RESUMO
BACKGROUND: Formation of tissue-specific transcriptional programs underlies multicellular development, including dorsoventral (DV) patterning of the Drosophila embryo. This involves interactions between transcriptional enhancers and promoters in a chromatin context, but how the chromatin landscape influences transcription is not fully understood. RESULTS: Here we comprehensively resolve differential transcriptional and chromatin states during Drosophila DV patterning. We find that RNA Polymerase II pausing is established at DV promoters prior to zygotic genome activation (ZGA), that pausing persists irrespective of cell fate, but that release into productive elongation is tightly regulated and accompanied by tissue-specific P-TEFb recruitment. DV enhancers acquire distinct tissue-specific chromatin states through CBP-mediated histone acetylation that predict the transcriptional output of target genes, whereas promoter states are more tissue-invariant. Transcriptome-wide inference of burst kinetics in different cell types revealed that while DV genes are generally characterized by a high burst size, either burst size or frequency can differ between tissues. CONCLUSIONS: The data suggest that pausing is established by pioneer transcription factors prior to ZGA and that release from pausing is imparted by enhancer chromatin state to regulate bursting in a tissue-specific manner in the early embryo. Our results uncover how developmental patterning is orchestrated by tissue-specific bursts of transcription from Pol II primed promoters in response to enhancer regulatory cues.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , RNA Polimerase II/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fatores de Transcrição/metabolismo , Cromatina/metabolismo , Transcrição GênicaRESUMO
The ability of animals to perceive and respond to sensory information is essential for their survival in diverse environments. While much progress has been made in understanding various sensory modalities, the sense of hygrosensation, which involves the detection and response to humidity, remains poorly understood. In this study, we focused on the hygrosensory, and closely related thermosensory, systems in the vinegar fly Drosophila melanogaster to unravel the molecular profile of the cells of these senses. Using a transcriptomic analysis of over 37,000 nuclei, we identified twelve distinct clusters of cells corresponding to temperature-sensing arista neurons, humidity-sensing sacculus neurons, and support cells relating to these neurons. By examining the expression of known and novel marker genes, we validated the identity of these clusters and characterized their gene expression profiles. We found that each cell type could be characterized by a unique expression profile of ion channels, GPCR signaling molecules, synaptic vesicle cycle proteins, and cell adhesion molecules. Our findings provide valuable insights into the molecular basis of hygro- and thermosensation. Understanding the mechanisms underlying hygro- and thermosensation may shed light on the broader understanding of sensory systems and their adaptation to different environmental conditions in animals.
Assuntos
Ascomicetos , Drosophilidae , Animais , Drosophila melanogaster/genética , Ácido Acético , Neurônios , Núcleo CelularRESUMO
Heterokaryosis is a system in which genetically distinct nuclei coexist within the same cytoplasm. While heterokaryosis dominates the life cycle of many fungal species, the transcriptomic changes associated with the transition from homokaryosis to heterokaryosis is not well understood. Here, we analyse gene expression profiles of homokaryons and heterokaryons from three phylogenetically and reproductively isolated lineages of the filamentous ascomycete Neurospora tetrasperma. We show that heterokaryons are transcriptionally distinct from homokaryons in the sexual stage of development, but not in the vegetative stage, suggesting that the phenotypic switch to fertility in heterokaryons is associated with major changes in gene expression. Heterokaryon expression is predominantly defined by additive effects of its two nuclear components. Furthermore, allele-specific expression analysis of heterokaryons with varying nuclear ratios show patterns of expression ratios strongly dependent on nuclear ratios in the vegetative stage. By contrast, in the sexual stage, strong deviations of expression ratios indicate a co-regulation of nuclear gene expression in all three lineages. Taken together, our results show two levels of expression control: additive effects suggest a nuclear level of expression, whereas co-regulation of gene expression indicate a heterokaryon level of control.
