RESUMO
This year marks the 150(th) anniversary of the presentation by Gregor Mendel of his studies of plant hybridization to the Brunn Natural History Society. Their nature and meaning have been discussed many times. However, on this occasion, we reflect on the scientific enterprise and the perception of new discoveries.
Assuntos
Genética/história , Modelos Genéticos , Animais , Galinhas/genética , Cruzamentos Genéticos , História do Século XVIII , Pisum sativum/genética , Zea mays/genéticaRESUMO
The cellular and genetic networks that contribute to the development of the zeugopod (radius and ulna of the forearm, tibia and fibula of the leg) are not well understood, although these bones are susceptible to loss in congenital human syndromes and to the action of teratogens such as thalidomide. Using a new fate-mapping approach with the Chameleon transgenic chicken line, we show that there is a small contribution of SHH-expressing cells to the posterior ulna, posterior carpals and digit 3. We establish that although the majority of the ulna develops in response to paracrine SHH signalling in both the chicken and mouse, there are differences in the contribution of SHH-expressing cells between mouse and chicken as well as between the chicken ulna and fibula. This is evidence that, although zeugopod bones are clearly homologous according to the fossil record, the gene regulatory networks that contribute to their development and evolution are not fixed.
Assuntos
Animais Geneticamente Modificados , Galinhas , Proteínas Hedgehog , Animais , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Galinhas/genética , Camundongos , Evolução Biológica , Embrião de Galinha , Ulna , Regulação da Expressão Gênica no Desenvolvimento , Fíbula/metabolismo , Rádio (Anatomia)/metabolismo , Humanos , Extremidades/embriologiaRESUMO
DNA methylation is a key regulator of eukaryote genomes, and is of particular relevance in the regulation of gene expression on the sex chromosomes, with a key role in dosage compensation in mammalian XY systems. In the case of birds, dosage compensation is largely absent, with it being restricted to two small Male Hyper-Methylated (MHM) regions on the Z chromosome. To investigate how variation in DNA methylation is regulated on the Z chromosome we utilised a wild x domestic advanced intercross in the chicken, with both hypothalamic methylomes and transcriptomes assayed in 124 individuals. The relatively large numbers of individuals allowed us to identify additional genomic MHM regions on the Z chromosome that were significantly differentially methylated between the sexes. These regions appear to down-regulate local gene expression in males, but not remove it entirely (unlike the lncRNAs identified in the initial MHM regions). These MHM regions were further tested and the most balanced genes appear to show decreased expression in males, whilst methylation appeared to be far more correlated with gene expression in the less balanced, as compared to the most balanced genes. In addition, quantitative trait loci (QTL) that regulate variation in methylation on the Z chromosome, and those loci that regulate methylation on the autosomes that derive from the Z chromosome were mapped. Trans-effect hotspots were also identified that were based on the autosomes but affected the Z, and also one that was based on the Z chromosome but that affected both autosomal and sex chromosome DNA methylation regulation. We show that both cis and trans loci that originate from the Z chromosome never exhibit an interaction with sex, whereas trans loci originating from the autosomes but affecting the Z chromosome always display such an interaction. Our results highlight how additional MHM regions are actually present on the Z chromosome, and they appear to have smaller-scale effects on gene expression in males. Quantitative variation in methylation is also regulated both from the autosomes to the Z chromosome, and from the Z chromosome to the autosomes.
Assuntos
Galinhas , Cromossomos Sexuais , Animais , Masculino , Galinhas/genética , Metilação de DNA/genética , Mecanismo Genético de Compensação de Dose , Genoma , Mamíferos/genética , Cromossomos Sexuais/genéticaRESUMO
Heteromorphic sex chromosomes (XY or ZW) present problems of gene dosage imbalance between sexes and with autosomes. A need for dosage compensation has long been thought to be critical in vertebrates. However, this was questioned by findings of unequal mRNA abundance measurements in monotreme mammals and birds. Here, we demonstrate unbalanced mRNA levels of X genes in platypus males and females and a correlation with differential loading of histone modifications. We also observed unbalanced transcripts of Z genes in chicken. Surprisingly, however, we found that protein abundance ratios were 1:1 between the sexes in both species, indicating a post-transcriptional layer of dosage compensation. We conclude that sex chromosome output is maintained in chicken and platypus (and perhaps many other non therian vertebrates) via a combination of transcriptional and post-transcriptional control, consistent with a critical importance of sex chromosome dosage compensation.
