RESUMEN
BACKGROUND: Transposable elements (TEs) widely contribute to the evolution of genomes allowing genomic innovations, generating germinal and somatic heterogeneity, and giving birth to long non-coding RNAs (lncRNAs). These features have been associated to the evolution, functioning, and complexity of the nervous system at such a level that somatic retrotransposition of long interspersed element (LINE) L1 has been proposed to be associated to human cognition. Among invertebrates, octopuses are fascinating animals whose nervous system reaches a high level of complexity achieving sophisticated cognitive abilities. The sequencing of the genome of the Octopus bimaculoides revealed a striking expansion of TEs which were proposed to have contributed to the evolution of its complex nervous system. We recently found a similar expansion also in the genome of Octopus vulgaris. However, a specific search for the existence and the transcription of full-length transpositionally competent TEs has not been performed in this genus. RESULTS: Here, we report the identification of LINE elements competent for retrotransposition in Octopus vulgaris and Octopus bimaculoides and show evidence suggesting that they might be transcribed and determine germline and somatic polymorphisms especially in the brain. Transcription and translation measured for one of these elements resulted in specific signals in neurons belonging to areas associated with behavioral plasticity. We also report the transcription of thousands of lncRNAs and the pervasive inclusion of TE fragments in the transcriptomes of both Octopus species, further testifying the crucial activity of TEs in the evolution of the octopus genomes. CONCLUSIONS: The neural transcriptome of the octopus shows the transcription of thousands of putative lncRNAs and of a full-length LINE element belonging to the RTE class. We speculate that a convergent evolutionary process involving retrotransposons activity in the brain has been important for the evolution of sophisticated cognitive abilities in this genus.
Asunto(s)
Octopodiformes , ARN Largo no Codificante , Animales , Encéfalo , Elementos Transponibles de ADN , Femenino , Genoma , Octopodiformes/genética , Embarazo , ARN Largo no Codificante/genética , Retroelementos/genéticaRESUMEN
Development of the Drosophila visceral muscle depends on Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) signaling, which specifies founder cells (FCs) in the circular visceral mesoderm (VM). Although Alk activation by its ligand Jelly Belly (Jeb) is well characterized, few target molecules have been identified. Here, we used targeted DamID (TaDa) to identify Alk targets in embryos overexpressing Jeb versus embryos with abrogated Alk activity, revealing differentially expressed genes, including the Snail/Scratch family transcription factor Kahuli (Kah). We confirmed Kah mRNA and protein expression in the VM, and identified midgut constriction defects in Kah mutants similar to those of pointed (pnt). ChIP and RNA-Seq data analysis defined a Kah target-binding site similar to that of Snail, and identified a set of common target genes putatively regulated by Kah and Pnt during midgut constriction. Taken together, we report a rich dataset of Alk-responsive loci in the embryonic VM and functionally characterize the role of Kah in the regulation of embryonic midgut morphogenesis.
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Quinasa de Linfoma Anaplásico , Proteínas de Unión al ADN , Proteínas de Drosophila , Desarrollo Embrionario , Proteínas del Tejido Nervioso , Proteínas Proto-Oncogénicas , Factores de Transcripción , Animales , Quinasa de Linfoma Anaplásico/genética , Diferenciación Celular/genética , Proteínas de Unión al ADN/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Proteínas de Drosophila/genética , Desarrollo Embrionario/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Mesodermo/crecimiento & desarrollo , Mesodermo/metabolismo , Desarrollo de Músculos/genética , Músculos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , RNA-Seq , Transducción de Señal/genética , Análisis de la Célula Individual , Factores de Transcripción/genéticaRESUMEN
Pentameric ligand-gated ion channels (LGICs) play conserved, critical roles in both excitatory and inhibitory synaptic transmission and can be activated by diverse neurochemical ligands. We have performed a characterization of orphan channels from the nematode C. elegans, identifying five new monoamine-gated LGICs with diverse functional properties and expression postsynaptic to aminergic neurons. These include polymodal anion channels activated by both dopamine and tyramine, which may mediate inhibitory transmission by both molecules in vivo. Intriguingly, we also find that a novel serotonin-gated cation channel, LGC-50, is essential for aversive olfactory learning of pathogenic bacteria, a process known to depend on serotonergic neurotransmission. Remarkably, the redistribution of LGC-50 to neuronal processes is modulated by olfactory conditioning, and lgc-50 point mutations that cause misregulation of receptor membrane expression interfere with olfactory learning. Thus, the intracellular trafficking and localization of these receptors at synapses may represent a molecular cornerstone of the learning mechanism.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Aminas Biogénicas/metabolismo , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/metabolismo , Canales Iónicos/metabolismo , Receptores de Serotonina/metabolismoRESUMEN
