RESUMEN
Neural differentiation requires a multifaceted program to alter gene expression along the proliferation to differentiation axis. While critical changes occur at the level of transcription, post-transcriptional mechanisms allow fine-tuning of protein output. We investigated the role of tRNAs in regulating gene expression during neural differentiation by quantifying tRNA abundance in neural progenitor-biased and neuron-biased Drosophila larval brains. We found that tRNA profiles are largely consistent between progenitor-biased and neuron-biased brains but significant variation occurs for 10 cytoplasmic isodecoders (individual tRNA genes) and this establishes differential tRNA levels for 8 anticodon groups. We used these tRNA data to investigate relationships between tRNA abundance, codon optimality-mediated mRNA decay, and translation efficiency in progenitors and neurons. Our data reveal that tRNA levels strongly correlate with codon optimality-mediated mRNA decay within each cell type but generally do not explain differences in stabilizing versus destabilizing codons between cell types. Regarding translation efficiency, we found that tRNA expression in neural progenitors preferentially supports translation of mRNAs whose products are in high demand in progenitors, such as those associated with protein synthesis. In neurons, tRNA expression shifts to disfavor translation of proliferation-related transcripts and preferentially support translation of transcripts tied to neuron-specific functions like axon pathfinding and synapse formation. Overall, our analyses reveal that changes in tRNA levels along the neural differentiation axis support optimal gene expression in progenitors and neurons.
RESUMEN
N6-methyladenosine (m6A) is the most prevalent internal mRNA modification in metazoans and is particularly abundant in the central nervous system. The extent to which m6A is dynamically regulated and whether m6A contributes to cell type-specific mRNA metabolism in the nervous system, however, is largely unknown. To address these knowledge gaps, we mapped m6A and measured mRNA decay in neural progenitors (neuroblasts) and neurons of the Drosophila melanogaster larval brain. We identified 867 m6A targets; 233 of these are novel and preferentially encode regulators of neuroblast proliferation, cell fate-specification and synaptogenesis. Comparison of the neuroblast and neuron m6A transcriptomes revealed that m6A stoichiometry is largely uniform; we did not find evidence of neuroblast-specific or neuron-specific m6A modification. While m6A stoichiometry is constant, m6A targets are significantly less stable in neuroblasts than in neurons, potentially due to m6A-independent stabilization in neurons. We used in vivo quantitative imaging of m6A target proteins in Mettl3 methyltransferase null brains and Ythdf m6A reader overexpressing brains to assay metabolic effects of m6A. Target protein levels decreased in Mettl3 null brains and increased in Ythdf overexpressing brains, supporting a previously proposed model in which m6A enhances translation of target mRNAs. We conclude that m6A does not directly regulate mRNA stability during Drosophila neurogenesis but is rather deposited on neurodevelopmental transcripts that have intrinsic low stability in order to augment protein output.
Asunto(s)
Drosophila melanogaster , Drosophila , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Animales , Metiltransferasas/genética , Metiltransferasas/metabolismo , Neuronas/metabolismo , ARN Mensajero/metabolismoRESUMEN
Obtaining neuron transcriptomes is challenging; their complex morphology and interconnected microenvironments make it difficult to isolate neurons without potentially altering gene expression. Multidendritic sensory neurons (md neurons) of Drosophila larvae are commonly used to study peripheral nervous system biology, particularly dendrite arborization. We sought to test if EC-tagging, a biosynthetic RNA tagging and purification method that avoids the caveats of physical isolation, would enable discovery of novel regulators of md neuron dendrite arborization. Our aims were twofold: discover novel md neuron transcripts and test the sensitivity of EC-tagging. RNAs were biosynthetically tagged by expressing CD:UPRT (a nucleobase-converting fusion enzyme) in md neurons and feeding 5-ethynylcytosine (EC) to larvae. Only CD:UPRT-expressing cells are competent to convert EC into 5-ethynyluridine-monophosphate which is subsequently incorporated into nascent RNA transcripts. Tagged RNAs were purified and used for RNA-sequencing. Reference RNA was prepared in a similar manner using 5-ethynyluridine (EUd) to tag RNA in all cells and negative control RNA-seq was performed on "mock tagged" samples to identify non-specifically purified transcripts. Differential expression analysis identified md neuron enriched and depleted transcripts. Three candidate genes encoding RNA-binding proteins (RBPs) were tested for a role in md neuron dendrite arborization. Loss-of-function for the m6A-binding factor Ythdc1 did not cause any dendrite arborization defects while RNAi of the other two candidates, the poly(A) polymerase Hiiragi and the translation regulator Hephaestus, caused significant defects in dendrite arborization. This work provides an expanded view of transcription in md neurons and a technical framework for combining EC-tagging with RNA-seq to profile transcription in cells that may not be amenable to physical isolation.