Assuntos
Galinhas , Mecanismo Genético de Compensação de Dose , Ornitorrinco , Cromossomos Sexuais , Animais , Galinhas/genética , Cromossomos Sexuais/genética , Masculino , Feminino , Ornitorrinco/genética , Transcrição Gênica , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Chicken embryos are a powerful and widely used animal model in developmental biology studies. Since the development of CRISPR technology, gene-edited chickens have been generated by transferring primordial germ cells (PGCs) into recipients after genetic modifications. However, low inheritance caused by competition between host germ cells and the transferred cells is a common complication and greatly reduces production efficiency. Here, we generated a gene-edited chicken, in which germ cells can be ablated in a drug-dependent manner, as recipients for gene-edited PGC transfer. We used the nitroreductase/metronidazole (NTR/Mtz) system for cell ablation, in which nitroreductase produces cytotoxic alkylating agents from administered metronidazole, causing cell apoptosis. The chicken Vasa homolog (CVH) gene locus was used to drive the expression of the nitroreductase gene in a germ cell-specific manner. In addition, a fluorescent protein gene, mCherry, was also placed in the CVH locus to visualize the PGCs. We named this system 'germ cell-specific autonomous removal induction' (gSAMURAI). gSAMURAI chickens will be an ideal recipient to produce offspring derived from transplanted exogenous germ cells.
Assuntos
Galinhas , Metronidazol , Embrião de Galinha , Animais , Galinhas/genética , Células Germinativas/metabolismo , Nitrorredutases/metabolismoRESUMO
During gonadal sex determination, the supporting cell lineage differentiates into Sertoli cells in males and pre-granulosa cells in females. Recently, single cell RNA-seq data have indicated that chicken steroidogenic cells are derived from differentiated supporting cells. This differentiation process is achieved by a sequential upregulation of steroidogenic genes and downregulation of supporting cell markers. The exact mechanism regulating this differentiation process remains unknown. We have identified TOX3 as a previously unreported transcription factor expressed in embryonic Sertoli cells of the chicken testis. TOX3 knockdown in males resulted in increased CYP17A1-positive Leydig cells. TOX3 overexpression in male and female gonads resulted in a significant decline in CYP17A1-positive steroidogenic cells. In ovo knockdown of the testis determinant DMRT1 in male gonads resulted in a downregulation of TOX3 expression. Conversely, DMRT1 overexpression caused an increase in TOX3 expression. Taken together, these data indicate that DMRT1-mediated regulation of TOX3 modulates expansion of the steroidogenic lineage, either directly, via cell lineage allocation, or indirectly, via signaling from the supporting to steroidogenic cell populations.
Assuntos
Galinhas , Processos de Determinação Sexual , Animais , Embrião de Galinha , Masculino , Feminino , Galinhas/genética , Linhagem da Célula , Fatores de Transcrição/metabolismo , Gônadas/metabolismo , Testículo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Diferenciação Sexual/genéticaRESUMO
During avian influenza virus (AIV) infection, host defensive proteins promote antiviral innate immunity or antagonize viral components to limit viral replication. UFM1-specific ligase 1 (UFL1) is involved in regulating innate immunity and DNA virus replication in mammals, but the molecular mechanism by which chicken (ch)UFL1 regulates AIV replication is unclear. In this study, we first identified chUFL1 as a negative regulator of AIV replication by enhancing innate immunity and disrupting the assembly of the viral polymerase complex. Mechanistically, chUFL1 interacted with chicken stimulator of IFN genes (chSTING) and contributed to chSTING dimerization and the formation of the STING-TBK1-IRF7 complex. We further demonstrated that chUFL1 promoted K63-linked polyubiquitination of chSTING at K308 to facilitate chSTING-mediated type I IFN production independent of UFMylation. Additionally, chUFL1 expression was upregulated in response to AIV infection. Importantly, chUFL1 also interacted with the AIV PA protein to inhibit viral polymerase activity. Furthermore, chUFL1 impeded the nuclear import of the AIV PA protein and the assembly of the viral polymerase complex to suppress AIV replication. Collectively, these findings demonstrate that chUFL1 restricts AIV replication by disrupting the viral polymerase complex and facilitating type I IFN production, which provides new insights into the regulation of AIV replication in chickens.