Muscle pathology in inclusion body myositis (IBM) typically includes inflammatory cell infiltration, muscle fibers with rimmed vacuoles and cytochrome c oxidase (COX)-deficient fibers. Previous studies have revealed clonal expansion of large mitochondrial DNA (mtDNA) deletions in the COX-deficient muscle fibers. Technical limitations have prevented complete investigations of the mtDNA deletions and other mtDNA variants. Detailed characterization by deep sequencing of mtDNA in muscle samples from 21 IBM patients and 10 age-matched controls was performed after whole genome sequencing with a mean depth of mtDNA coverage of 46,000x. Multiple large mtDNA deletions and duplications were identified in all IBM and control muscle samples. In general, the IBM muscles demonstrated a larger number of deletions and duplications with a mean heteroplasmy level of 10% (range 1%-35%) compared to controls (1%, range 0.2%-3%). There was also a small increase in the number of somatic single nucleotide variants in IBM muscle. More than 200 rearrangements were recurrent in at least two or more IBM muscles while 26 were found in both IBM and control muscles. The deletions and duplications, with a high recurrence rate, were mainly observed in three mtDNA regions, m.534-4429, m.6330-13993, and m.8636-16072, where some were flanked by repetitive sequences. The mtDNA copy number in IBM muscle was reduced to 42% of controls. Immunohistochemical and western blot analyses of IBM muscle revealed combined complex I and complex IV deficiency affecting the COX-deficient fibers. In conclusion, deep sequencing and quantitation of mtDNA variants revealed that IBM muscles had markedly increased levels of large deletions and duplications, and there were also indications of increased somatic single nucleotide variants and reduced mtDNA copy numbers compared to age-matched controls. The distribution and type of variants were similar in IBM muscle and controls indicating an accelerated aging process in IBM muscle, possibly associated with chronic inflammation.
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ADN Mitocondrial/genética , Fibras Musculares Esqueléticas/patología , Miositis por Cuerpos de Inclusión/genética , Miositis por Cuerpos de Inclusión/patología , Anciano , Deficiencia de Citocromo-c Oxidasa/genética , Deficiencia de Citocromo-c Oxidasa/metabolismo , Deficiencia de Citocromo-c Oxidasa/patología , Femenino , Reordenamiento Génico/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Mitocondrias/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Miositis por Cuerpos de Inclusión/metabolismoRESUMEN
Deletions and duplications in mitochondrial DNA (mtDNA) cause mitochondrial disease and accumulate in conditions such as cancer and age-related disorders, but validated high-throughput methodology that can readily detect and discriminate between these two types of events is lacking. Here we establish a computational method, MitoSAlt, for accurate identification, quantification and visualization of mtDNA deletions and duplications from genomic sequencing data. Our method was tested on simulated sequencing reads and human patient samples with single deletions and duplications to verify its accuracy. Application to mouse models of mtDNA maintenance disease demonstrated the ability to detect deletions and duplications even at low levels of heteroplasmy.
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ADN Mitocondrial/genética , Eliminación de Gen , Duplicación de Gen , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Animales , ADN Mitocondrial/química , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Ratones , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN/normasRESUMEN
OBJECTIVE: To determine the pathogenicity of a novel POLG mutation in a man with late-onset autosomal recessive progressive external ophthalmoplegia using clinical, molecular, and biochemical analyses. METHODS: A multipronged approach with detailed neurologic examinations, muscle biopsy analyses, molecular genetic studies, and in vitro biochemical characterization. RESULTS: The patient had slowly progressive bilateral ptosis and severely reduced horizontal and vertical gaze. Muscle biopsy showed slight variability in muscle fiber size, scattered ragged red fibers, and partial cytochrome c oxidase deficiency. Biallelic mutations were identified in the POLG gene encoding the catalytic A subunit of POLγ. One allele carried a novel mutation in the exonuclease domain (c.590T>C; p.F197S), and the other had a previously characterized null mutation in the polymerase domain (c.2740A>C; p.T914P). Biochemical characterization revealed that the novel F197S mutant protein had reduced exonuclease and DNA polymerase activities and confirmed that T914P was inactive. By deep sequencing of mitochondrial DNA (mtDNA) extracted from muscle, multiple large-scale rearrangements were mapped and quantified. CONCLUSIONS: The patient's phenotype was caused by biallelic POLG mutations, resulting in one inactive POLγA protein (T914P) and one with decreased polymerase and exonuclease activity (F197S). The reduction in polymerase activity explains the presence of multiple pathogenic large-scale deletions in the patient's mtDNA.