Assuntos
Neurospora , Alelos , Núcleo Celular/genética , Expressão Gênica , Neurospora/genéticaRESUMO
Combined measurements of mRNA and protein expression in single cells enable in-depth analysis of cellular states. We present SPARC, an approach that combines single-cell RNA-sequencing with proximity extension essays to simultaneously measure global mRNA and 89 intracellular proteins in individual cells. We show that mRNA expression fails to accurately reflect protein abundance at the time of measurement, although the direction of changes is in agreement during neuronal differentiation. Moreover, protein levels of transcription factors better predict their downstream effects than do their corresponding transcripts. Finally, we highlight that protein expression variation is overall lower than mRNA variation, but relative protein variation does not reflect the mRNA level. Our results demonstrate that mRNA and protein measurements in single cells provide different and complementary information regarding cell states. SPARC presents a state-of-the-art co-profiling method that overcomes current limitations in throughput and protein localization, including removing the need for cell fixation.
Assuntos
Perfilação da Expressão Gênica/métodos , Proteômica/métodos , Análise de Célula Única/métodos , Humanos , Proteínas/genética , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA/métodos , Transcrição Gênica/genéticaRESUMO
Aggregative multicellularity has evolved multiple times in diverse groups of eukaryotes, exemplified by the well-studied development of dictyostelid social amoebas, for example, Dictyostelium discoideum However, it is still poorly understood why multicellularity emerged in these amoebas while the majority of other members of Amoebozoa are unicellular. Previously, a novel type of noncoding RNA, Class I RNAs, was identified in D. discoideum and shown to be important for normal multicellular development. Here, we investigated Class I RNA evolution and its connection to multicellular development. We identified a large number of new Class I RNA genes by constructing a covariance model combined with a scoring system based on conserved upstream sequences. Multiple genes were predicted in representatives of each major group of Dictyostelia and expression analysis confirmed that our search approach identifies expressed Class I RNA genes with high accuracy and sensitivity and that the RNAs are developmentally regulated. Further studies showed that Class I RNAs are ubiquitous in Dictyostelia and share highly conserved structure and sequence motifs. In addition, Class I RNA genes appear to be unique to dictyostelid social amoebas because they could not be identified in outgroup genomes, including their closest known relatives. Our results show that Class I RNA is an ancient class of ncRNAs, likely to have been present in the last common ancestor of Dictyostelia dating back at least 600 million years. Based on previous functional analyses and the presented evolutionary investigation, we hypothesize that Class I RNAs were involved in evolution of multicellularity in Dictyostelia.
Assuntos
Dictyostelium/citologia , Dictyostelium/genética , Evolução Molecular , Filogenia , RNA não Traduzido/genética , Dictyostelium/classificaçãoRESUMO
DNA methylation is an epigenetic mark that plays an important role in genetic regulation in eukaryotes. Major progress has been made in dissecting the molecular pathways that regulate DNA methylation. Yet, little is known about DNA methylation variation over evolutionary time. Here we present an investigation of the variation of DNA methylation and transposable element (TE) content in species of the filamentous ascomycetes Neurospora. We generated genome-wide DNA methylation data at single-base resolution, together with genomic TE content and gene expression data, of 10 individuals representing five closely related Neurospora species. We found that the methylation levels were low (ranging from 1.3% to 2.5%) and varied among the genomes in a species-specific way. Furthermore, we found that the TEs over 400 bp long were targeted by DNA methylation, and in all genomes, high methylation correlated with low GC, confirming a conserved link between DNA methylation and Repeat Induced Point (RIP) mutations in this group of fungi. Both TE content and DNA methylation pattern showed phylogenetic signal, and the species with the highest TE load (N. crassa) also exhibited the highest methylation level per TE. Our results suggest that DNA methylation is an evolvable trait and indicate that the genomes of Neurospora are shaped by an evolutionary arms race between TEs and host defence.