Asunto(s)
Dendritas/fisiología , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Neurogénesis/genética , Polinucleotido Adenililtransferasa/metabolismo , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Células Receptoras Sensoriales/fisiología , Animales , Animales Modificados Genéticamente , Citosina/administración & dosificación , Citosina/análogos & derivados , Citosina/metabolismo , Nucleótidos de Desoxiuracil/química , Nucleótidos de Desoxiuracil/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Mutación con Pérdida de Función , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Polinucleotido Adenililtransferasa/genética , Proteína de Unión al Tracto de Polipirimidina/genética , ARN/química , ARN/metabolismo , Interferencia de ARN , RNA-Seq , Células Receptoras Sensoriales/citología , Coloración y Etiquetado/métodosRESUMEN
Tissue-specific mRNA stability is important for cell fate and physiology, but the mechanisms involved are not fully understood. We found that zygotic mRNA stability in Drosophila correlates with codon content: optimal codons are enriched in stable transcripts associated with metabolic functions like translation, while non-optimal codons are enriched in unstable transcripts, including those associated with neural development. Bioinformatic analyses and reporter assays revealed that similar codons stabilize or destabilize mRNAs in the nervous system and other tissues, but the link between codon content and stability is attenuated in the nervous system. We confirmed that optimal codons are decoded by abundant tRNAs while non-optimal codons are decoded by less abundant tRNAs in embryos and in the nervous system. We conclude that codon optimality is a general determinant of zygotic mRNA stability, and attenuation of codon optimality allows trans-acting factors to exert greater influence over mRNA decay in the nervous system.
Asunto(s)
Codón/química , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN de Transferencia/genética , Animales , Animales Modificados Genéticamente , Codón/metabolismo , Biología Computacional/métodos , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Embrión no Mamífero , Semivida , Humanos , Neurogénesis/genética , Estabilidad del ARN , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Cigoto/crecimiento & desarrollo , Cigoto/metabolismoRESUMEN
Cell type-specific transcription is a key determinant of cell fate and function. An ongoing challenge in biology is to develop robust and stringent biochemical methods to explore gene expression with cell type specificity. This challenge has become even greater as researchers attempt to apply high-throughput RNA analysis methods under in vivo conditions. TU-tagging and EC-tagging are in vivo biosynthetic RNA tagging techniques that allow spatial and temporal specificity in RNA purification. Spatial specificity is achieved through targeted expression of pyrimidine salvage enzymes (uracil phosphoribosyltransferase and cytosine deaminase) and temporal specificity is achieved by controlling exposure to bioorthogonal substrates of these enzymes (4-thiouracil and 5-ethynylcytosine). Tagged RNAs can be purified from total RNA extracted from an animal or tissue and used in transcriptome profiling analyses. In addition to identifying cell type-specific mRNA profiles, these techniques are applicable to noncoding RNAs and can be used to measure RNA transcription and decay. Potential applications of TU-tagging and EC-tagging also include fluorescent RNA imaging and selective definition of RNA-protein interactions. TU-tagging and EC-tagging hold great promise for supporting research at the intersection of RNA biology and developmental biology. This article is categorized under: Technologies > Analysis of the Transcriptome.