RESUMEN
Klhl14-AS is a long noncoding RNA expressed since early specification of thyroid bud and is the most enriched gene in the mouse thyroid primordium at E10.5. Here, we studied its involvement in thyroid carcinogenesis by analyzing its expression in cancer tissues and different models of neoplastic transformation. Compared with normal thyroid tissue and cells, Klhl14-AS was significantly downregulated in human thyroid carcinoma tissue specimens, particularly the anaplastic histotype, thyroid cancer cell lines, and rodent models of thyroid cancer. Downregulating the expression of Klhl14-AS in normal thyroid cells decreased the expression of thyroid differentiation markers and cell death and increased cell viability. These effects were mediated by the binding of Klhl14-AS to two miRNAs, Mir182-5p and Mir20a-5p, which silenced Pax8 and Bcl2, both essential players of thyroid differentiation. MIR182-5p and MIR20a-5p were upregulated in human thyroid cancer and thyroid cancer experimental models and their effects on Pax8 and Bcl2 were rescued by Klhl14-AS overexpression, confirming Klhl14-AS as a ceRNA for both Pax8 and Bcl2. This work connects deregulation of differentiation with increased proliferation and survival in thyroid neoplastic cells and highlights a novel ceRNA circuitry involving key regulators of thyroid physiology. SIGNIFICANCE: This study describes a new ceRNA with potential tumor suppression activity and helps us better understand the regulatory mechanisms during thyroid differentiation and carcinogenesis.
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Carcinogénesis/genética , Factor de Transcripción PAX8/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Largo no Codificante/genética , Glándula Tiroides/patología , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , Animales , Biomarcadores de Tumor/genética , Carcinogénesis/patología , Muerte Celular/genética , Diferenciación Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Supervivencia Celular/genética , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica/genética , Células HeLa , Humanos , Ratones , Ratones Transgénicos , MicroARNs/genética , Regulación hacia Arriba/genéticaRESUMEN
Mitochondrial DNA (mtDNA) deletions are associated with mitochondrial disease, and also accumulate during normal human ageing. The mechanisms underlying mtDNA deletions remain unknown although several models have been proposed. Here we use deep sequencing to characterize abundant mtDNA deletions in patients with mutations in mitochondrial DNA replication factors, and show that these have distinct directionality and repeat characteristics. Furthermore, we recreate the deletion formation process in vitro using only purified mitochondrial proteins and defined DNA templates. Based on our in vivo and in vitro findings, we conclude that mtDNA deletion formation involves copy-choice recombination during replication of the mtDNA light strand.
Asunto(s)
ADN Mitocondrial/genética , Eliminación de Secuencia/genética , Southern Blotting , Replicación del ADN/genética , Humanos , Proteínas Mitocondriales/genética , Mutación/genéticaRESUMEN
Host specific resistance and non-host resistance are two plant immune responses to counter pathogen invasion. Gene network organizing principles leading to quantitative differences in resistant and susceptible host during host specific resistance are poorly understood. Vascular wilt caused by root pathogen Fusarium species is complex and governed by host specific resistance in crop plants, including chickpea. Here, we temporally profiled two contrasting chickpea genotypes in disease and immune state to better understand gene expression switches in host specific resistance. Integrative gene-regulatory network elucidated tangible insight into interaction coordinators leading to pathway determination governing distinct (disease or immune) phenotypes. Global network analysis identified five major hubs with 389 co-regulated genes. Functional enrichment revealed immunome containing three subnetworks involving CTI, PTI and ETI and wilt diseasome encompassing four subnetworks highlighting pathogen perception, penetration, colonization and disease establishment. These subnetworks likely represent key components that coordinate various biological processes favouring defence or disease. Furthermore, we identified core 76 disease/immunity related genes through subcellular analysis. Our regularized network with robust statistical assessment captured known and unexpected gene interaction, candidate novel regulators as future biomarkers and first time showed system-wide quantitative architecture corresponding to genotypic characteristics in wilt landscape.
