22-like is ubiquitously expressed, but highly enriched in the peripodial epithelium (PE). In early pupae, eRpL22-like is broadly distributed in multiple cell types, but later, is primarily enriched in interommatidial hair cells (IoHC). Adult patterns include the ring of accessory cells around ommatidia. Adult retinae IoHC patterning phenotypes (shown by scanning electron microscopy) may be linked to RNAi-mediated eRpL22-like depletion within larval PE. Immunoblots and polysome profile analyses show multiple variants of eRpL22-like across development, with the variant at the expected molecular mass co-sedimenting with active ribosomes. CONCLUSION: Our data reveal differential patterns of eRpL22-like expression relative to eRpL22 and suggest a specific role for eRpL22-like in developmental patterning of the eye.