RESUMEN
Differentiation of stem and progenitor cells is a highly regulated process that involves the coordinated action of multiple layers of regulation. Here we show how the post-transcriptional regulatory layer instructs the level of chromatin regulation via miR-144 and its targets to orchestrate chromatin condensation during erythropoiesis. The loss of miR-144 leads to impaired chromatin condensation during erythrocyte maturation. Among the several targets of miR-144 that influence chromatin organization, the miR-144-dependent regulation of Hmgn2 is conserved from fish to humans. Our genetic probing of the miR-144/Hmgn2 regulatory axis establish that intact miR-144 target sites in the Hmgn2 3'UTR are necessary for the proper maturation of erythrocytes in both zebrafish and human iPSC-derived erythroid cells while loss of Hmgn2 rescues in part the miR-144 null phenotype. Altogether, our results uncover miR-144 and its target Hmgn2 as the backbone of the genetic regulatory circuit that controls the terminal differentiation of erythrocytes in vertebrates.
Asunto(s)
Cromatina , Eritropoyesis , MicroARNs , Pez Cebra , MicroARNs/metabolismo , MicroARNs/genética , Eritropoyesis/genética , Pez Cebra/genética , Pez Cebra/metabolismo , Humanos , Animales , Cromatina/metabolismo , Cromatina/genética , Eritrocitos/metabolismo , Regiones no Traducidas 3'/genética , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Diferenciación Celular/genéticaRESUMEN
As part of the risk assessment (RA) requirements for genetically modified (GM) plants, according to Regulation (EU) No 503/2013 and the EFSA guidance on the RA of food and feed from GM plants (EFSA GMO Panel 2011), applicants need to perform a molecular characterisation of the DNA sequences inserted in the GM plant genome. This Technical Note to the applicants puts together requirements and recommendations for the quality assessment of the methodology, analysis and reporting when DNA sequencing is used for the molecular characterisation of GM plants. In particular, it applies to the use of Sanger sequencing and next-generation sequencing for the characterisation of the inserted genetic material and its flanking regions at each insertion site, the determination of the copy number of all detectable inserts and the analysis of the genetic stability of the inserts. This updated document replaces the EFSA 2018 Technical Note and reflects the current knowledge in scientific-technical methods for generating and verifying, in a standardised manner, DNA sequencing data in the context of RA of GM plants. It does not take into consideration the verification and validation of the detection method which remains under the remit of the Joint Research Centre (JRC).
RESUMEN
Differentiation of stem and progenitor cells is a highly regulated process that involves the coordinated action of multiple layers of regulation. Here we show how the post-transcriptional regulatory layer instructs the level of chromatin regulation via miR-144 and its targets to orchestrate chromatin condensation during erythropoiesis. The loss of miR-144 leads to impaired chromatin condensation during erythrocyte maturation. Among the several targets of miR-144 that influence chromatin organization, the miR-144-dependent regulation of Hmgn2 is conserved from fish to humans. Our genetic probing of the miR-144/Hmgn2 regulatory axis established that intact miR-144 target sites in the Hmgn2 3'UTR are necessary for the proper maturation of erythrocytes in both zebrafish and human iPSC-derived erythroid cells while loss of Hmgn2 rescues in part the miR-144 null phenotype. Altogether, our results uncover miR-144 and its target Hmgn2 as the backbone of the genetic regulatory circuit that controls the terminal differentiation of erythrocytes in vertebrates.
RESUMEN
Differences in lifespan between males and females are found across many taxa and may be determined, at least in part, by differential responses to diet. Here we tested the hypothesis that the higher dietary sensitivity of female lifespan is mediated by higher and more dynamic expression in nutrient-sensing pathways in females. We first reanalysed existing RNA-seq data, focusing on 17 nutrient-sensing genes with reported lifespan effects. This revealed, consistent with the hypothesis, a dominant pattern of female-biased gene expression, and among sex-biased genes there tended to be a loss of female-bias after mating. We then tested directly the expression of these 17 nutrient-sensing genes in wild-type third instar larvae, once-mated 5- and 16-day-old adults. This confirmed sex-biased gene expression and showed that it was generally absent in larvae, but frequent and stable in adults. Overall, the findings suggest a proximate explanation for the sensitivity of female lifespan to dietary manipulations. We suggest that the contrasting selective pressures to which males and females are subject create differing nutritional demands and requirements, resulting in sex differences in lifespan. This underscores the potential importance of the health impacts of sex-specific dietary responses.