Asunto(s)
Cicer/genética , Cicer/inmunología , Redes Reguladoras de Genes/genética , Inmunidad de la Planta/genética , Transcriptoma/genética , Cicer/microbiología , Fusarium/inmunología , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes/inmunología , Genes de Plantas/genética , Genes de Plantas/inmunología , Genotipo , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Fenotipo , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta/inmunología , Raíces de Plantas/genética , Raíces de Plantas/inmunología , Transcriptoma/inmunologíaRESUMEN
Antisense transcripts and other long non-coding RNAs are pervasive in mammalian cells, and some of these molecules have been proposed to regulate proximal protein-coding genes in cis For example, non-coding transcription can contribute to inactivation of tumor suppressor genes in cancer, and antisense transcripts have been implicated in the epigenetic inactivation of imprinted genes. However, our knowledge is still limited and more such regulatory interactions likely await discovery. Here, we make use of available gene expression data from a large compendium of human tumors to generate hypotheses regarding non-coding-to-coding cis-regulatory relationships with emphasis on negative associations, as these are less likely to arise for reasons other than cis-regulation. We document a large number of possible regulatory interactions, including 193 coding/non-coding pairs that show expression patterns compatible with negative cis-regulation. Importantly, by this approach we capture several known cases, and many of the involved coding genes have known roles in cancer. Our study provides a large catalog of putative non-coding/coding cis-regulatory pairs that may serve as a basis for further experimental validation and characterization.
Asunto(s)
Neoplasias/genética , Sistemas de Lectura Abierta/genética , ARN Largo no Codificante/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Metilación de ADN/genética , Regulación Neoplásica de la Expresión Génica , Humanos , ARN Largo no Codificante/metabolismo , Sitio de Iniciación de la TranscripciónRESUMEN
Diatoms and copepods are main actors in marine food webs. The prey-predator interactions between them affect bloom dynamics, shape marine ecosystems and impact the energy transfer to higher trophic levels. Recently it has been demonstrated that the presence of grazers may affect the diatom prey beyond the direct effect of grazing. Here, we investigated the response of the chain-forming centric diatom Skeletonema marinoi to grazer cues, including changes in morphology, gene expression and metabolic profile. S. marinoi cells were incubated with Calanus finmarchicus or with Centropages typicus and in both cases responded by reducing the chain length, whereas changes in gene expression indicated an activation of stress response, changes in the lipid and nitrogen metabolism, in cell cycle regulation and in frustule formation. Transcripts linked to G protein-coupled receptors and to nitric oxide synthesis were differentially expressed suggesting involvement of these signalling transduction pathways in the response. Downregulation of a lipoxygenase in the transcriptomic data and of its products in the metabolomic data also indicate an involvement of oxylipins. Our data contribute to a better understanding of the gene function in diatoms, providing information on the nature of genes implicated in the interaction with grazers, a crucial process in marine ecosystems.
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Copépodos/metabolismo , Diatomeas/metabolismo , Transcriptoma , Animales , Ciclo Celular , Regulación hacia Abajo , Ecosistema , Cadena Alimentaria , Perfilación de la Expresión Génica , Metabolismo de los Lípidos , Lípidos/química , Metaboloma , Nitrógeno/química , Oxilipinas/metabolismo , Fenotipo , Filogenia , Receptores Acoplados a Proteínas G/metabolismo , Transducción de SeñalRESUMEN
How mtDNA replication is terminated and the newly formed genomes are separated remain unknown. We here demonstrate that the mitochondrial isoform of topoisomerase 3α (Top3α) fulfills this function, acting independently of its nuclear role as a component of the Holliday junction-resolving BLM-Top3α-RMI1-RMI2 (BTR) complex. Our data indicate that mtDNA replication termination occurs via a hemicatenane formed at the origin of H-strand replication and that Top3α is essential for resolving this structure. Decatenation is a prerequisite for separation of the segregating unit of mtDNA, the nucleoid, within the mitochondrial network. The importance of this process is highlighted in a patient with mitochondrial disease caused by biallelic pathogenic variants in TOP3A, characterized by muscle-restricted mtDNA deletions and chronic progressive external ophthalmoplegia (CPEO) plus syndrome. Our work establishes Top3α as an essential component of the mtDNA replication machinery and as the first component of the mtDNA separation machinery.