Assuntos
Vírus da Influenza A , Influenza Aviária , Interferon Tipo I , Ubiquitina-Proteína Ligases , Replicação Viral , Animais , Galinhas/genética , Imunidade Inata , Vírus da Influenza A/metabolismo , Vírus da Influenza A/fisiologia , Influenza Aviária/metabolismo , Nucleotidiltransferases , Replicação Viral/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Whole-genome sequencing projects are increasingly populating the tree of life and characterizing biodiversity1-4. Sparse taxon sampling has previously been proposed to confound phylogenetic inference5, and captures only a fraction of the genomic diversity. Here we report a substantial step towards the dense representation of avian phylogenetic and molecular diversity, by analysing 363 genomes from 92.4% of bird families-including 267 newly sequenced genomes produced for phase II of the Bird 10,000 Genomes (B10K) Project. We use this comparative genome dataset in combination with a pipeline that leverages a reference-free whole-genome alignment to identify orthologous regions in greater numbers than has previously been possible and to recognize genomic novelties in particular bird lineages. The densely sampled alignment provides a single-base-pair map of selection, has more than doubled the fraction of bases that are confidently predicted to be under conservation and reveals extensive patterns of weak selection in predominantly non-coding DNA. Our results demonstrate that increasing the diversity of genomes used in comparative studies can reveal more shared and lineage-specific variation, and improve the investigation of genomic characteristics. We anticipate that this genomic resource will offer new perspectives on evolutionary processes in cross-species comparative analyses and assist in efforts to conserve species.
Assuntos
Aves/classificação , Aves/genética , Genoma/genética , Genômica/métodos , Genômica/normas , Filogenia , Animais , Galinhas/genética , Conservação dos Recursos Naturais , Conjuntos de Dados como Assunto , Tentilhões/genética , Humanos , Seleção Genética/genética , Sintenia/genéticaRESUMO
Gene-expression programs define shared and species-specific phenotypes, but their evolution remains largely uncharacterized beyond the transcriptome layer1. Here we report an analysis of the co-evolution of translatomes and transcriptomes using ribosome-profiling and matched RNA-sequencing data for three organs (brain, liver and testis) in five mammals (human, macaque, mouse, opossum and platypus) and a bird (chicken). Our within-species analyses reveal that translational regulation is widespread in the different organs, in particular across the spermatogenic cell types of the testis. The between-species divergence in gene expression is around 20% lower at the translatome layer than at the transcriptome layer owing to extensive buffering between the expression layers, which especially preserved old, essential and housekeeping genes. Translational upregulation specifically counterbalanced global dosage reductions during the evolution of sex chromosomes and the effects of meiotic sex-chromosome inactivation during spermatogenesis. Despite the overall prevalence of buffering, some genes evolved faster at the translatome layer-potentially indicating adaptive changes in expression; testis tissue shows the highest fraction of such genes. Further analyses incorporating mass spectrometry proteomics data establish that the co-evolution of transcriptomes and translatomes is reflected at the proteome layer. Together, our work uncovers co-evolutionary patterns and associated selective forces across the expression layers, and provides a resource for understanding their interplay in mammalian organs.
Assuntos
Evolução Molecular , Mamíferos/genética , Biossíntese de Proteínas , Transcriptoma/genética , Animais , Encéfalo/metabolismo , Galinhas/genética , Feminino , Genes Ligados ao Cromossomo X/genética , Humanos , Fígado/metabolismo , Macaca/genética , Masculino , Camundongos , Gambás/genética , Especificidade de Órgãos/genética , Ornitorrinco/genética , Biossíntese de Proteínas/genética , RNA-Seq , Ribossomos/metabolismo , Cromossomos Sexuais/genética , Especificidade da Espécie , Espermatogênese/genética , Testículo/metabolismo , Regulação para CimaRESUMO
Gene expression is temporally and spatially regulated by the interaction of transcription factors (TFs) and cis-regulatory elements (CREs). The uneven distribution of TF binding sites across the genome poses challenges in understanding how this distribution evolves to regulate spatio-temporal gene expression and consequent heritable phenotypic variation. In this study, chromatin accessibility profiles and gene expression profiles were collected from several species including mammals (human, mouse, bovine), fish (zebrafish and medaka), and chicken. Transcription factor binding sites clustered regions (TFCRs) at different embryonic stages were characterized to investigate regulatory evolution. The study revealed dynamic changes in TFCR distribution during embryonic development and species evolution. The synchronization between TFCR complexity and gene expression was assessed across species using RegulatoryScore. Additionally, an explainable machine learning model highlighted the importance of the distance between TFCR and promoter in the coordinated regulation of TFCRs on gene expression. Our results revealed the developmental and evolutionary dynamics of TFCRs during embryonic development from fish, chicken to mammals. These data provide valuable resources for exploring the relationship between transcriptional regulation and phenotypic differences during embryonic development.