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Citosina/metabolismo , Perfilación de la Expresión Génica/métodos , ARN/genética , ARN/metabolismo , Tiouracilo/análogos & derivados , Animales , Citosina/análogos & derivados , Humanos , Modelos Genéticos , Tiouracilo/metabolismo , Transcripción GenéticaRESUMEN
Purification of cell type-specific RNAs remains a significant challenge. One solution involves biosynthetic tagging of target RNAs. RNA tagging via incorporation of 4-thiouracil (TU) in cells expressing transgenic uracil phosphoribosyltransferase (UPRT), a method known as TU-tagging, has been used in multiple systems but can have limited specificity due to endogenous pathways of TU incorporation. Here, we describe an alternative method that requires the activity of two enzymes: cytosine deaminase (CD) and UPRT. We found that the sequential activity of these enzymes converts 5-ethynylcytosine (EC) to 5-ethynyluridine monophosphate that is subsequently incorporated into nascent RNAs. The ethynyl group allows efficient detection and purification of tagged RNAs. We show that 'EC-tagging' occurs in tissue culture cells and Drosophila engineered to express CD and UPRT. Additional control can be achieved through a split-CD approach in which functional CD is reconstituted from independently expressed fragments. We demonstrate the sensitivity and specificity of EC-tagging by obtaining cell type-specific gene expression data from intact Drosophila larvae, including transcriptome measurements from a small population of central brain neurons. EC-tagging provides several advantages over existing techniques and should be broadly useful for investigating the role of differential RNA expression in cell identity, physiology and pathology.
Asunto(s)
Linaje de la Célula/genética , Citosina/análogos & derivados , ARN/análisis , Coloración y Etiquetado/métodos , Animales , Animales Modificados Genéticamente , Células Cultivadas , Citosina/metabolismo , Citosina/farmacología , Citosina Desaminasa/metabolismo , Drosophila melanogaster , Perfilación de la Expresión Génica/métodos , Células HeLa , Humanos , Especificidad de Órganos/genética , Pentosiltransferasa/metabolismo , ARN/genéticaRESUMEN
BACKGROUND: Gene expression patterns are determined by rates of mRNA transcription and decay. While transcription is known to regulate many developmental processes, the role of mRNA decay is less extensively defined. A critical step toward defining the role of mRNA decay in neural development is to measure genome-wide mRNA decay rates in neural tissue. Such information should reveal the degree to which mRNA decay contributes to differential gene expression and provide a foundation for identifying regulatory mechanisms that affect neural mRNA decay. RESULTS: We developed a technique that allows genome-wide mRNA decay measurements in intact Drosophila embryos, across all tissues and specifically in the nervous system. Our approach revealed neural-specific decay kinetics, including stabilization of transcripts encoding regulators of axonogenesis and destabilization of transcripts encoding ribosomal proteins and histones. We also identified correlations between mRNA stability and physiologic properties of mRNAs; mRNAs that are predicted to be translated within axon growth cones or dendrites have long half-lives while mRNAs encoding transcription factors that regulate neurogenesis have short half-lives. A search for candidate cis-regulatory elements identified enrichment of the Pumilio recognition element (PRE) in mRNAs encoding regulators of neurogenesis. We found that decreased expression of the RNA-binding protein Pumilio stabilized predicted neural mRNA targets and that a PRE is necessary to trigger reporter-transcript decay in the nervous system. CONCLUSIONS: We found that differential mRNA decay contributes to the relative abundance of transcripts involved in cell-fate decisions, axonogenesis, and other critical events during Drosophila neural development. Neural-specific decay kinetics and the functional specificity of mRNA decay suggest the existence of a dynamic neurodevelopmental mRNA decay network. We found that Pumilio is one component of this network, revealing a novel function for this RNA-binding protein.