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Segregación Cromosómica/genética , Replicación del ADN/genética , ADN-Topoisomerasas de Tipo I/metabolismo , ADN Mitocondrial/biosíntesis , Dinámicas Mitocondriales/genética , Línea Celular Tumoral , ADN Mitocondrial/genética , Células HeLa , Humanos , Mitocondrias/genética , Enfermedades Mitocondriales/genética , Oftalmoplejía Externa Progresiva Crónica/genéticaRESUMEN
lncRNAs are acquiring increasing relevance as regulators in a wide spectrum of biological processes. The extreme heterogeneity in the mechanisms of action of these molecules, however, makes them very difficult to study, especially regarding their molecular function. A novel lncRNA has been recently identified as the most enriched transcript in mouse developing thyroid. Due to its genomic localization antisense to the protein-encoding Klhl14 gene, we named it Klhl14-AS. In this paper, we highlight that mouse Klhl14-AS produces at least five splicing variants, some of which have not been previously described. Klhl14-AS is expressed with a peculiar pattern, characterized by diverse relative abundance of its isoforms in different mouse tissues. We examine the whole expression level of Klhl14-AS in a panel of adult mouse tissues, showing that it is expressed in the thyroid, lung, kidney, testis, ovary, brain, and spleen, although at different levels. In situ hybridization analysis reveals that, in the context of each organ, Klhl14-AS shows a cell type-specific expression. Interestingly, databases report a similar expression profile for human Klhl14-AS. Our observations suggest that this lncRNA could play cell type-specific roles in several organs and pave the way for functional characterization of this gene in appropriate biological contexts.
RESUMEN
Microalgae play a major role as primary producers in aquatic ecosystems. Cell signalling regulates their interactions with the environment and other organisms, yet this process in phytoplankton is poorly defined. Using the marine planktonic diatom Pseudo-nitzschia multistriata, we investigated the cell response to cues released during sexual reproduction, an event that demands strong regulatory mechanisms and impacts on population dynamics. We sequenced the genome of P. multistriata and performed phylogenomic and transcriptomic analyses, which allowed the definition of gene gains and losses, horizontal gene transfers, conservation and evolutionary rate of sex-related genes. We also identified a small number of conserved noncoding elements. Sexual reproduction impacted on cell cycle progression and induced an asymmetric response of the opposite mating types. G protein-coupled receptors and cyclic guanosine monophosphate (cGMP) are implicated in the response to sexual cues, which overall entails a modulation of cell cycle, meiosis-related and nutrient transporter genes, suggesting a fine control of nutrient uptake even under nutrient-replete conditions. The controllable life cycle and the genome sequence of P. multistriata allow the reconstruction of changes occurring in diatoms in a key phase of their life cycle, providing hints on the evolution and putative function of their genes and empowering studies on sexual reproduction.
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Evolución Biológica , Diatomeas/fisiología , Transporte Biológico/genética , Ciclo Celular , Diatomeas/genética , Regulación del Desarrollo de la Expresión Génica , Filogenia , Dinámica Poblacional , Reproducción/genética , Transducción de SeñalRESUMEN
Identification of cancer driver genes using somatic mutation patterns indicative of positive selection has become a major goal in cancer genomics. However, cancer cells additionally depend on a large number of genes involved in basic cellular processes. While such genes should in theory be subject to strong purifying (negative) selection against damaging somatic mutations, these patterns have been elusive and purifying selection remains inadequately explored in cancer. Here, we hypothesized that purifying selection should be evident in hemizygous genomic regions, where damaging mutations cannot be compensated for by healthy alleles. Using a 7,781-sample pan-cancer dataset, we first confirmed this in POLR2A, an essential gene where hemizygous deletions are known to confer elevated sensitivity to pharmacological suppression. We next used this principle to identify several genes and pathways that show patterns indicative of purifying selection to avoid deleterious mutations. These include the POLR2A interacting protein INTS10 as well as genes involved in mRNA splicing, nonsense-mediated mRNA decay and other RNA processing pathways. Many of these genes belong to large protein complexes, and strong overlaps were observed with recent functional screens for gene essentiality in human cells. Our analysis supports that purifying selection acts to preserve the remaining function of many hemizygously deleted essential genes in tumors, indicating vulnerabilities that might be exploited by future therapeutic strategies.