Assuntos
Metilação de DNA , Epigenoma , Neurospora crassa/genética , Elementos de DNA Transponíveis , Regulação Fúngica da Expressão Gênica , Genoma FúngicoRESUMO
The process of cardiac morphogenesis in humans is incompletely understood. Its full characterization requires a deep exploration of the organ-wide orchestration of gene expression with a single-cell spatial resolution. Here, we present a molecular approach that reveals the comprehensive transcriptional landscape of cell types populating the embryonic heart at three developmental stages and that maps cell-type-specific gene expression to specific anatomical domains. Spatial transcriptomics identified unique gene profiles that correspond to distinct anatomical regions in each developmental stage. Human embryonic cardiac cell types identified by single-cell RNA sequencing confirmed and enriched the spatial annotation of embryonic cardiac gene expression. In situ sequencing was then used to refine these results and create a spatial subcellular map for the three developmental phases. Finally, we generated a publicly available web resource of the human developing heart to facilitate future studies on human cardiogenesis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Miócitos Cardíacos/metabolismo , Análise de Célula Única , Transcriptoma , Feminino , Humanos , Masculino , Morfogênese , Miócitos Cardíacos/citologia , RNA-SeqRESUMO
Understanding complex biological systems requires functional characterization of specialized tissue domains. However, existing strategies for generating and analysing high-throughput spatial expression profiles were developed for a limited range of organisms, primarily mammals. Here we present the first available approach to generate and study high-resolution, spatially resolved functional profiles in a broad range of model plant systems. Our process includes high-throughput spatial transcriptome profiling followed by spatial gene and pathway analyses. We first demonstrate the feasibility of the technique by generating spatial transcriptome profiles from model angiosperms and gymnosperms microsections. In Arabidopsis thaliana we use the spatial data to identify differences in expression levels of 141 genes and 189 pathways in eight inflorescence tissue domains. Our combined approach of spatial transcriptomics and functional profiling offers a powerful new strategy that can be applied to a broad range of plant species, and is an approach that will be pivotal to answering fundamental questions in developmental and evolutionary biology.
Assuntos
Arabidopsis/genética , Perfilação da Expressão Gênica/métodos , Genes de Plantas , Picea/genética , Populus/genética , Estudos de Viabilidade , Reprodutibilidade dos TestesRESUMO
Co-expression of physically linked genes occurs surprisingly frequently in eukaryotes. Such chromosomal clustering may confer a selective advantage as it enables coordinated gene regulation at the chromatin level. We studied the chromosomal organization of genes involved in male reproductive development in Arabidopsis thaliana. We developed an in-silico tool to identify physical clusters of co-regulated genes from gene expression data. We identified 17 clusters (96 genes) involved in stamen development and acting downstream of the transcriptional activator MS1 (MALE STERILITY 1), which contains a PHD domain associated with chromatin re-organization. The clusters exhibited little gene homology or promoter element similarity, and largely overlapped with reported repressive histone marks. Experiments on a subset of the clusters suggested a link between expression activation and chromatin conformation: qRT-PCR and mRNA in situ hybridization showed that the clustered genes were up-regulated within 48 h after MS1 induction; out of 14 chromatin-remodeling mutants studied, expression of clustered genes was consistently down-regulated only in hta9/hta11, previously associated with metabolic cluster activation; DNA fluorescence in situ hybridization confirmed that transcriptional activation of the clustered genes was correlated with open chromatin conformation. Stamen development thus appears to involve transcriptional activation of physically clustered genes through chromatin de-condensation.
Assuntos
Arabidopsis/genética , Cromatina/metabolismo , Regulação da Expressão Gênica de Plantas , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Flores/genética , Flores/crescimento & desenvolvimento , Duplicação Gênica , Genes de Plantas , Genoma de Planta , Código das Histonas , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
A better understanding of transcriptional and post-transcriptional regulation of gene expression in bacteria relies on studying their transcriptome. RNA sequencing methods are used not only to assess RNA abundance but also the exact boundaries of primary and processed transcripts. Here, we developed a method, called identification of specific cleavage position (ISCP), which enables the identification of direct endoribonuclease targets in vivo by comparing the 5Î and 3Î ends of processed transcripts between wild type and RNase deficient strains. To demonstrate the ISCP method, we used as a model the double-stranded specific RNase III in the human pathogen Streptococcus pyogenes. We mapped 92 specific cleavage positions (SCPs) among which, 48 were previously described and 44 are new, with the characteristic 2 nucleotides 3Î overhang of RNase III. Most SCPs were located in untranslated regions of RNAs. We screened for RNase III targets using transcriptomic differential expression analysis (DEA) and compared those with the RNase III targets identified using the ISCP method. Our study shows that in S. pyogenes, under standard growth conditions, RNase III has a limited impact both on antisense transcripts and on global gene expression with the expression of most of the affected genes being downregulated in an RNase III deletion mutant.
