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BACKGROUND: During its life cycle, the human pathogen Trypanosoma cruzi must quickly adapt to different environments, in which the variation in the gene expression of the regulatory U-rich RNA-binding protein 1 (TcUBP1) plays a crucial role. We have previously demonstrated that the overexpression of TcUBP1 in insect-dwelling epimastigotes orchestrates an RNA regulon to promote differentiation to infective forms. METHODS: In an attempt to generate TcUBP1 knockout parasites by using CRISPR-Cas9 technology, in the present study, we obtained a variant transcript that encodes a protein with 95% overall identity and a modified N-terminal sequence. The expression of this mutant protein, named TcUBP1mut, was notably reduced compared to that of the endogenous form found in normal cells. TcUBP1mut-knockdown epimastigotes exhibited normal growth and differentiation into infective metacyclic trypomastigotes and were capable of infecting mammalian cells. RESULTS: We analyzed the RNA-Seq expression profiles of these parasites and identified 276 up- and 426 downregulated genes with respect to the wildtype control sample. RNA-Seq comparison across distinct developmental stages revealed that the transcriptomic profile of these TcUBP1mut-knockdown epimastigotes significantly differs not only from that of epimastigotes in the stationary phase but also from the gene expression landscape characteristic of infective forms. This is both contrary to and consistent with the results of our recent study involving TcUBP1-overexpressing cells. CONCLUSION: Together, our findings demonstrate that the genes exhibiting opposite changes under overexpression and knockdown conditions unveil key mRNA targets regulated by TcUBP1. These mostly encompass transcripts that encode for trypomastigote-specific surface glycoproteins and ribosomal proteins, supporting a role for TcUBP1 in determining the molecular characteristics of the infective stage.
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Proteínas de Protozoários , Proteínas de Ligação a RNA , Trypanosoma cruzi , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Perfilação da Expressão Gênica , Animais , Técnicas de Silenciamento de Genes , Transcriptoma , Humanos , Mutação , Estágios do Ciclo de Vida/genéticaRESUMO
Infectious bovine keratoconjunctivitis (IBK) is an ocular disease that affects bovines and has significant economic and health effects worldwide. Gram negative bacteria Moraxella bovis and Moraxella bovoculi are its main etiological agents. Antimicrobial therapy against IBK is often difficult in beef and dairy herds and, although vaccines are commercially available, their efficacy is variable and dependent on local strains. The aim of this study was to analyze for the first time the genomes of Uruguayan clinical isolates of M. bovis and M. bovoculi. The genomes were de novo assembled and annotated; the genetic basis of fimbrial synthesis was analyzed and virulence factors were identified. A 94% coverage in the reference genomes of both species, and more than 80% similarity to the reference genomes were observed. The mechanism of fimbrial phase variation in M. bovis was detected, and the tfpQ orientation of these genes confirmed, in an inversion region of approximately 2.18kb. No phase variation was determined in the fimbrial gene of M. bovoculi. When virulence factors were compared between strains, it was observed that fimbrial genes have 36.2% sequence similarity. In contrast, the TonB-dependent lactoferrin/transferrin receptor exhibited the highest percentage of amino acid similarity (97.7%) between strains, followed by cytotoxins MbxA/MbvA and the ferric uptake regulator. The role of these virulence factors in the pathogenesis of IBK and their potential as vaccine components should be explored.
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Doenças dos Bovinos , Genoma Bacteriano , Ceratoconjuntivite Infecciosa , Moraxella bovis , Moraxella , Animais , Moraxella/genética , Moraxella/isolamento & purificação , Bovinos , Moraxella bovis/genética , Ceratoconjuntivite Infecciosa/microbiologia , Doenças dos Bovinos/microbiologia , Infecções por Moraxellaceae/microbiologia , Infecções por Moraxellaceae/veterinária , Uruguai , Fatores de Virulência/genéticaRESUMO
Previous studies have shown that overexpression of the Trypanosoma cruzi U-rich RNA-binding protein 1 (TcUBP1) in insect-dwelling epimastigotes results in a gene expression pattern resembling that of the infective form of the pathogen. Here, we used CRISPR-Cas9-induced edition of TcUBP1 and full-length protein overexpression in epimastigote cells to monitor transcriptomic changes during the epimastigote-to-metacyclic trypomastigote stage transition of T. cruzi. This dataset includes the bioinformatics analysis of three different RNA-seq samples, each with three biological replicates, showing differential mRNA abundances. The current transcriptome report has the potential to shed light on the quantitative variances in the expression of significant up- or down-regulated mRNAs as a consequence of the levels of the UBP1 protein. Raw data files were deposited at the NCBI Sequence Read Archive - SRA at http://ncbi.nlm.nih.gov/Traces/sra/sra.cgi with accession numbers PRJNA907231 and PRJNA949967.