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Carcinogénesis/genética , Proteínas Portadoras/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , ARN Polimerasa II/genética , Selección Genética/genética , Alelos , Evolución Molecular , Genoma Humano , Genómica , Humanos , Mutación , Proteínas de Neoplasias/biosíntesis , Neoplasias/patología , Transducción de Señal/genéticaRESUMEN
Pufferfish such as fugu and tetraodon carry the smallest genomes among all vertebrates and are ideal for studying genome evolution. However, comparative genomics using these species is hindered by the poor annotation of their genomes. We performed RNA sequencing during key stages of maternal to zygotic transition of Tetraodon nigroviridis and report its first developmental transcriptome. We assembled 61,033 transcripts (23,837 loci) representing 80% of the annotated gene models and 3816 novel coding transcripts from 2667 loci. We demonstrate the similarities of gene expression profiles between pufferfish and zebrafish during maternal to zygotic transition and annotated 1120 long non-coding RNAs (lncRNAs) many of which differentially expressed during development. The promoters for 60% of the assembled transcripts result validated by CAGE-seq. Despite the extreme compaction of the tetraodon genome and the dramatic loss of transposons, the length of lncRNA exons remain comparable to that of other vertebrates and a small set of lncRNAs appears enriched for transposable elements suggesting a selective pressure acting on lncRNAs length and composition. Finally, a set of lncRNAs are microsyntenic between teleost and vertebrates, which indicates potential regulatory interactions between lncRNAs and their flanking coding genes. Our work provides a fundamental molecular resource for vertebrate comparative genomics and embryogenesis studies.
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Proteínas de Peces/genética , Regulación del Desarrollo de la Expresión Génica , Genoma , ARN Largo no Codificante/genética , Tetraodontiformes/genética , Transcriptoma , Animales , Genómica , Tetraodontiformes/crecimiento & desarrolloRESUMEN
BACKGROUND: Crocus sativus stigmas form rich source of apocarotenoids like crocin, picrocrocin and saffranal which besides imparting color, flavour and aroma to saffron spice also have tremendous pharmacological properties. Inspite of their importance, the biosynthetic pathway of Crocus apocarotenoids is not fully elucidated. Moreover, the mechanism of their stigma specific accumulation remains unknown. Therefore, deep transcriptome sequencing of Crocus stigma and rest of the flower tissue was done to identify the genes and transcriptional regulators involved in the biosynthesis of these compounds. RESULTS: Transcriptome of stigma and rest of the flower tissue was sequenced using Illumina Genome Analyzer IIx platform which generated 64,604,402 flower and 51,350,714 stigma reads. Sequences were assembled de novo using trinity resulting in 64,438 transcripts which were classified into 32,204 unigenes comprising of 9853 clusters and 22,351 singletons. A comprehensive functional annotation and gene ontology (GO) analysis was carried out. 58.5 % of the transcripts showed similarity to sequences present in public databases while rest could be specific to Crocus. 5789 transcripts showed similarity to transcription factors representing 76 families out of which Myb family was most abundant. Many genes involved in carotenoid/apocarotenoid pathway were identified for the first time in this study which includes zeta-carotene isomerase and desaturase, carotenoid isomerase and lycopene epsilon-cyclase. GO analysis showed that the predominant classes in biological process category include metabolic process followed by cellular process and primary metabolic process. KEGG mapping analysis indicated that pathways involved in ribosome, carbon and starch and sucrose metabolism were highly represented. Differential expression analysis indicated that key carotenoid/apocarotenoid pathway genes including phytoene synthase, phytoene desaturase and carotenoid cleavage dioxygenase 2 are enriched in stigma thereby providing molecular proof for stigma to be the site of apocarotenoid biosynthesis. CONCLUSIONS: This data would provide a rich source for understanding the carotenoid/apocarotenoid metabolism in Crocus. The database would also help in investigating many questions related to saffron biology including flower development.
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Carotenoides/biosíntesis , Crocus/genética , Crocus/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Transcriptoma , Análisis por Conglomerados , Biología Computacional/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Anotación de Secuencia Molecular , Filogenia , Reproducibilidad de los Resultados , Factores de Transcripción/genéticaRESUMEN
UNLABELLED: The eukaryotic transcriptome is composed of thousands of coding and long non-coding RNAs (lncRNAs). However, we lack a software platform to identify both RNA classes in a given transcriptome. Here we introduce Annocript, a pipeline that combines the annotation of protein coding transcripts with the prediction of putative lncRNAs in whole transcriptomes. It downloads and indexes the needed databases, runs the analysis and produces human readable and standard outputs together with summary statistics of the whole analysis. AVAILABILITY AND IMPLEMENTATION: Annocript is distributed under the GNU General Public License (version 3 or later) and is freely available at https://github.com/frankMusacchia/Annocript. CONTACT: remo.sanges@szn.it.