Asunto(s)
Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica/genética , Sistema Nervioso/embriología , Neurogénesis/genética , Estabilidad del ARN/fisiología , ARN Mensajero/metabolismo , Regiones no Traducidas 3'/genética , Animales , Dactinomicina/farmacología , Dendritas/metabolismo , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiología , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Embrión no Mamífero/metabolismo , Ontología de Genes , Conos de Crecimiento/metabolismo , Semivida , Sistema Nervioso/metabolismo , Proteínas de Unión al ARN/biosíntesis , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/fisiología , Secuencias Reguladoras de Ácido Ribonucleico/genética , Tiouridina/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Cigoto/metabolismoRESUMEN
Neural progenitors of the Drosophila larval brain, called neuroblasts, can be divided into distinct populations based on patterns of proliferation and differentiation. Type I neuroblasts produce ganglion mother cells (GMCs) that divide once to produce differentiated progeny, while type II neuroblasts produce self-renewing intermediate neural progenitors (INPs) and thus generate lineages containing many more progeny. We identified Taranis (Tara) as an important determinant of type I lineage-specific neural progenitor proliferation patterns. Tara is an ortholog of mammalian SERTAD proteins that are known to regulate cell cycle progression. Tara is differentially-expressed in neural progenitors, with high levels of expression in proliferating type I neuroblasts but no detectable expression in type II lineage INPs. Tara is necessary for cell cycle reactivation in quiescent neuroblasts and for cell cycle progression in type I lineages. Cell cycle defects in tara mutant neuroblasts are due to decreased activation of the E2F1/Dp transcription factor complex and delayed progression through S-phase. Mis-expression of tara in type II lineages delays INP cell cycle progression and induces premature differentiation of INPs into GMCs. Premature INP differentiation can also be induced by loss of E2F1/Dp function and elevated E2F1/Dp expression suppresses Tara-induced INP differentiation. Our results show that lineage-specific Tara expression is necessary for proper brain development and suggest that distinct cell cycle regulatory mechanisms exist in type I versus type II neural progenitors.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Neuronas/citología , Células Madre/citología , Animales , Ciclo Celular , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Cruzamientos Genéticos , Drosophila melanogaster , Perfilación de la Expresión Génica , Inmunohistoquímica/métodos , Hibridación in Situ , Células-Madre Neurales/citología , Neuronas/metabolismo , Factores de TiempoRESUMEN
Transcriptional profiling is a powerful approach for understanding development and disease. Current cell type-specific RNA purification methods have limitations, including cell dissociation trauma or inability to identify all RNA species. Here, we describe "mouse thiouracil (TU) tagging," a genetic and chemical intersectional method for covalent labeling and purification of cell type-specific RNA in vivo. Cre-induced expression of uracil phosphoribosyltransferase (UPRT) provides spatial specificity; injection of 4-thiouracil (4TU) provides temporal specificity. Only UPRT(+) cells exposed to 4TU produce thio-RNA, which is then purified for RNA sequencing (RNA-seq). This method can purify transcripts from spatially complex and rare (<5%) cells, such as Tie2:Cre(+) brain endothelia/microglia (76% validated by expression pattern), or temporally dynamic transcripts, such as those acutely induced by lipopolysaccharide (LPS) injection. Moreover, generating chimeric mice via UPRT(+) bone marrow transplants identifies immune versus niche spleen RNA. TU tagging provides a novel method for identifying actively transcribed genes in specific cells at specific times within intact mice.
Asunto(s)
Biología Molecular/métodos , ARN/aislamiento & purificación , Coloración y Etiquetado/métodos , Tiouracilo/metabolismo , Animales , Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea , Encéfalo/embriología , Encéfalo/metabolismo , Quimera , Perfilación de la Expresión Génica , Ratones , Transgenes/genéticaRESUMEN
Similar to mammalian neural progenitors, Drosophila neuroblasts progressively lose competence to make early-born neurons. In neuroblast 7-1 (NB7-1), Kruppel (Kr) specifies the third-born U3 motoneuron and Kr misexpression induces ectopic U3 cells. However, competence to generate U3 cells is limited to early divisions, when the Eve(+) U motoneurons are produced, and competence is lost when NB7-1 transitions to making interneurons. We have found that Polycomb repressor complexes (PRCs) are necessary and sufficient to restrict competence in NB7-1. PRC loss of function extends the ability of Kr to induce U3 fates and PRC gain of function causes precocious loss of competence to make motoneurons. PRCs also restrict competence to make HB9(+) Islet(+) motoneurons in another neuroblast that undergoes a motoneuron-to-interneuron transition, NB3-1. In contrast to the regulation of motoneuron competence, PRC activity does not affect the production of Eve(+) interneurons by NB3-3, HB9(+) Islet(+) interneurons by NB7-3, or Dbx(+) interneurons by multiple neuroblasts. These findings support a model in which PRCs establish motoneuron-specific competence windows in neuroblasts that transition from motoneuron to interneuron production.