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BACKGROUND: Defining cellular mechanisms that drive Alzheimer's disease (AD) pathogenesis and progression will be aided by studies defining how gene expression patterns change during pre-symptomatic AD and ensuing periods of declining cognition. Previous studies have emphasized changes in transcriptome, but not translatome regulation, leaving the ultimate results of gene expression alterations relatively unexplored in the context of AD. OBJECTIVE: To identify genes whose expression might be regulated at the transcriptome and translatome levels in AD, we analyzed gene expression in cerebral cortex of two AD model mouse strains, CVN (APPSwDI;NOS2 -/- ) and Tg2576 (APPSw), and their companion wild type (WT) strains at 6 months of age by tandem RNA-Seq and Ribo-Seq (ribosome profiling). METHODS: Identical starting pools of bulk RNA were used for RNA-Seq and Ribo-Seq. Differential gene expression analysis was performed at the transcriptome, translatome, and translational efficiency levels. Regulated genes were functionally evaluated by gene ontology tools. RESULTS: Compared to WT mice, AD model mice had similar levels of transcriptome regulation, but differences in translatome regulation. A microglial signature associated with early stages of Aß accumulation was upregulated at both levels in CVN mice. Although the two mice strains did not share many regulated genes, they showed common regulated pathways related to AßPP metabolism associated with neurotoxicity and neuroprotection. CONCLUSION: This work represents the first genome-wide study of brain translatome regulation in animal models of AD and provides evidence of a tight and early translatome regulation of gene expression controlling the balance between neuroprotective and neurodegenerative processes in brain.
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Doença de Alzheimer , Doença de Alzheimer/patologia , Animais , Modelos Animais de Doenças , Estudo de Associação Genômica Ampla , Humanos , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , TranscriptomaRESUMO
Trypanosoma cruzi is the etiological agent for Chagas disease, a neglected parasitic disease in Latin America. Gene transcription control governs the eukaryotic cell replication but is absent in trypanosomatids; thus, it must be replaced by posttranscriptional regulatory events. We investigated the entrance into the T. cruzi replicative cycle using ribosome profiling and proteomics on G1/S epimastigote cultures synchronized with hydroxyurea. We identified 1,784 translationally regulated genes (change > 2, false-discovery rate [FDR] < 0.05) and 653 differentially expressed proteins (change > 1.5, FDR < 0.05), respectively. A major translational remodeling accompanied by an extensive proteome change is found, while the transcriptome remains largely unperturbed at the replicative entrance of the cell cycle. The differentially expressed genes comprise specific cell cycle processes, confirming previous findings while revealing candidate cell cycle regulators that undergo previously unnoticed translational regulation. Clusters of genes showing a coordinated regulation at translation and protein abundance share related biological functions such as cytoskeleton organization and mitochondrial metabolism; thus, they may represent posttranscriptional regulons. The translatome and proteome of the coregulated clusters change in both coupled and uncoupled directions, suggesting that complex cross talk between the two processes is required to achieve adequate protein levels of different regulons. This is the first simultaneous assessment of the transcriptome, translatome, and proteome of trypanosomatids, which represent a paradigm for the absence of transcriptional control. The findings suggest that gene expression chronology along the T. cruzi cell cycle is controlled mainly by translatome and proteome changes coordinated using different mechanisms for specific gene groups. IMPORTANCE Trypanosoma cruzi is an ancient eukaryotic unicellular parasite causing Chagas disease, a potentially life-threatening illness that affects 6 to 7 million people, mostly in Latin America. The antiparasitic treatments for the disease have incomplete efficacy and adverse reactions; thus, improved drugs are needed. We study the mechanisms governing the replication of the parasite, aiming to find differences with the human host, valuable for the development of parasite-specific antiproliferative drugs. Transcriptional regulation is essential for replication in most eukaryotes, but in trypanosomatids, it must be replaced by subsequent gene regulation steps since they lack transcription initiation control. We identified the genome-wide remodeling of mRNA translation and protein abundance during the entrance to the replicative phase of the cell cycle. We found that translation is strongly regulated, causing variation in protein levels of specific cell cycle processes, representing the first simultaneous study of the translatome and proteome in trypanosomatids.