Assuntos
Regulação Bacteriana da Expressão Gênica , RNA Antissenso/genética , RNA Bacteriano/genética , Ribonuclease III/genética , Streptococcus pyogenes/genética , Proteínas de Bactérias , Pareamento de Bases , Sequência de Bases , Deleção de Genes , Conformação de Ácido Nucleico , Clivagem do RNA , RNA Antissenso/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonuclease III/deficiência , Streptococcus pyogenes/metabolismo , Transcriptoma , Regiões não TraduzidasRESUMO
Understanding the causes of cis-regulatory variation is a long-standing aim in evolutionary biology. Although cis-regulatory variation has long been considered important for adaptation, we still have a limited understanding of the selective importance and genomic determinants of standing cis-regulatory variation. To address these questions, we studied the prevalence, genomic determinants, and selective forces shaping cis-regulatory variation in the outcrossing plant Capsella grandiflora We first identified a set of 1,010 genes with common cis-regulatory variation using analyses of allele-specific expression (ASE). Population genomic analyses of whole-genome sequences from 32 individuals showed that genes with common cis-regulatory variation (i) are under weaker purifying selection and (ii) undergo less frequent positive selection than other genes. We further identified genomic determinants of cis-regulatory variation. Gene body methylation (gbM) was a major factor constraining cis-regulatory variation, whereas presence of nearby transposable elements (TEs) and tissue specificity of expression increased the odds of ASE. Our results suggest that most common cis-regulatory variation in C. grandiflora is under weak purifying selection, and that gene-specific functional constraints are more important for the maintenance of cis-regulatory variation than genome-scale variation in the intensity of selection. Our results agree with previous findings that suggest TE silencing affects nearby gene expression, and provide evidence for a link between gbM and cis-regulatory constraint, possibly reflecting greater dosage sensitivity of body-methylated genes. Given the extensive conservation of gbM in flowering plants, this suggests that gbM could be an important predictor of cis-regulatory variation in a wide range of plant species.
Assuntos
Capsella/genética , Regulação da Expressão Gênica de Plantas/genética , Genes de Plantas , Genoma de Planta , Metilação de DNA , Elementos de DNA Transponíveis , DNA de Plantas/genética , DNA de Plantas/metabolismo , Perfilação da Expressão Gênica , Inativação Gênica , Variação Genética , Grécia , Metagenômica/métodos , RNA de Plantas/genética , Seleção GenéticaRESUMO
BACKGROUND: Although most insect species are specialized on one or few groups of plants, there are phytophagous insects that seem to use virtually any kind of plant as food. Understanding the nature of this ability to feed on a wide repertoire of plants is crucial for the control of pest species and for the elucidation of the macroevolutionary mechanisms of speciation and diversification of insect herbivores. Here we studied Vanessa cardui, the species with the widest diet breadth among butterflies and a potential insect pest, by comparing tissue-specific transcriptomes from caterpillars that were reared on different host plants. We tested whether the similarities of gene-expression response reflect the evolutionary history of adaptation to these plants in the Vanessa and related genera, against the null hypothesis of transcriptional profiles reflecting plant phylogenetic relatedness. RESULT: Using both unsupervised and supervised methods of data analysis, we found that the tissue-specific patterns of caterpillar gene expression are better explained by the evolutionary history of adaptation of the insects to the plants than by plant phylogeny. CONCLUSION: Our findings suggest that V. cardui may use two sets of expressed genes to achieve polyphagy, one associated with the ancestral capability to consume Rosids and Asterids, and another allowing the caterpillar to incorporate a wide range of novel host-plants.