Assuntos
Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Aprendizado de Máquina , Fatores de Transcrição , Animais , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Sítios de Ligação , Humanos , Camundongos , Bovinos , Oryzias/genética , Oryzias/metabolismo , Oryzias/embriologia , Galinhas/genética , Desenvolvimento Embrionário/genética , Regiões Promotoras Genéticas , Cromatina/metabolismo , Cromatina/genéticaRESUMO
Microchromosomes are prevalent in nonmammalian vertebrates [P. D. Waters et al., Proc. Natl. Acad. Sci. U.S.A. 118 (2021)], but a few of them are missing in bird genome assemblies. Here, we present a new chicken reference genome containing all autosomes, a Z and a W chromosome, with all gaps closed except for the W. We identified ten small microchromosomes (termed dot chromosomes) with distinct sequence and epigenetic features, among which six were newly assembled. Those dot chromosomes exhibit extremely high GC content and a high level of DNA methylation and are enriched for housekeeping genes. The pericentromeric heterochromatin of dot chromosomes is disproportionately large and continues to expand with the proliferation of satellite DNA and testis-expressed genes. Our analyses revealed that the 41-bp CNM repeat frequently forms higher-order repeats (HORs) at the centromeres of acrocentric chromosomes. The centromere core regions where the kinetochore attaches often encompass telomeric sequence (TTAGGG)n, and in a one of the dot chromosomes, the centromere core recruits an endogenous retrovirus (ERV). We further demonstrate that the W chromosome shares some common features with dot chromosomes, having large arrays of hypermethylated tandem repeats. Finally, using the complete chicken chromosome models, we reconstructed a fine picture of chordate karyotype evolution, revealing frequent chromosomal fusions before and after vertebrate whole-genome duplications. Our sequence and epigenetic characterization of chicken chromosomes shed insights into the understanding of vertebrate genome evolution and chromosome biology.
Assuntos
Centrômero , Galinhas , Animais , Masculino , Galinhas/genética , Centrômero/genética , Telômero , Heterocromatina , Sequências de Repetição em TandemRESUMO
Human activities have precipitated a rise in the levels of introgressive gene flow among animals. The investigation of conspecific populations at different time points may shed light on the magnitude of human-mediated introgression. We used the red junglefowl Gallus gallus, the wild ancestral form of the chicken, as our study system. As wild junglefowl and domestic chickens readily admix, conservationists fear that domestic introgression into junglefowl may compromise their wild genotype. By contrasting the whole genomes of 51 chickens with 63 junglefowl from across their natural range, we found evidence of a loss of the wild genotype across the Anthropocene. When comparing against the genomes of junglefowl from approximately a century ago using rigorous ancient-DNA protocols, we discovered that levels of domestic introgression are not equal among and within modern wild populations, with the percentage of domestic ancestry around 20-50%. We identified a number of domestication markers in which chickens are deeply differentiated from historic junglefowl regardless of breed and/or geographic provenance, with eight genes under selection. The latter are involved in pathways dealing with development, reproduction and vision. The wild genotype is an allelic reservoir that holds most of the genetic diversity of G. gallus, a species which is immensely important to human society. Our study provides fundamental genomic infrastructure to assist in efforts to prevent a further loss of the wild genotype through introgression of domestic alleles.