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Diferenciación Celular/fisiología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomía & histología , Neuronas Motoras/fisiología , Complejos Multiproteicos/metabolismo , Animales , Linaje de la Célula , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Epigénesis Genética , Interneuronas/citología , Interneuronas/fisiología , Neuronas Motoras/citología , Complejos Multiproteicos/química , Mutación , Complejo Represivo Polycomb 1RESUMEN
We found that the combination of spatially restricted uracil phosphoribosyltransferase (UPRT) expression with 4-thiouracil delivery can be used to label and purify cell type-specific RNA from intact complex tissues in Drosophila melanogaster. This method is useful for isolating RNA from cell types that are difficult to isolate by dissection or dissociation methods and should work in many organisms, including mammals and other vertebrates.
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Drosophila melanogaster/genética , Pentosiltransferasa/química , ARN/genética , ARN/aislamiento & purificación , Tiouracilo/análogos & derivados , Extractos de Tejidos/química , Extractos de Tejidos/aislamiento & purificación , Animales , Drosophila melanogaster/química , Manejo de Especímenes/métodos , Coloración y Etiquetado , Tiouracilo/químicaRESUMEN
Microarray-based analysis of mRNA expression has provided a genome-wide understanding of the genes and pathways involved in many biological processes. However, two limitations are often associated with traditional microarray experiments. First, standard methods of microarray analysis measure mRNA abundance, not mRNA synthesis or mRNA decay, and, therefore, do not provide any information regarding the mechanisms regulating transcript levels. Second, microarrays are often performed with mRNA from a mixed population of cells, and data for a specific cell-type of interest can be difficult to obtain. This chapter describes a method, referred to here as "4TU-tagging," which can be used to overcome these limitations. 4TU-Tagging uses cell type-specific expression of the uracil phosphoribosyltransferase gene of Toxoplasma gondii and the uracil analog 4-thiouracil (4TU) to selectively tag and purify RNA. Pulse-labeling of newly synthesized RNA with 4TU followed by a "chase" with unmodified uracil allows in vivo measurements of mRNA synthesis and decay in specific cells. Experimental design considerations for applying 4TU-tagging to different systems and protocols for cell type-specific RNA tagging, purification, and microarray analysis are covered in this chapter.
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Pentosiltransferasa/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Tiouracilo/análogos & derivados , Animales , Expresión Génica , Humanos , Especificidad de Órganos , ARN Mensajero/análisis , ARN Mensajero/genética , Tiouracilo/metabolismoRESUMEN
RNA analysis by biosynthetic tagging (RABT) enables sensitive and specific queries of (a) how gene expression is regulated on a genome-wide scale and (b) transcriptional profiling of a single cell or tissue type in vivo. RABT can be achieved by exploiting unique properties of Toxoplasma gondii uracil phosphoribosyltransferase (TgUPRT), a pyrimidine salvage enzyme that couples ribose-5-phosphate to the N1 nitrogen of uracil to yield uridine monophosphate (UMP). When 4-thiouracil is provided as a TgUPRT substrate, the resultant product is 4-thiouridine monophosphate which can, ultimately, be incorporated into RNA. Thio-substituted nucleotides are not a natural component of nucleic acids and are readily tagged, detected, and purified with commercially available reagents. Thus, one can do pulse/chase experiments to measure synthesis and decay rates and/or use cell-specific expression of the TgUPRT to tag only RNA synthesized in a given cell type. This chapter updates the original RABT protocol (1) and addresses methodological details associated with RABT that were beyond the scope or space allotment of the initial report.
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Pentosiltransferasa/metabolismo , ARN/análisis , ARN/biosíntesis , Tiouracilo/análogos & derivados , Animales , Biotinilación , Northern Blotting , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Biología Molecular/métodos , ARN/genética , Especificidad por Sustrato , Tionucleótidos/análisis , Tionucleótidos/biosíntesis , Tiouracilo/metabolismo , Toxoplasma/enzimologíaRESUMEN
Apicomplexans are responsible for significant human and animal disease worldwide, including malaria and toxoplasmosis. Herein we summarize recent advances in gene expression analysis in these eukaryotic pathogens, especially with respect to their developmental biology, and discuss the impact this work may have on the development of new vaccines and chemotherapeutics.