Asunto(s)
Comunicación Celular , Longevidad , Femenino , Masculino , Animales , Larva/genética , Expresión Génica , NutrientesRESUMEN
Synechococcus sp. PCC 11901 reportedly demonstrates the highest, most sustained growth of any known cyanobacterium under optimized conditions. Due to its recent discovery, our knowledge of its biology, including the factors underlying sustained, fast growth, is limited. Furthermore, tools specific for genetic manipulation of PCC 11901 are not established. Here, we demonstrate that PCC 11901 shows faster growth than other model cyanobacteria, including the fast-growing species Synechococcuselongatus UTEX 2973, under optimal growth conditions for UTEX 2973. Comparative genomics between PCC 11901 and Synechocystis sp. PCC 6803 reveal conservation of most metabolic pathways but PCC 11901 has a simplified electron transport chain and reduced light harvesting complex. This may underlie its superior light use, reduced photoinhibition, and higher photosynthetic and respiratory rates. To aid biotechnology applications, we developed a vitamin B12 auxotrophic mutant but were unable to generate unmarked knockouts using two negative selectable markers, suggesting that recombinase- or CRISPR-based approaches may be required for repeated genetic manipulation. Overall, this study establishes PCC 11901 as one of the most promising species currently available for cyanobacterial biotechnology and provides a useful set of bioinformatics tools and strains for advancing this field, in addition to insights into the factors underlying its fast growth phenotype.
Asunto(s)
Synechococcus , Synechocystis , Biotecnología , Redes y Vías Metabólicas , Fotosíntesis , Synechococcus/genética , Synechococcus/metabolismo , Synechocystis/genéticaRESUMEN
Y RNAs (84-112 nt) are non-coding RNAs transcribed by RNA polymerase III and are characterized by a distinctive secondary structure. Human Y RNAs interact with the autoimmune proteins SSB and RO60 that together form a ribonucleoprotein (RNP) complex termed RoRNP and Y RNAs also perform regulatory roles in DNA and RNA replication and stability, which has major implications for diseases including cancer. During cellular stress and apoptosis, Y RNAs are cleaved into 3' and 5' end fragments termed Y RNA-derived small RNAs (ysRNAs). Although some ysRNA functions in stress, apoptosis and cancer have been reported, their fundamental biogenesis has not been described. Here we report that 3' end RNY5 cleavage is structure dependent. In high throughput mutagenesis experiments, cleavage occurred between the 2nd and 3rd nt above a double stranded stem comprising high GC content. We demonstrate that an internal loop above stem S3 is critical for producing 3' end ysRNAs (31 nt) with mutants resulting in longer or no ysRNAs. We show a UGGGU sequence motif at position 22 of RNY5 is critical for producing 5' end ysRNAs (22-25 nt). We show that intact RO60 is critical for ysRNA biogenesis. We conclude that ribonuclease L (RNASEL) contributes to Y RNA cleavage in mouse embryonic fibroblasts but is not the only endoribonuclease important in human cells.
Asunto(s)
ARN no Traducido , Ribonucleoproteínas , Animales , Fibroblastos/metabolismo , Ratones , Conformación de Ácido Nucleico , Procesamiento Postranscripcional del ARN , ARN no Traducido/genética , Ribonucleoproteínas/metabolismoRESUMEN
The presence of small RNAs in sperm is a relatively recent discovery and little is currently known about their importance and functions. Environmental changes including social conditions and dietary manipulations are known to affect the composition and expression of some small RNAs in sperm and may elicit a physiological stress response resulting in an associated change in gamete miRNA profiles. Here, we tested how microRNA profiles in sperm are affected by variation in both sexual selection and dietary regimes in Drosophila melanogaster selection lines. The selection lines were exposed to standard versus low yeast diet treatments and three different population sex ratios (male-biased, female-biased, or equal sex) in a full-factorial design. After 38 generations of selection, all males were maintained on their selected diet and in a common garden male-only environment prior to sperm sampling. We performed transcriptome analyses on miRNAs in purified sperm samples. We found 11 differentially expressed miRNAs with the majority showing differences between male- and female-biased lines. Dietary treatment only had a significant effect on miRNA expression levels in interaction with sex ratio. Our findings suggest that long-term adaptation may affect miRNA profiles in sperm and that these may show varied interactions with short-term environmental changes.