Assuntos
Evolução Biológica , Borboletas/genética , Animais , Borboletas/crescimento & desenvolvimento , Borboletas/fisiologia , Herbivoria , Larva/fisiologia , Magnoliopsida/genética , Magnoliopsida/fisiologia , Oviposição , Filogenia , TranscriptomaRESUMO
Streptococcus pyogenes is a human pathogen responsible for a wide spectrum of diseases ranging from mild to life-threatening infections. During the infectious process, the temporal and spatial expression of pathogenicity factors is tightly controlled by a complex network of protein and RNA regulators acting in response to various environmental signals. Here, we focus on the class of small RNA regulators (sRNAs) and present the first complete analysis of sRNA sequencing data in S. pyogenes. In the SF370 clinical isolate (M1 serotype), we identified 197 and 428 putative regulatory RNAs by visual inspection and bioinformatics screening of the sequencing data, respectively. Only 35 from the 197 candidates identified by visual screening were assigned a predicted function (T-boxes, ribosomal protein leaders, characterized riboswitches or sRNAs), indicating how little is known about sRNA regulation in S. pyogenes. By comparing our list of predicted sRNAs with previous S. pyogenes sRNA screens using bioinformatics or microarrays, 92 novel sRNAs were revealed, including antisense RNAs that are for the first time shown to be expressed in this pathogen. We experimentally validated the expression of 30 novel sRNAs and antisense RNAs. We show that the expression profile of 9 sRNAs including 2 predicted regulatory elements is affected by the endoribonucleases RNase III and/or RNase Y, highlighting the critical role of these enzymes in sRNA regulation.
Assuntos
RNA Antissenso/genética , Pequeno RNA não Traduzido/genética , Riboswitch/genética , Streptococcus pyogenes/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Sequência de RNA , Streptococcus pyogenes/patogenicidadeRESUMO
The selfing syndrome constitutes a suite of floral and reproductive trait changes that have evolved repeatedly across many evolutionary lineages in response to the shift to selfing. Convergent evolution of the selfing syndrome suggests that these changes are adaptive, yet our understanding of the detailed molecular genetic basis of the selfing syndrome remains limited. Here, we investigate the role of cis-regulatory changes during the recent evolution of the selfing syndrome in Capsella rubella, which split from the outcrosser Capsella grandiflora less than 200 ka. We assess allele-specific expression (ASE) in leaves and flower buds at a total of 18,452 genes in three interspecific F1 C. grandiflora x C. rubella hybrids. Using a hierarchical Bayesian approach that accounts for technical variation using genomic reads, we find evidence for extensive cis-regulatory changes. On average, 44% of the assayed genes show evidence of ASE; however, only 6% show strong allelic expression biases. Flower buds, but not leaves, show an enrichment of cis-regulatory changes in genomic regions responsible for floral and reproductive trait divergence between C. rubella and C. grandiflora. We further detected an excess of heterozygous transposable element (TE) insertions near genes with ASE, and TE insertions targeted by uniquely mapping 24-nt small RNAs were associated with reduced expression of nearby genes. Our results suggest that cis-regulatory changes have been important during the recent adaptive floral evolution in Capsella and that differences in TE dynamics between selfing and outcrossing species could be important for rapid regulatory divergence in association with mating system shifts.
Assuntos
Adaptação Fisiológica/genética , Capsella/genética , Capsella/fisiologia , Flores/genética , Flores/fisiologia , Sequências Reguladoras de Ácido Nucleico/genética , Alelos , Elementos de DNA Transponíveis/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Estudos de Associação Genética , Heterozigoto , Hibridização Genética , Mutagênese Insercional/genética , Fenótipo , Folhas de Planta/genética , Locos de Características Quantitativas/genética , RNA de Plantas/genética , RNA de Plantas/metabolismo , Reprodução/genética , AutofertilizaçãoRESUMO
We have adapted a method to map cell surface proteins and to monitor the effect of specific regulatory RNAs on the surface composition of the bacteria. This method involves direct labeling of surface proteins of living bacteria using fluorescent dyes and a subsequent separation of the crude extract by 2D gel electrophoresis. The strategy yields a substantial enrichment in surface proteins over cytoplasmic proteins. We validated this method by monitoring the effect of the regulatory RNA MicA in Escherichia coli, which regulates the synthesis of several outer membrane proteins, and highlighted the role of Staphylococcus aureus RNAIII for the maintenance of cell wall integrity.
Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Escherichia coli/metabolismo , RNA Bacteriano/metabolismo , Staphylococcus aureus/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Sequência de Bases , Carbocianinas/metabolismo , Parede Celular/metabolismo , Eletroforese em Gel Bidimensional/métodos , Escherichia coli/genética , Microscopia Confocal , Dados de Sequência Molecular , RNA Bacteriano/genética , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Coloração e Rotulagem/métodos , Staphylococcus aureus/genéticaRESUMO
Dictyostelium intermediate repeat sequence 1 (DIRS-1) is the founding member of a poorly characterized class of retrotransposable elements that contain inverse long terminal repeats and tyrosine recombinase instead of DDE-type integrase enzymes. In Dictyostelium discoideum, DIRS-1 forms clusters that adopt the function of centromeres, rendering tight retrotransposition control critical to maintaining chromosome integrity. We report that in deletion strains of the RNA-dependent RNA polymerase RrpC, full-length and shorter DIRS-1 messenger RNAs are strongly enriched. Shorter versions of a hitherto unknown long non-coding RNA in DIRS-1 antisense orientation are also enriched in rrpC- strains. Concurrent with the accumulation of long transcripts, the vast majority of small (21 mer) DIRS-1 RNAs vanish in rrpC- strains. RNASeq reveals an asymmetric distribution of the DIRS-1 small RNAs, both along DIRS-1 and with respect to sense and antisense orientation. We show that RrpC is required for post-transcriptional DIRS-1 silencing and also for spreading of RNA silencing signals. Finally, DIRS-1 mis-regulation in the absence of RrpC leads to retrotransposon mobilization. In summary, our data reveal RrpC as a key player in the silencing of centromeric retrotransposon DIRS-1. RrpC acts at the post-transcriptional level and is involved in spreading of RNA silencing signals, both in the 5' and 3' directions.
Assuntos
Dictyostelium/genética , Interferência de RNA , RNA Polimerase Dependente de RNA/fisiologia , Retroelementos , Núcleo Celular/genética , Dictyostelium/enzimologia , Genoma , Regiões Promotoras Genéticas , RNA Antissenso/metabolismo , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/metabolismo , RNA Polimerase Dependente de RNA/genética , Sequências Repetidas TerminaisRESUMO
We present here a method that enables functional screening of large number of mutations in a single experiment through the combination of random mutagenesis, phenotypic cell sorting and high-throughput sequencing. As a test case, we studied post-transcriptional gene regulation of the bacterial csgD messenger RNA, which is regulated by a small RNA (sRNA). A 109 bp sequence within the csgD 5'-UTR, containing all elements for expression and sRNA-dependent control, was mutagenized close to saturation. We monitored expression from a translational gfp fusion and collected fractions of cells with distinct expression levels by fluorescence-activated cell sorting. Deep sequencing of mutant plasmids from cells in different activity-sorted fractions identified functionally important positions in the messenger RNA that impact on intrinsic (translational activity per se) and extrinsic (sRNA-based) gene regulation. The results obtained corroborate previously published data. In addition to pinpointing nucleotide positions that change expression levels, our approach also reveals mutations that are silent in terms of gene expression and/or regulation. This method provides a simple and informative tool for studies of regulatory sequences in RNA, in particular addressing RNA structure-function relationships (e.g. sRNA-mediated control, riboswitch elements). However, slight protocol modifications also permit mapping of functional DNA elements and functionally important regions in proteins.