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Perfilação da Expressão Gênica/métodos , Proteômica/métodos , Ribossomos/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Estágios do Ciclo de Vida , Processamento de Proteína Pós-Traducional , Proteoma/genética , Proteínas de Protozoários/análise , RNA de Protozoário/análise , Transcriptoma , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismoRESUMO
Programmed cell death 4 (PDCD4) protein is a tumor suppressor that inhibits translation through the mTOR-dependent initiation factor EIF4A, but its functional role and mRNA targets in neurons remain largely unknown. Our work identified that PDCD4 is highly expressed in axons and dendrites of CNS and PNS neurons. Using loss- and gain-of-function experiments in cortical and dorsal root ganglia primary neurons, we demonstrated the capacity of PDCD4 to negatively control axonal growth. To explore PDCD4 transcriptome and translatome targets, we used Ribo-seq and uncovered a list of potential targets with known functions as axon/neurite outgrowth regulators. In addition, we observed that PDCD4 can be locally synthesized in adult axons in vivo, and its levels decrease at the site of peripheral nerve injury and before nerve regeneration. Overall, our findings demonstrate that PDCD4 can act as a new regulator of axonal growth via the selective control of translation, providing a target mechanism for axon regeneration and neuronal plasticity processes in neurons.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Axônios/metabolismo , Dendritos/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Células Cultivadas , Mutação com Ganho de Função , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Mutação com Perda de Função , Masculino , Camundongos , Células PC12 , Cultura Primária de Células , Biossíntese de Proteínas , Proteínas de Ligação a RNA/genética , Ratos , Regulação para CimaRESUMO
Transference of RNAs and ribosomes from Schwann cell-to-axon was demonstrated in normal and regenerating peripheral nerves. Previously, we have shown that RNAs transfer is dependent on F-actin cytoskeleton and Myosin Va. Here, we explored the contribution of microtubules to newly synthesized RNAs transport from Schwann cell nuclei up to nodal microvilli in sciatic nerves. Results using immunohistochemistry and quantitative confocal FRET analysis indicate that Schwann cell-derived RNAs co-localize with microtubules in Schwann cell cytoplasm. Additionally, transport of Schwann cell-derived RNAs is nocodazole and colchicine sensitive demonstrating its dependence on microtubule network integrity. Moreover, mRNAs codifying neuron-specific proteins are among Schwann cell newly synthesized RNAs population, and some of them are associated with KIF1B and KIF5B microtubules-based motors.