Asunto(s)
Drosophila , MicroARNs , Animales , Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Masculino , MicroARNs/genética , MicroARNs/metabolismo , Selección Sexual , Espermatozoides/fisiologíaRESUMEN
Maternal diet during gestation and lactation affects the development of skeletal muscles in offspring and determines muscle health in later life. In this paper, we describe the association between maternal low protein diet-induced changes in offspring skeletal muscle and the differential expression (DE) of small non-coding RNAs (sncRNAs). We used a mouse model of maternal protein restriction, where dams were fed either a normal (N, 20%) or a low protein (L, 8%) diet during gestation and newborns were cross-fostered to N or L lactating dams, resulting in the generation of NN, NL and LN offspring groups. Total body and tibialis anterior (TA) weights were decreased in weanling NL male offspring but were not different in the LN group, as compared to NN. However, histological evaluation of TA muscle revealed reduced muscle fibre size in both groups at weaning. Small RNA-sequencing demonstrated DE of multiple miRs, snoRNAs and snRNAs. Bioinformatic analyses of miRs-15a, -34a, -122 and -199a, in combination with known myomiRs, confirmed their implication in key muscle-specific biological processes. This is the first comprehensive report for the DE of sncRNAs in nutrition-associated programming of skeletal muscle development, highlighting the need for further research to unravel the detailed molecular mechanisms.
Asunto(s)
Dieta con Restricción de Proteínas , Lactancia/metabolismo , Músculo Esquelético/metabolismo , ARN Nucleolar Pequeño/metabolismo , ARN Pequeño no Traducido , Animales , Proteínas Bacterianas/metabolismo , Biología Computacional , Femenino , Proteínas Luminiscentes/metabolismo , Masculino , Fenómenos Fisiologicos Nutricionales Maternos , Ratones , MicroARNs/genética , Desarrollo de Músculos , Análisis de Secuencia de ADN , DesteteRESUMEN
Somites arising from paraxial mesoderm are a hallmark of the segmented vertebrate body plan. They form sequentially during axis extension and generate musculoskeletal cell lineages. How paraxial mesoderm becomes regionalised along the axis and how this correlates with dynamic changes of chromatin accessibility and the transcriptome remains unknown. Here, we report a spatiotemporal series of ATAC-seq and RNA-seq along the chick embryonic axis. Footprint analysis shows differential coverage of binding sites for several key transcription factors, including CDX2, LEF1 and members of HOX clusters. Associating accessible chromatin with nearby expressed genes identifies cis-regulatory elements (CRE) for TCF15 and MEOX1. We determine their spatiotemporal activity and evolutionary conservation in Xenopus and human. Epigenome silencing of endogenous CREs disrupts TCF15 and MEOX1 gene expression and recapitulates phenotypic abnormalities of anterior-posterior axis extension. Our integrated approach allows dissection of paraxial mesoderm regulatory circuits in vivo and has implications for investigating gene regulatory networks.