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Anotación de Secuencia Molecular , ARN Largo no Codificante/genética , Análisis de Secuencia de ARN/métodos , Programas Informáticos , Transcriptoma , HumanosRESUMEN
Seed storage proteins are known to be utilized as carbon and nitrogen source for growing seedlings and thus are considered as potential candidates for nutritional improvement. However, their precise function remains unknown. We have earlier shown that ectopic expression of a seed storage protein, AmA1, leads to increase in protein besides high tuber yield in potato. To elucidate the AmA1-regulated molecular mechanism affecting increased protein synthesis, reserve accumulation, and enhanced growth, a comparative proteomics approach has been applied to tuber life-cycle between wild-type and AmA1 potato. The differential display of proteomes revealed 150 AmA1-responsive protein spots (ARPs) that change their intensities more than 2.5-fold. The LC-ESI-MS/MS analyses led to the identification of 80 ARPs presumably associated with cell differentiation, regulating diverse functions, viz., protein biogenesis and storage, bioenergy and metabolism, and cell signaling. Metabolome study indicated up-regulation of amino acids paralleling the proteomics analysis. To validate this, we focused our attention on anatomical study that showed differences in cell size in the cortex, premedullary zone and pith of the tuber, coinciding with AmA1 expression and localization. Further, we interrogated the proteome data using one-way analysis of variance, cluster, and partial correlation analysis that identified two significant protein modules and six small correlation groups centered around isoforms of cysteine protease inhibitor, actin, heat shock cognate protein 83 and 14-3-3, pointing toward AmA1-regulated overlapping processes of protein enhancement and cell growth perhaps through a common mechanism of function. A model network was constructed using the protein data sets, which aim to show how target proteins might work in coordinated fashion and attribute to increased protein synthesis and storage reserve accumulation in AmA1 tubers on one hand and organ development on the other.
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Biosíntesis de Proteínas/genética , Proteómica/métodos , Proteínas de Almacenamiento de Semillas/metabolismo , Plantones/crecimiento & desarrollo , Solanum tuberosum/genética , Análisis de Varianza , Proliferación Celular , Cromatografía Liquida , Análisis por Conglomerados , Electroforesis en Gel Bidimensional , Metabolómica/métodos , Plantas Modificadas Genéticamente , Proteínas de Almacenamiento de Semillas/genética , Solanum tuberosum/crecimiento & desarrollo , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Long non-coding RNAs (lncRNA) are a major class of non-coding RNAs. They are involved in diverse intra-cellular mechanisms like molecular scaffolding, splicing and DNA methylation. Through these mechanisms they are reported to play a role in cellular differentiation and development. They show an enriched expression in the brain where they are implicated in maintaining cellular identity, homeostasis, stress responses and plasticity. Low sequence conservation and lack of functional annotations make it difficult to identify homologs of mammalian lncRNAs in other vertebrates. A computational evaluation of the lncRNAs through systematic conservation analyses of both sequences as well as their genomic architecture is required. RESULTS: Our results show that a subset of mouse candidate lncRNAs could be distinguished from random sequences based on their alignment with zebrafish phastCons elements. Using ROC analyses we were able to define a measure to select significantly conserved lncRNAs. Indeed, starting from ~2,800 mouse lncRNAs we could predict that between 4 and 11% present conserved sequence fragments in fish genomes. Gene ontology (GO) enrichment analyses of protein coding genes, proximal to the region of conservation, in both organisms highlighted similar GO classes like regulation of transcription and central nervous system development. The proximal coding genes in both the species show enrichment of their expression in brain. In summary, we show that interesting genomic regions in zebrafish could be marked based on their sequence homology to a mouse lncRNA, overlap with ESTs and proximity to genes involved in nervous system development. CONCLUSIONS: Conservation at the sequence level can identify a subset of putative lncRNA orthologs. The similar protein-coding neighborhood and transcriptional information about the conserved candidates provide support to the hypothesis that they share functional homology. The pipeline herein presented represents a proof of principle showing that a portion between 4 and 11% of lncRNAs retains region of conservation between mammals and fishes. We believe this study will result useful as a reference to analyze the conservation of lncRNAs in newly sequenced genomes and transcriptomes.