Assuntos
Axônios/metabolismo , Microtúbulos/metabolismo , RNA/metabolismo , Células de Schwann/metabolismo , Nervo Isquiático/metabolismo , Animais , Masculino , Bainha de Mielina/metabolismo , Regeneração Nervosa , RNA/análise , Transporte de RNA , Ratos , Ratos Sprague-DawleyRESUMO
Axonal protein synthesis has been shown to play a role in developmental and regenerative growth, as well as in the maintenance of the axoplasm in a steady state. Recent studies have begun to identify the mRNAs localized in axons, which could be translated locally under different conditions. Despite that by now hundreds or thousands of mRNAs have been shown to be localized into the axonal compartment of cultured neurons in vitro, knowledge of which mRNAs are localized in mature myelinated axons is quite limited. With the purpose of characterizing the transcriptome of mature myelinated motor axons of peripheral nervous systems, we modified the axon microdissection method devised by Koenig, enabling the isolation of the axoplasm RNA to perform RNA-seq analysis. The transcriptome analysis indicates that the number of RNAs detected in mature axons is lower in comparison with in vitro data, depleted of glial markers, and enriched in neuronal markers. The mature myelinated axons are enriched for mRNAs related to cytoskeleton, translation, and oxidative phosphorylation. Moreover, it was possible to define core genes present in axons when comparing our data with transcriptomic data of axons grown in different conditions. This work provides evidence that axon microdissection is a valuable method to obtain genome-wide data from mature and myelinated axons of the peripheral nervous system, and could be especially useful for the study of axonal involvement in neurodegenerative pathologies of motor neurons such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophies (SMA).
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Esclerose Lateral Amiotrófica/genética , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/genética , RNA/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Axônios/metabolismo , Axônios/patologia , Diferenciação Celular/genética , Perfilação da Expressão Gênica , Humanos , Microdissecção , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Sistema Nervoso Periférico/metabolismo , Sistema Nervoso Periférico/patologia , RNA Mensageiro/genética , RNA-Seq , Transcriptoma/genéticaRESUMO
The discovery of a large number of long noncoding RNAs (lncRNAs), and the finding that they may play key roles in different biological processes, have started to provide a new perspective in the understanding of gene regulation. It has been shown that the testes express the highest amount of lncRNAs among different vertebrate tissues. However, although some studies have addressed the characterization of lncRNAs along spermatogenesis, an exhaustive analysis of the differential expression of lncRNAs at its different stages is still lacking. Here, we present the results for lncRNA transcriptome profiling along mouse spermatogenesis, employing highly pure flow sorted spermatogenic stage-specific cell populations, strand-specific RNAseq, and a combination of up-to-date bioinformatic pipelines for analysis. We found that the vast majority of testicular lncRNA genes are expressed at post-meiotic stages (i.e. spermiogenesis), which are characterized by extensive post-transcriptional regulation. LncRNAs at different spermatogenic stages shared common traits in terms of transcript length, exon number, and biotypes. Most lncRNAs were lincRNAs, followed by a high representation of antisense (AS) lncRNAs. Co-expression analyses showed a high correlation along the different spermatogenic stage transitions between the expression patterns of AS lncRNAs and their overlapping protein-coding genes, raising possible clues about lncRNA-related regulatory mechanisms. Interestingly, we observed the co-localization of an AS lncRNA and its host sense mRNA in the chromatoid body, a round spermatids-specific organelle that has been proposed as a reservoir of RNA-related regulatory machinery. An additional, intriguing observation is the almost complete lack of detectable expression for Y-linked testicular lncRNAs, despite that a high number of lncRNA genes are annotated for this chromosome.