Assuntos
Galinhas , Genética Populacional , Genoma , Animais , Galinhas/genética , Fluxo Gênico , Genoma/genética , Genótipo , FilogeniaRESUMO
The biochemical pathway regulating the synthesis of yellow/red pheomelanin is less well characterized than the synthesis of black/brown eumelanin. Inhibitor of gold (IG phenotype) is a plumage colour variant in chicken that provides an opportunity to further explore this pathway since the recessive allele (IG) at this locus is associated with a defect in the production of pheomelanin. IG/IG homozygotes display a marked dilution of red pheomelanin pigmentation, whilst black pigmentation (eumelanin) is only slightly affected. Here we show that a 2-base pair insertion (frame-shift mutation) in the 5th exon of the Catechol-O-methyltransferase containing domain 1 gene (COMTD1), expected to cause a complete or partial loss-of-function of the COMTD1 enzyme, shows complete concordance with the IG phenotype within and across breeds. We show that the COMTD1 protein is localized to mitochondria in pigment cells. Knockout of Comtd1 in a mouse melanocytic cell line results in a reduction in pheomelanin metabolites and significant alterations in metabolites of glutamate/glutathione, riboflavin, and the tricarboxylic acid cycle. Furthermore, COMTD1 overexpression enhanced cellular proliferation following chemical-induced transfection, a potential inducer of oxidative stress. These observations suggest that COMTD1 plays a protective role for melanocytes against oxidative stress and that this supports their ability to produce pheomelanin.
Assuntos
Catecol O-Metiltransferase , Galinhas , Camundongos , Animais , Galinhas/genética , Catecol O-Metiltransferase/genética , Camundongos Knockout , Melaninas/metabolismo , Pigmentação/genética , Mutação da Fase de LeituraRESUMO
Retroviral-mediated misexpression in chicken embryos has been a powerful research tool for developmental biologists in the last two decades. In the RCASBP retroviral vectors that are widely used for in vivo somatic transgenesis, a coding sequence of interest is under the transcriptional control of a strong viral promoter in the long terminal repeat. While this has proven to be effective for studying secreted signalling proteins, interpretation of the mechanisms of action of nuclear factors is more difficult using this system since it is not clear whether phenotypic effects are cell-autonomous or not, and therefore whether they represent a function of the endogenous protein. Here, we report the consequences of retroviral expression using the RCANBP backbone, in which the transcription factor Dlx5 is expressed under the control of chondrocyte-specific regulatory sequences from the Col2a1 gene. To our knowledge, this is the first demonstration of a tissue-specific phenotype in the chicken embryo.
Assuntos
Galinhas , Fatores de Transcrição , Animais , Embrião de Galinha , Galinhas/genética , Fatores de Transcrição/genética , Técnicas de Transferência de Genes , Retroviridae/genética , Regulação da Expressão Gênica , Vetores GenéticosRESUMO
RNA Binding Proteins regulate, in part, alternative pre-mRNA splicing and, in turn, gene expression patterns. Polypyrimidine tract binding proteins PTBP1 and PTBP2 are paralogous RNA binding proteins sharing 74% amino acid sequence identity. Both proteins contain four structured RNA-recognition motifs (RRMs) connected by linker regions and an N-terminal region. Despite their similarities, the paralogs have distinct tissue-specific expression patterns and can regulate discrete sets of target exons. How two highly structurally similar proteins can exert different splicing outcomes is not well understood. Previous studies revealed that PTBP2 is post-translationally phosphorylated in the unstructured N-terminal, Linker 1, and Linker 2 regions that share less sequence identity with PTBP1 signifying a role for these regions in dictating the paralog's distinct splicing activities. To this end, we conducted bioinformatics analysis to determine the evolutionary conservation of RRMs versus linker regions in PTBP1 and PTBP2 across species. To determine the role of PTBP2 unstructured regions in splicing activity, we created hybrid PTBP1-PTBP2 constructs that had counterpart PTBP1 regions swapped to an otherwise PTBP2 protein and assayed on differentially regulated exons. We also conducted molecular dynamics studies to investigate how negative charges introduced by phosphorylation in PTBP2 unstructured regions can alter their physical properties. Collectively, results from our studies reveal an important role for PTBP2 unstructured regions and suggest a role for phosphorylation in the differential splicing activities of the paralogs on certain regulated exons.