Asunto(s)
Embrión de Pollo/fisiología , Cromatina , Regulación del Desarrollo de la Expresión Génica , Mesodermo/fisiología , Secuencias Reguladoras de Ácidos Nucleicos/fisiología , Transcriptoma , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factor de Transcripción CDX2/genética , Factor de Transcripción CDX2/metabolismo , Linaje de la Célula , Femenino , Gastrulación/genética , Gastrulación/fisiología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Factor de Unión 1 al Potenciador Linfoide/genética , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Somitos/metabolismo , Factores de Transcripción/metabolismo , Xenopus laevisRESUMEN
Nile tilapia is the third most cultivated fish worldwide and a novel model species for evolutionary studies. Aiming to improve productivity and contribute to the selection of traits of economic impact, biotechnological approaches have been intensively applied to species enhancement. In this sense, recent studies have focused on the multiple roles played by microRNAs (miRNAs) in the post-transcriptional regulation of protein-coding genes involved in the emergence of phenotypes with relevance for aquaculture. However, there is still a growing demand for a reference resource dedicated to integrating Nile Tilapia miRNA information, obtained from both experimental and in silico approaches, and facilitating the analysis and interpretation of RNA sequencing data. Here, we present an open repository dedicated to Nile Tilapia miRNAs: the "miRTil database". The database stores data on 734 mature miRNAs identified in 11 distinct tissues and five key developmental stages. The database provides detailed information about miRNA structure, genomic context, predicted targets, expression profiles, and relative 5p/3p arm usage. Additionally, miRTil also includes a comprehensive pre-computed miRNA-target interaction network containing 4936 targets and 19,580 interactions.
Asunto(s)
Cíclidos/genética , Cíclidos/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Transcriptoma , Animales , Secuencia de Bases , Bases de Datos Genéticas , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Mapas de Interacción de Proteínas , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN/métodos , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
MicroRNAs (miRNAs) are sequentially processed by two RNase III enzymes, Drosha and Dicer. miR-451 is the only known miRNA whose processing bypasses Dicer and instead relies on the slicer activity of Argonaute-2 (Ago2). miR-451 is highly conserved in vertebrates and regulates erythrocyte maturation, where it becomes the most abundant miRNA. However, the basis for the non-canonical biogenesis of miR-451 is unclear. Here, we show that Ago2 is less efficient than Dicer in processing pre-miRNAs, but this deficit is overcome when miR-144 represses Dicer in a negative-feedback loop during erythropoiesis. Loss of miR-144-mediated Dicer repression in zebrafish embryos and human cells leads to increased canonical miRNA production and impaired miR-451 maturation. Overexpression of Ago2 rescues some of the defects of miR-451 processing. Thus, the evolution of Ago2-dependent processing allows miR-451 to circumvent the global repression of canonical miRNAs elicited, in part, by the miR-144 targeting of Dicer during erythropoiesis.
Asunto(s)
Proteínas Argonautas/genética , Eritropoyesis/genética , MicroARNs/genética , Animales , ARN Helicasas DEAD-box/genética , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Interferencia de ARN , Ribonucleasa III/genética , Pez Cebra/genética , Pez Cebra/crecimiento & desarrolloRESUMEN
MOTIVATION: MicroRNA (miRNA) target prediction algorithms do not generally consider biological context and therefore generic target prediction based on seed binding can lead to a high level of false-positive predictions. Here, we present FilTar, a method that incorporates RNA-Seq data to make miRNA target prediction specific to a given cell type or tissue of interest. RESULTS: We demonstrate that FilTar can be used to: (i) provide sample specific 3'-UTR reannotation; extending or truncating default annotations based on RNA-Seq read evidence and (ii) filter putative miRNA target predictions by transcript expression level, thus removing putative interactions where the target transcript is not expressed in the tissue or cell line of interest. We test the method on a variety of miRNA transfection datasets and demonstrate increased accuracy versus generic miRNA target prediction methods. AVAILABILITY AND IMPLEMENTATION: FilTar is freely available and can be downloaded from https://github.com/TBradley27/FilTar. The tool is implemented using the Python and R programming languages, and is supported on GNU/Linux operating systems. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
MicroARNs , Algoritmos , Animales , MicroARNs/genética , RNA-Seq , Programas InformáticosRESUMEN
Mating induces extensive physiological, biochemical and behavioural changes in female animals of many taxa. In contrast, the overall phenotypic and transcriptomic consequences of mating for males, hence how they might differ from those of females, are poorly described. Post mating responses in each sex are rapidly initiated, predicting the existence of regulatory mechanisms in addition to transcriptional responses involving de novo gene expression. That post mating responses appear different for each sex also predicts that the genome-wide signatures of mating should show evidence of sex-specific specialisation. In this study, we used high resolution RNA sequencing to provide the first direct comparisons of the transcriptomic responses of male and female Drosophila to mating, and the first comparison of mating-responsive miRNAs in both sexes in any species. As predicted, the results revealed the existence of sex- and body part-specific mRNA and miRNA expression profiles. More genes were differentially expressed in the female head-thorax than the abdomen following mating, whereas the opposite was true in males. Indeed, the transcriptional profile of male head-thorax tissue was largely unaffected by mating, and no differentially expressed genes were detected at the most stringent significance threshold. A subset of ribosomal genes in females were differentially expressed in both body parts, but in opposite directions, consistent with the existence of body part-specific resource allocation switching. Novel, mating-responsive miRNAs in each sex were also identified, and a miRNA-mRNA interactions analysis revealed putative targets among mating-responsive genes. We show that the structure of genome-wide responses by each sex to mating is strongly divergent, and provide new insights into how shared genomes can achieve characteristic distinctiveness.