Assuntos
RNA Longo não Codificante/genética , Espermatogênese/fisiologia , Animais , Regulação da Expressão Gênica , Masculino , Camundongos , RNA Antissenso , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Espermátides/citologia , Espermátides/fisiologia , Espermatogênese/genética , Testículo/citologia , Testículo/fisiologiaRESUMO
The protein Deleted in Breast Cancer-1 is a regulator of several transcription factors and epigenetic regulators, including HDAC3, Rev-erb-alpha, PARP1 and SIRT1. It is well known that DBC1 regulates its targets, including SIRT1, by protein-protein interaction. However, little is known about how DBC1 biological activity is regulated. In this work, we show that in quiescent cells DBC1 is proteolytically cleaved, producing a protein (DN-DBC1) that misses the S1-like domain and no longer binds to SIRT1. DN-DBC1 is also found in vivo in mouse and human tissues. Interestingly, DN-DBC1 is cleared once quiescent cells re-enter to the cell cycle. Using a model of liver regeneration after partial hepatectomy, we found that DN-DBC1 is down-regulated in vivo during regeneration. In fact, WT mice show a decrease in SIRT1 activity during liver regeneration, coincidentally with DN-DBC1 downregulation and the appearance of full length DBC1. This effect on SIRT1 activity was not observed in DBC1 KO mice. Finally, we found that DBC1 KO mice have altered cell cycle progression and liver regeneration after partial hepatectomy, suggesting that DBC1/DN-DBC1 transitions play a role in normal cell cycle progression in vivo after cells leave quiescence. We propose that quiescent cells express DN-DBC1, which either replaces or coexist with the full-length protein, and that restoring of DBC1 is required for normal cell cycle progression in vitro and in vivo. Our results describe for the first time in vivo a naturally occurring form of DBC1, which does not bind SIRT1 and is dynamically regulated, thus contributing to redefine the knowledge about its function.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Técnicas de Inativação de Genes , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Ciclo Celular/genética , Humanos , Regeneração Hepática/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peso Molecular , Ligação Proteica/genética , Domínios Proteicos , Proteólise , Sirtuína 1/metabolismoRESUMO
Protein translation is a key step in gene expression. The development of Ribosome Profiling has allowed the global analysis of this process at sub-codon resolution. In the last years the method has been applied to several models ranging from bacteria to mammalian cells yielding a surprising amount of insight on the mechanism and the regulation of translation. In this review we describe the key aspects of the experimental protocol and comment on the main conclusions raised in different models.
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After linear sequences of genomes and epigenomic landscape data, the 3D organization of chromatin in the nucleus is the next level to be explored. Different organisms present a general hierarchical organization, with chromosome territories at the top. Chromatin interaction maps, obtained by chromosome conformation capture (3C)-based methodologies, for eight plant species reveal commonalities, but also differences, among them and with animals. The smallest structures, found in high-resolution maps of the Arabidopsis genome, are single genes. Epigenetic marks (histone modification and DNA methylation), transcriptional activity, and chromatin interaction appear to be correlated, and whether structure is the cause or consequence of the function of interacting regions is being actively investigated.
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Cromossomos de Plantas/genética , Genoma de Planta/genética , Cromatina/genética , Epigênese Genética/genéticaRESUMO
Cell and developmental processes are complex, and profoundly dependent on spatial relationships that change over time. Innovative educational or teaching strategies are always needed to foster deep comprehension of these processes and their dynamic features. However, laboratory exercises in cell and developmental biology at the undergraduate level do not often take into account the time dimension. In this article, we provide a laboratory exercise focused in cell migration, aiming to stimulate thinking in time and space dimensions through a simplification of more complex processes occurring in cell or developmental biology. The use of open-source tools for the analysis, as well as the whole package of raw results (available at http://github.com/danielprieto/keratocyte) make it suitable for its implementation in courses with very diverse budgets. Aiming to facilitate the student's transition from science-students to science-practitioners we propose an exercise of scientific thinking, and an evaluation method. This in turn is communicated here to facilitate the finding of common caveats and weaknesses in the process of producing simple scientific communications describing the results achieved. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(6):475-482, 2017.
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Escamas de Animais/citologia , Movimento Celular , Ceratócitos da Córnea/citologia , Biologia do Desenvolvimento/educação , Avaliação Educacional , Peixes , Laboratórios/economia , Animais , Técnicas de Cultura de Células/economiaRESUMO
We present the draft genome of Cupriavidus UYMMa02A, a rhizobium strain isolated from root nodules of Mimosa magentea The assembly has approximately 8.1 million bp with an average G+C of 64.1%. Symbiotic and metal-resistance genes were identified. The study of this genome will contribute to the understanding of rhizobial evolution.