Assuntos
Processamento Alternativo , Proteína de Ligação a Regiões Ricas em Polipirimidinas , Vertebrados , Animais , Humanos , Camundongos , Ratos , Éxons/genética , Ribonucleoproteínas Nucleares Heterogêneas/química , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Simulação de Dinâmica Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Especificidade de Órgãos , Fosforilação , Proteína de Ligação a Regiões Ricas em Polipirimidinas/química , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Especificidade da Espécie , Vertebrados/genética , Galinhas/genéticaRESUMO
The outstanding human cognitive capacities are computed in the cerebral cortex, a mammalian-specific brain region and the place of massive biological innovation. Long noncoding RNAs have emerged as gene regulatory elements with higher evolutionary turnover than mRNAs. The many long noncoding RNAs identified in neural tissues make them candidates for molecular sources of cerebral cortex evolution and disease. Here, we characterized the genomic and cellular shifts that occurred during the evolution of the long noncoding RNA repertoire expressed in the developing cerebral cortex and explored putative roles for these long noncoding RNAs in the evolution of the human brain. Using transcriptomics and comparative genomics, we comprehensively annotated the cortical transcriptomes of humans, rhesus macaques, mice, and chickens and classified human cortical long noncoding RNAs into evolutionary groups as a function of their predicted minimal ages. Long noncoding RNA evolutionary groups showed differences in expression levels, splicing efficiencies, transposable element contents, genomic distributions, and transcription factor binding to their promoters. Furthermore, older long noncoding RNAs showed preferential expression in germinative zones, outer radial glial cells, and cortical inhibitory (GABAergic) neurons. In comparison, younger long noncoding RNAs showed preferential expression in cortical excitatory (glutamatergic) neurons, were enriched in primate and human-specific gene co-expression modules, and were dysregulated in neurodevelopmental disorders. These results suggest different evolutionary routes for older and younger cortical long noncoding RNAs, highlighting old long noncoding RNAs as a possible source of molecular evolution of conserved developmental programs; conversely, we propose that the de novo expression of primate- and human-specific young long noncoding RNAs is a putative source of molecular evolution and dysfunction of cortical excitatory neurons, warranting further investigation.
Assuntos
Córtex Cerebral , Macaca mulatta , Neurônios , RNA Longo não Codificante , RNA Longo não Codificante/genética , Humanos , Córtex Cerebral/metabolismo , Animais , Camundongos , Neurônios/metabolismo , Galinhas/genética , Evolução Molecular , TranscriptomaRESUMO
Modifications in gene regulation are driving forces in the evolution of organisms. Part of these changes involve cis-regulatory elements (CREs), which contact their target genes through higher-order chromatin structures. However, how such architectures and variations in CREs contribute to transcriptional evolvability remains elusive. We use Hoxd genes as a paradigm for the emergence of regulatory innovations, as many relevant enhancers are located in a regulatory landscape highly conserved in amniotes. Here, we analysed their regulation in murine vibrissae and chicken feather primordia, two skin appendages expressing different Hoxd gene subsets, and compared the regulation of these genes in these appendages with that in the elongation of the posterior trunk. In the two former structures, distinct subsets of Hoxd genes are contacted by different lineage-specific enhancers, probably as a result of using an ancestral chromatin topology as an evolutionary playground, whereas the gene regulation that occurs in the mouse and chicken embryonic trunk partially relies on conserved CREs. A high proportion of these non-coding sequences active in the trunk have functionally diverged between species, suggesting that transcriptional robustness is maintained, despite considerable divergence in enhancer sequences.
Assuntos
Galinhas , Sequências Reguladoras de Ácido Nucleico , Animais , Galinhas/genética , Cromatina/genética , Desenvolvimento Embrionário/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
N6-methyladenosine (m6A) in mRNA regulates almost every stage in the mRNA life cycle, and the development of methodologies for the high-throughput detection of methylated sites in mRNA using m6A-specific methylated RNA immunoprecipitation with next-generation sequencing (MeRIPSeq) or m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) have revolutionized the m6A research field. Both of these methods are based on immunoprecipitation of fragmented mRNA. However, it is well documented that antibodies often have nonspecific activities, thus verification of identified m6A sites using an antibody-independent method would be highly desirable. We mapped and quantified the m6A site in the chicken ß-actin zipcode based on the data from chicken embryo MeRIPSeq results and our RNA-Epimodification Detection and Base-Recognition (RedBaron) antibody-independent assay. We also demonstrated that methylation of this site in the ß-actin zipcode enhances ZBP1 binding in vitro, while methylation of a nearby adenosine abolishes binding. This suggests that m6A may play a role in regulating localized translation of ß-actin mRNA, and the ability of m6A to enhance or inhibit a reader protein's RNA binding highlights the importance of m6A detection at nucleotide resolution.