Assuntos
Regiões 5' não Traduzidas , Mutagênese , Sequências Reguladoras de Ácido Ribonucleico , Proteínas de Escherichia coli/genética , Citometria de Fluxo , Regulação Bacteriana da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Conformação de Ácido Nucleico , Fenótipo , Biossíntese de Proteínas , RNA Mensageiro/química , Pequeno RNA não Traduzido/metabolismo , Análise de Sequência de DNA , Transativadores/genéticaRESUMO
High-throughput sequencing methods have become invaluable for detection and analysis of small RNAs. The results are millions of sequences that need to be carefully analyzed by computational methods and preferentially verified by different experimental techniques. Here we describe how to use high-throughput sequencing followed by bioinformatics and northern blot to identify one particular class of small RNA, microRNAs.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , MicroRNAs/genética , RNA de Protozoário/genética , Animais , Northern Blotting/métodos , Biologia Computacional , Dictyostelium/genética , Eletroforese em Gel de Poliacrilamida , MicroRNAs/isolamento & purificação , RNA de Protozoário/isolamento & purificação , Análise de Sequência de RNA/métodosRESUMO
Conifers normally go through a long juvenile period, for Norway spruce (Picea abies) around 20 to 25 years, before developing male and female cones. We have grown plants from inbred crosses of a naturally occurring spruce mutant (acrocona). One-fourth of the segregating acrocona plants initiate cones already in their second growth cycle, suggesting control by a single locus. The early cone-setting properties of the acrocona mutant were utilized to identify candidate genes involved in vegetative-to-reproductive phase change in Norway spruce. Poly(A(+)) RNA samples from apical and basal shoots of cone-setting and non-cone-setting plants were subjected to high-throughput sequencing (RNA-seq). We assembled and investigated 33,383 expressed putative protein-coding acrocona transcripts. Eight transcripts were differentially expressed between selected sample pairs. One of these (Acr42124_1) was significantly up-regulated in apical shoot samples from cone-setting acrocona plants, and the encoded protein belongs to the MADS box gene family of transcription factors. Using quantitative real-time polymerase chain reaction with independently derived plant material, we confirmed that the MADS box gene is up-regulated in both needles and buds of cone-inducing shoots when reproductive identity is determined. Our results constitute important steps for the development of a rapid cycling model system that can be used to study gene function in conifers. In addition, our data suggest the involvement of a MADS box transcription factor in the vegetative-to-reproductive phase change in Norway spruce.
Assuntos
Perfilação da Expressão Gênica , Proteínas de Domínio MADS/genética , Picea/genética , Proteínas de Plantas/genética , Cruzamentos Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Domínio MADS/classificação , Proteínas de Domínio MADS/metabolismo , Mutação , Noruega , Fenótipo , Filogenia , Picea/crescimento & desenvolvimento , Picea/metabolismo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/classificação , Proteínas de Plantas/metabolismo , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SuéciaRESUMO
Phytophthora infestans is the oomycete pathogen responsible for the devastating late blight disease on potato and tomato. There is presently an intense research focus on the role(s) of effectors in promoting late blight disease development. However, little is known about how they are regulated, or how diversity in their expression may be generated among different isolates. Here we present data from investigation of RNA silencing processes, characterized by non-coding small RNA molecules (sRNA) of 19-40 nt. From deep sequencing of sRNAs we have identified sRNAs matching numerous RxLR and Crinkler (CRN) effector protein genes in two isolates differing in pathogenicity. Effector gene-derived sRNAs were present in both isolates, but exhibited marked differences in abundance, especially for CRN effectors. Small RNAs in P. infestans grouped into three clear size classes of 21, 25/26 and 32 nt. Small RNAs from all size classes mapped to RxLR effector genes, but notably 21 nt sRNAs were the predominant size class mapping to CRN effector genes. Some effector genes, such as PiAvr3a, to which sRNAs were found, also exhibited differences in transcript accumulation between the two isolates. The P. infestans genome is rich in transposable elements, and the majority of sRNAs of all size classes mapped to these sequences, predominantly to long terminal repeat (LTR) retrotransposons. RNA silencing of Dicer and Argonaute genes provided evidence that generation of 21 nt sRNAs is Dicer-dependent, while accumulation of longer sRNAs was impacted by silencing of Argonaute genes. Additionally, we identified six microRNA (miRNA) candidates from our sequencing data, their precursor sequences from the genome sequence, and target mRNAs. These miRNA candidates have features characteristic of both plant and metazoan miRNAs.