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BACKGROUND: Due to the absence of transcription initiation regulation of protein coding genes transcribed by RNA polymerase II, posttranscriptional regulation is responsible for the majority of gene expression changes in trypanosomatids. Therefore, cataloging the abundance of mRNAs (transcriptome) and the level of their translation (translatome) is a key step to understand control of gene expression in these organisms. RESULTS: Here we assess the extent of regulation of the transcriptome and the translatome in the Chagas disease causing agent, Trypanosoma cruzi, in both the non-infective (epimastigote) and infective (metacyclic trypomastigote) insect's life stages using RNA-seq and ribosome profiling. The observed steady state transcript levels support constitutive transcription and maturation implying the existence of distinctive posttranscriptional regulatory mechanisms controlling gene expression levels at those parasite stages. Meanwhile, the downregulation of a large proportion of the translatome indicates a key role of translation control in differentiation into the infective form. The previously described proteomic data correlate better with the translatomes than with the transcriptomes and translational efficiency analysis shows a wide dynamic range, reinforcing the importance of translatability as a regulatory step. Translation efficiencies for protein families like ribosomal components are diminished while translation of the transialidase virulence factors is upregulated in the quiescent infective metacyclic trypomastigote stage. CONCLUSIONS: A large subset of genes is modulated at the translation level in two different stages of Trypanosoma cruzi life cycle. Translation upregulation of virulence factors and downregulation of ribosomal proteins indicates different degrees of control operating to prepare the parasite for an infective life form. Taking together our results show that translational regulation, in addition to regulation of steady state level of mRNA, is a major factor playing a role during the parasite differentiation.
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Perfilação da Expressão Gênica/métodos , Proteômica/métodos , Ribossomos/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimento , Regulação para Baixo , Regulação da Expressão Gênica no Desenvolvimento , Estágios do Ciclo de Vida , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/análise , RNA de Protozoário/análise , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Regulação para CimaRESUMO
Sorting of specific mRNAs to particular cellular locations and regulation of their translation is an essential mechanism underlying cell polarization. The transport of RNAs by kinesins and dyneins has been clearly established in several cell models, including neurons in culture. A similar role appears to exist in higher eukaryotes for the myosins. Myosin Va (Myo5a) has been described as a component of ribonucleoprotein particles (RNPs) in the adult rat nervous system and associated to ZBP1 and ribosomes in ribosomal periaxoplasmic plaques (PARPs), making it a likely candidate for mediating some aspects of RNA transport in neurons. To test this hypothesis, we have characterized RNPs containing Myo5a in adult brains of rats and mice. Microarray analysis of RNAs co-immunoprecipitated with Myo5a indicates that this motor may associate with a specific subpopulation of neuronal mRNAs. We found mRNAs encoding α-synuclein and several proteins with functions in translation in these RNPs. Immunofluorescence analyses of RNPs showed apparent co-localization of Myo5a with ribosomes, mRNA and RNA-binding proteins in discrete structures present both in axons of neurons in culture and in myelinated fibers of medullary roots. Our data suggest that PARPs include RNPs bearing the mRNA coding for Myo5a and are equipped with kinesin and Myo5a molecular motors. In conclusion, we suggest that Myo5a is involved in mRNA trafficking both in the central and peripheral nervous systems.
Assuntos
Axônios/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Actinas/metabolismo , Animais , Encéfalo/metabolismo , Células Cultivadas , Gânglios Espinais/metabolismo , Bulbo , Camundongos , Camundongos Endogâmicos C57BL , Fibras Nervosas Mielinizadas/metabolismo , Ratos , Ratos Sprague-Dawley , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismoRESUMO
To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration.
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Actinas/metabolismo , Axônios/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , RNA/metabolismo , Células de Schwann/metabolismo , Nervo Isquiático/metabolismo , Actinas/antagonistas & inibidores , Actinas/genética , Animais , Transporte Biológico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Comunicação Celular , Expressão Gênica , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Células de Schwann/citologia , Nervo Isquiático/citologia , Nervo Isquiático/lesões , Tiazolidinas/farmacologiaRESUMO
We studied the subcellular distribution of mitochondria and superoxide dismutase-1 (SOD1) in whole mounts of microdissected motor axons of rats expressing the ALS-linked SOD1-G93A mutation. The rationale was to determine whether physical interactions between the enzyme and mitochondria were linked to the axonopathy of motor fibers occurring in amyotrophic lateral sclerosis (ALS). Mitochondria and SOD1 displayed a homogeneous distribution along motor axons both in nontransgenic rats and in those overexpressing wild-type SOD1. In contrast, axons from SOD1-G93A rats (older than 35 days) showed accumulation of mitochondria in discrete clusters located at regular intervals. Most of SOD1 immunoreactivity was enriched in these clusters and colocalized with mitochondria, suggesting a recruitment of SOD1-G93A to the organelle. The SOD1/mitochondrial clusters were abundant in motor axons but scarcely seen in sensory axons. Clusters also were stained for neuronal nitric oxide synthase, nitrotyrosine, and cytochrome c. The later also was detected surrounding clusters. Ubiquitin colocalized with clusters only at late stages of the disease. The cytoskeleton was not overtly altered in clusters. These results suggest that mutant SOD1 and defective mitochondria create localized dysfunctional domains in motor axons, which may lead to progressive axonopathy in ALS.