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Apicomplexa/genética , Regulación del Desarrollo de la Expresión Génica , Animales , Apicomplexa/enzimología , Etiquetas de Secuencia Expresada , Inhibidores de Histona Desacetilasas , Interacciones Huésped-Parásitos/genética , Humanos , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Cellular competence is an essential but poorly understood aspect of development. Is competence a general property that affects multiple signaling pathways (e.g., chromatin state), or is competence specific for each signaling pathway (e.g., availability of cofactors)? Here we find that Drosophila neuroblast 7-1 (NB7-1) has a single early window of competence to respond to four different temporal identity genes (Hunchback, Krüppel, Pdm, and Castor); that each of these factors specifies distinct motor neuron identities within this competence window but not outside it; and that progressive restriction to respond to Hunchback and Krüppel occurs within this window. Our work raises the possibility that multiple competence windows may allow the same factors to generate different cell types within the same lineage.
Asunto(s)
Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Drosophila/embriología , Neuronas Motoras/citología , Transducción de Señal/fisiología , Animales , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Homeodominio/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Microscopía Fluorescente , Neuronas Motoras/metabolismo , Factores del Dominio POU/metabolismo , Factores de Tiempo , Factores de Transcripción/metabolismoRESUMEN
Standard microarrays measure mRNA abundance, not mRNA synthesis, and therefore cannot identify the mechanisms that regulate gene expression. We have developed a method to overcome this limitation by using the salvage enzyme uracil phosphoribosyltransferase (UPRT) from the protozoan Toxoplasma gondii. T. gondii UPRT has been well characterized because of its application in monitoring parasite growth: mammals lack this enzyme activity and thus only the parasite incorporates (3)H-uracil into its nucleic acids. In this study we used RNA labeling by UPRT to determine the roles of mRNA synthesis and decay in the control of gene expression during T. gondii asexual development. We also used this approach to specifically label parasite RNA during a mouse infection and to incorporate thio-substituted uridines into the RNA of human cells engineered to express T. gondii UPRT, indicating that engineered UPRT expression will allow cell-specific analysis of gene expression in organisms other than T. gondii.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Pentosiltransferasa/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional/fisiología , Animales , Humanos , Tasa de Depuración Metabólica , Pentosiltransferasa/química , ARN Mensajero/química , Transducción de Señal/fisiología , Coloración y Etiquetado/métodos , Toxoplasma/genética , Toxoplasma/metabolismoRESUMEN
Asexual development in Toxoplasma gondii is a vital aspect of the parasite's life cycle, allowing transmission and avoidance of the host immune response. Differentiation of rapidly dividing tachyzoites into slowly growing, encysted bradyzoites involves significant changes in both physiology and morphology. We generated microarrays of approximately 4,400 Toxoplasma cDNAs, representing a minimum of approximately 600 genes (based on partial sequencing), and used these microarrays to study changes in transcript levels during tachyzoite-to-bradyzoite differentiation. This approach has allowed us to (i) determine expression profiles of previously described developmentally regulated genes, (ii) identify novel developmentally regulated genes, and (iii) identify distinct classes of genes based on the timing and magnitude of changes in transcript levels. Whereas microarray analysis typically involves comparisons of mRNA levels at different time points, we have developed a method to measure relative transcript abundance between genes at a given time point. This method was used to determine transcript levels in parasites prior to differentiation and to further classify bradyzoite-induced genes, thus allowing a more comprehensive view of changes in gene expression than is provided by standard expression profiles. Newly identified developmentally regulated genes include putative surface proteins (a SAG1-related protein, SRS9, and a mucin-domain containing protein), regulatory and metabolic enzymes (methionine aminopeptidase, oligopeptidase, aminotransferase, and glucose-6-phosphate dehydrogenase homologues), and a subset of genes encoding secretory organelle proteins (MIC1, ROP1, ROP2, ROP4, GRA1, GRA5, and GRA8). This analysis permits the first in-depth look at changes in gene expression during development of this complex protozoan parasite.