Asunto(s)
Drosophila melanogaster/genética , Conducta Sexual Animal , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiología , Femenino , Perfilación de la Expresión Génica , Masculino , MicroARNs/genética , MicroARNs/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ReproducciónRESUMEN
In the version of this Letter originally published, the Methods incorrectly stated that all phytoplankton cultures were sampled in mid-exponential phase. The low-nitrogen cultures were sampled in early stationary phase and at the point at which Fv/Fm values decreased, to indicate that cultures were experiencing low-nitrogen conditions. All other phytoplankton cultures were sampled in exponential phase. Growth and Fv/Fm data are provided here on high- and low-nitrogen cultures (Figs 1, 2 and 3) to clarify and support this correction. The Methods also stated that cell counting was done using a Beckman Multisizer 3 Coulter Counter, but a CASY Model TT Cell Counter was used.
RESUMEN
MiRNAs are crucial regulators of gene expression found across both the plant and animal kingdoms. While the number of annotated miRNAs deposited in miRBase has greatly increased in recent years, few studies provided comparative analyses across sets of related species, or investigated the role of miRNAs in the evolution of gene regulation. We generated small RNA libraries across 5 mammalian species (cow, dog, horse, pig and rabbit) from 4 different tissues (brain, heart, kidney and testis). We identified 1676 miRBase and 413 novel miRNAs by manually curating the set of computational predictions obtained from miRCat and miRDeep2. Our dataset spanning five species has enabled us to investigate the molecular mechanisms and selective pressures driving the evolution of miRNAs in mammals. We highlight the important contributions of intronic sequences (366 orthogroups), duplication events (135 orthogroups) and repetitive elements (37 orthogroups) in the emergence of new miRNA loci. We use this framework to estimate the patterns of gains and losses across the phylogeny, and observe high levels of miRNA turnover. Additionally, the identification of lineage-specific losses enables the characterisation of the selective constraints acting on the associated target sites. Compared to the miRBase subset, novel miRNAs tend to be more tissue specific. 20 percent of novel orthogroups are restricted to the brain, and their target repertoires appear to be enriched for neuron activity and differentiation processes. These findings may reflect an important role for young miRNAs in the evolution of brain expression plasticity. Many seed sequences appear to be specific to either the cow or the dog. Analyses on the associated targets highlight the presence of several genes under artificial positive selection, suggesting an involvement of these miRNAs in the domestication process. Altogether, we provide an overview on the evolutionary mechanisms responsible for miRNA turnover in 5 domestic species, and their possible contribution to the evolution of gene regulation.