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Esclerose Lateral Amiotrófica/genética , Axônios/metabolismo , Mitocôndrias/enzimologia , Mutação , Superóxido Dismutase/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Citocromos c/metabolismo , Modelos Animais de Doenças , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Tirosina/análogos & derivados , Tirosina/metabolismo , Ubiquitina/metabolismoRESUMO
BACKGROUND: Tc38 of Trypanosoma cruzi has been isolated as a single stranded DNA binding protein with high specificity for the poly [dT-dG] sequence. It is present only in Kinetoplastidae protozoa and its sequence lacks homology to known functional domains. Tc38 orthologues present in Trypanosoma brucei and Leishmania were proposed to participate in quite different cellular processes. To further understand the function of this protein in Trypanosoma cruzi, we examined its in vitro binding to biologically relevant [dT-dG] enriched sequences, its expression and subcellular localization during the cell cycle and through the parasite life stages. RESULTS: By using specific antibodies, we found that Tc38 protein from epimastigote extracts participates in complexes with the poly [dT-dG] probe as well as with the universal minicircle sequence (UMS), a related repeated sequence found in maxicircle DNA, and the telomeric repeat. However, we found that Tc38 predominantly localizes into the mitochondrion. Though Tc38 is constitutively expressed through non-replicating and replicating life stages of T. cruzi, its subcellular localization in the unique parasite mitochondrion changes according to the cell cycle stage. In epimastigotes, Tc38 is found only in association with kDNA in G1 phase. From the S to G2 phase the protein localizes in two defined and connected spots flanking the kDNA. These spots disappear in late G2 turning into a diffuse dotted signal which extends beyond the kinetoplast. This later pattern is more evident in mitosis and cytokinesis. Finally, late in cytokinesis Tc38 reacquires its association with the kinetoplast. In non-replicating parasite stages such as trypomastigotes, the protein is found only surrounding the entire kinetoplast structure. CONCLUSIONS: The dynamics of Tc38 subcellular localization observed during the cell cycle and life stages support a major role for Tc38 related to kDNA replication and maintenance.
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Ciclo Celular , Proteínas de Ligação a DNA/metabolismo , Mitocôndrias/química , Trypanosoma cruzi/fisiologia , Animais , DNA de Cinetoplasto/metabolismo , Ligação ProteicaRESUMO
The distribution of ribosomes and polysomes in uninjured myelinated axons of rat sciatic nerve was analyzed. Ribosomes were identified by immunocytochemistry at the light and electron microscopic levels. A polyclonal antibody developed against ribosomes recognized both rRNA and ribosomal proteins. The distribution of the immunoreaction product was similar to that obtained with human anti-ribosomal P protein. The immunoreaction product distributions were of two types in axons: 1) periodic localization in the cortical region of axoplasm that appeared as a compact structural aggregate, consistent with that described as a periaxoplasmic ribosomal plaques (PARP) domain (Koenig et al. [2000] J. Neurosci. 20:8390-8400), and 2) scattered small immuno-reactive clusters of varying sizes (RNP) within the central core of the axon. The latter observation suggested the possibility that RNP-like particles could be associated with the axonal transport system and in transit. Immunoreaction product was also associated with a novel structural inclusion, possibly multi-vesicular in makeup that was located in the axon and at the myelin-axon interface, and visible at the light and EM levels. The potential significance of this structural peculiarity is considered.