Assuntos
Actinas , Galinhas , Animais , Embrião de Galinha , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Actinas/genética , Galinhas/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Anticorpos , Nucleotídeos/metabolismoRESUMO
Influenza A viruses (IAVs) can overcome species barriers by adaptation of the receptor-binding site of the hemagglutinin (HA). To initiate infection, HAs bind to glycan receptors with terminal sialic acids, which are either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc); the latter is mainly found in horses and pigs but not in birds and humans. We investigated the influence of previously identified equine NeuGc-adapting mutations (S128T, I130V, A135E, T189A, and K193R) in avian H7 IAVs in vitro and in vivo. We observed that these mutations negatively affected viral replication in chicken cells but not in duck cells and positively affected replication in horse cells. In vivo, the mutations reduced virus virulence and mortality in chickens. Ducks excreted high viral loads longer than chickens, although they appeared clinically healthy. To elucidate why these viruses infected chickens and ducks despite the absence of NeuGc, we re-evaluated the receptor binding of H7 HAs using glycan microarray and flow cytometry studies. This re-evaluation demonstrated that mutated avian H7 HAs also bound to α2,3-linked NeuAc and sialyl-LewisX, which have an additional fucose moiety in their terminal epitope, explaining why infection of ducks and chickens was possible. Interestingly, the α2,3-linked NeuAc and sialyl-LewisX epitopes were only bound when presented on tri-antennary N-glycans, emphasizing the importance of investigating the fine receptor specificities of IAVs. In conclusion, the binding of NeuGc-adapted H7 IAV to tri-antennary N-glycans enables viral replication and shedding by chickens and ducks, potentially facilitating interspecies transmission of equine-adapted H7 IAVs.IMPORTANCEInfluenza A viruses (IAVs) cause millions of deaths and illnesses in birds and mammals each year. The viral surface protein hemagglutinin initiates infection by binding to host cell terminal sialic acids. Hemagglutinin adaptations affect the binding affinity to these sialic acids and the potential host species targeted. While avian and human IAVs tend to bind to N-acetylneuraminic acid (sialic acid), equine H7 viruses prefer binding to N-glycolylneuraminic acid (NeuGc). To better understand the function of NeuGc-specific adaptations in hemagglutinin and to elucidate interspecies transmission potential NeuGc-adapted viruses, we evaluated the effects of NeuGc-specific mutations in avian H7 viruses in chickens and ducks, important economic hosts and reservoir birds, respectively. We also examined the impact on viral replication and found a binding affinity to tri-antennary N-glycans containing different terminal epitopes. These findings are significant as they contribute to the understanding of the role of receptor binding in avian influenza infection.
Assuntos
Galinhas , Patos , Cavalos , Vírus da Influenza A , Influenza Aviária , Ácidos Neuramínicos , Animais , Humanos , Galinhas/genética , Galinhas/metabolismo , Galinhas/virologia , Patos/genética , Patos/metabolismo , Patos/virologia , Epitopos/química , Epitopos/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Cavalos/genética , Cavalos/metabolismo , Cavalos/virologia , Vírus da Influenza A/química , Vírus da Influenza A/classificação , Vírus da Influenza A/metabolismo , Influenza Aviária/genética , Influenza Aviária/transmissão , Influenza Aviária/virologia , Mutação , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Ácidos Neuramínicos/química , Ácidos Neuramínicos/metabolismo , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/metabolismo , Suínos/virologia , Zoonoses Virais/metabolismo , Zoonoses Virais/transmissão , Zoonoses Virais/virologiaRESUMO
The evolution of gene expression in mammalian organ development remains largely uncharacterized. Here we report the transcriptomes of seven organs (cerebrum, cerebellum, heart, kidney, liver, ovary and testis) across developmental time points from early organogenesis to adulthood for human, rhesus macaque, mouse, rat, rabbit, opossum and chicken. Comparisons of gene expression patterns identified correspondences of developmental stages across species, and differences in the timing of key events during the development of the gonads. We found that the breadth of gene expression and the extent of purifying selection gradually decrease during development, whereas the amount of positive selection and expression of new genes increase. We identified differences in the temporal trajectories of expression of individual genes across species, with brain tissues showing the smallest percentage of trajectory changes, and the liver and testis showing the largest. Our work provides a resource of developmental transcriptomes of seven organs across seven species, and comparative analyses that characterize the development and evolution of mammalian organs.