Asunto(s)
Animales Domésticos/genética , Evolución Molecular , MicroARNs/genética , Regiones no Traducidas 3' , Animales , Mamíferos/genética , Especificidad de la EspecieRESUMEN
Motivation: RNA interference, a highly conserved regulatory mechanism, is mediated via small RNAs (sRNA). Recent technical advances enabled the analysis of larger, complex datasets and the investigation of microRNAs and the less known small interfering RNAs. However, the size and intricacy of current data requires a comprehensive set of tools, able to discriminate the patterns from the low-level, noise-like, variation; numerous and varied suggestions from the community represent an invaluable source of ideas for future tools, the ability of the community to contribute to this software is essential. Results: We present a new version of the UEA sRNA Workbench, reconfigured to allow an easy insertion of new tools/workflows. In its released form, it comprises of a suite of tools in a user-friendly environment, with enhanced capabilities for a comprehensive processing of sRNA-seq data e.g. tools for an accurate prediction of sRNA loci (CoLIde) and miRNA loci (miRCat2), as well as workflows to guide the users through common steps such as quality checking of the input data, normalization of abundances or detection of differential expression represent the first step in sRNA-seq analyses. Availability and implementation: The UEA sRNA Workbench is available at: http://srna-workbench.cmp.uea.ac.uk. The source code is available at: https://github.com/sRNAworkbenchuea/UEA_sRNA_Workbench. Supplementary information: Supplementary data are available at Bioinformatics online.
Asunto(s)
MicroARNs/genética , ARN Interferente Pequeño/genética , Análisis de Secuencia de ARN/métodos , Programas Informáticos , Interferencia de ARN , Flujo de TrabajoRESUMEN
Skeletal myogenesis serves as a paradigm to investigate the molecular mechanisms underlying exquisitely regulated cell fate decisions in developing embryos. The evolutionarily conserved miR-133 family of microRNAs is expressed in the myogenic lineage, but how it acts remains incompletely understood. Here, we performed genome-wide differential transcriptomics of miR-133 knockdown (KD) embryonic somites, the source of vertebrate skeletal muscle. These analyses, performed in chick embryos, revealed extensive downregulation of Sonic hedgehog (Shh) pathway components: patched receptors, Hedgehog interacting protein and the transcriptional activator Gli1. By contrast, Gli3, a transcriptional repressor, was de-repressed and confirmed as a direct miR-133 target. Phenotypically, miR-133 KD impaired myotome formation and growth by disrupting proliferation, extracellular matrix deposition and epithelialization. Together, these observations suggest that miR-133-mediated Gli3 silencing is crucial for embryonic myogenesis. Consistent with this idea, we found that activation of Shh signalling by either purmorphamine, or KD of Gli3 by antisense morpholino, rescued the miR-133 KD phenotype. Thus, we identify a novel Shh/myogenic regulatory factor/miR-133/Gli3 axis that connects epithelial morphogenesis with myogenic fate specification.
Asunto(s)
Proteínas Portadoras/biosíntesis , Proteínas Hedgehog/metabolismo , Glicoproteínas de Membrana/biosíntesis , MicroARNs/genética , Desarrollo de Músculos/fisiología , Músculo Esquelético/embriología , Proteínas del Tejido Nervioso/biosíntesis , Receptores Patched/biosíntesis , Proteína Gli3 con Dedos de Zinc/biosíntesis , Animales , Diferenciación Celular/genética , Proliferación Celular/genética , Embrión de Pollo , Regulación hacia Abajo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Desarrollo de Músculos/genética , Músculo Esquelético/crecimiento & desarrollo , Cultivo Primario de Células , Proteína con Dedos de Zinc GLI1/biosíntesisRESUMEN
MicroRNAs (miRNAs) are key regulators of gene expression in multicellular organisms. The elucidation of miRNA function and evolution depends on the identification and characterization of miRNA repertoire of strategic organisms, as the fast-evolving cichlid fishes. Using RNA-seq and comparative genomics we carried out an in-depth report of miRNAs in Nile tilapia (Oreochromis niloticus), an emergent model organism to investigate evo-devo mechanisms. Five hundred known miRNAs and almost one hundred putative novel vertebrate miRNAs have been identified, many of which seem to be teleost-specific, cichlid-specific or tilapia-specific. Abundant miRNA isoforms (isomiRs) were identified with modifications in both 5p and 3p miRNA transcripts. Changes in arm usage (arm switching) of nine miRNAs were detected in early development, adult stage and even between male and female samples. We found an increasing complexity of miRNA expression during ontogenetic development, revealing a remarkable synchronism between the rate of new miRNAs recruitment and morphological changes. Overall, our results enlarge vertebrate miRNA collection and reveal a notable differential ratio of miRNA arms and isoforms influenced by sex and developmental life stage, providing a better picture of the evolutionary and spatiotemporal dynamics of miRNAs.
Asunto(s)
Cíclidos/genética , Genómica/métodos , MicroARNs/genética , Isoformas de Proteínas/genética , Animales , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Pruebas Genéticas , Estudio de Asociación del Genoma Completo , Estadios del Ciclo de Vida , Masculino , Análisis de Secuencia de ARN , Caracteres Sexuales , Transcripción GenéticaRESUMEN
Dimethylsulfoniopropionate (DMSP) is a globally important organosulfur molecule and the major precursor for dimethyl sulfide. These compounds are important info-chemicals, key nutrients for marine microorganisms, and are involved in global sulfur cycling, atmospheric chemistry and cloud formation1-3. DMSP production was thought to be confined to eukaryotes, but heterotrophic bacteria can also produce DMSP through the pathway used by most phytoplankton 4 , and the DsyB enzyme catalysing the key step of this pathway in bacteria was recently identified 5 . However, eukaryotic phytoplankton probably produce most of Earth's DMSP, yet no DMSP biosynthesis genes have been identified in any such organisms. Here we identify functional dsyB homologues, termed DSYB, in many phytoplankton and corals. DSYB is a methylthiohydroxybutryate methyltransferase enzyme localized in the chloroplasts and mitochondria of the haptophyte Prymnesium parvum, and stable isotope tracking experiments support these organelles as sites of DMSP synthesis. DSYB transcription levels increased with DMSP concentrations in different phytoplankton and were indicative of intracellular DMSP. Identification of the eukaryotic DSYB sequences, along with bacterial dsyB, provides the first molecular tools to predict the relative contributions of eukaryotes and prokaryotes to global DMSP production. Furthermore, evolutionary analysis suggests that eukaryotic DSYB originated in bacteria and was passed to eukaryotes early in their evolution.
Asunto(s)
Cloroplastos/enzimología , Haptophyta/enzimología , Metiltransferasas/genética , Mitocondrias/enzimología , Compuestos de Sulfonio/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Diatomeas/enzimología , Diatomeas/genética , Dinoflagelados/enzimología , Dinoflagelados/genética , Haptophyta/genética , Metiltransferasas/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Fitoplancton/metabolismoRESUMEN
BACKGROUND: The neural crest (NC) is a class of transitory stem cell-like cells unique to vertebrate embryos. NC cells arise within the dorsal neural tube where they undergo an epithelial to mesenchymal transition in order to migrate and differentiate throughout the developing embryo. The derivative cell types give rise to multiple tissues, including the craniofacial skeleton, peripheral nervous system and skin pigment cells. Several well-studied gene regulatory networks underpin NC development, which when disrupted can lead to various neurocristopathies such as craniofrontonasal dysplasia, DiGeorge syndrome and some forms of cancer. Small RNAs, such as microRNAs (miRNAs) are non-coding RNA molecules important in post-transcriptional gene silencing and critical for cellular regulation of gene expression. RESULTS: To uncover novel small RNAs in NC development we used high definition adapters and next generation sequencing of libraries derived from ectodermal explants of Xenopus laevis embryos induced to form neural and NC tissue. Ectodermal and blastula animal pole (blastula) stage tissues were also sequenced. We show that miR-427 is highly abundant in all four tissue types though in an isoform specific manner and we define a set of 11 miRNAs that are enriched in the NC. In addition, we show miR-301a and miR-338 are highly expressed in both the NC and blastula suggesting a role for these miRNAs in maintaining the stem cell-like phenotype of NC cells. CONCLUSION: We have characterised the miRNAs expressed in Xenopus embryonic explants treated to form ectoderm, neural or NC tissue. This has identified novel tissue specific miRNAs and highlighted differential expression of miR-427 isoforms.